A compound comprising a first and second antibody or antigen-binding fragment and methods of conjugating the same

Abstract

A compound comprising a first and second antibody or antigen-binding fragment and methods of conjugating the same A compound comprising the formula: A1-S'-L-S''-A2 wherein A1 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); A2 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.

Description

A compound comprising a first and second antibody or antigen-binding fragment and methods of conjugating the sameField

[0001] 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.Background

[0002] Therapeutic agents having both targeting and therapeutic effects are of increasing interest.

[0003] According to 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.

[0004] 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. For example, 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). Typically, 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.

[0005] The efficacy of ADCs as therapeutics may be improved by increasing the specificity for the target cell. In this regard, antibodies having bi- or multi-specificity are known in the art. For example, 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. Furthermore, antibodies have been engineered to express tandem three or four Fab repeats. However, it is often difficult to build a molecular construct with a particular combination of the targeting and effector elements either using chemical synthesis or recombinant technology.

[0006] Accordingly, there remains a need to provide more effective therapies with improved specificity for treating a wide range of diseases.Summary

[0007] In a first aspect, the invention relates to a compound comprising the formula: A1-S’-L-S”-A2,wherein A1represents 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);A2represents 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.

[0008] In some embodiments, the compound may consist essentially of the formula: A1-S’-L-S”- A2, wherein each of A1, A2, S’, S” and L are as defined herein.

[0009] In some embodiments, the compound may consist of the formula: A1-S’-L-S”-A2, wherein each of A1, A2, S’, S” and L are as defined herein.

[0010] In a second aspect, 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: A1-S’-Y’, wherein A1represents 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: A2-S”-Y”, wherein A2represents 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 aliphaticor 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 cyclic alkene or aliphatic or cyclic alkyne group, then Y” is hydrogen or -L”-X” and X” is an azide, nitrone, tetrazine or tetrazole group.

[0011] In a third aspect, 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 A1-S’-Y’, wherein A1represents 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: A2-S”-Y”, wherein A2represents 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 cyclic alkene or aliphatic or cyclic alkyne group, then Y” is hydrogen or -L”-X” and X” is an azide, nitrone, tetrazine or tetrazole group.Brief Description of Figures

[0012] Figure 1 shows a scheme of Bi-Fab formation steps and subsequent payload conjugation. Potential reactive pairs of functionalised Fabs are: R1= N3 and R2= DBCO(SPAAC) I BCN(SPAAC) / Alkyne(CuAAC) or R1= Tz and R2= BCN(iEDDA) / TCO(iEDDA).

[0013] 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.

[0014] Figure 3 shows dose response curves of cell kill assay conducted using a 9-point dose response of an (anti-CD33)-mal-PEG11-mal-(anti-CD33) homodimer conjugated to MMAF in KASUMI-3, HNT-34, MV4-11 and SHI-1 CD33+ cells and JURKAT and DND-39 CD33- cells. Error bars represent the standard error of the mean of 2 biological repeats (n=2) performed in duplicate.Detailed Description

[0015] The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0016] Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012); Therapeutic Monoclonal Antibodies: From Bench to Clinic, Zhiqiang An (Editor), Wiley, (2009); and Antibody Engineering, 2nd Ed., Vols 1 and 2, Ontermann and Dubel, eds., Springer-Verlag, Heidelberg (2010).

[0017] 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.Antibody or antigen-binding fragment

[0018] The compound comprises a first antibody or antigen-binding fragment, A1, and a second antibody or antigen-binding fragment, A2. Suitably, the first and second antibody or antigenbinding fragments each capable of binding to an antigen.

[0019] 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. As used herein, 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.

[0020] The term “epitope” 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.

[0021] 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. 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. In some aspects, 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.

[0022] The term "antibody" as used herein refers to an immunoglobulin (Ig) protein that is capable of binding an antigen. In particular, the term "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. The term antibody as used herein encompasses polyclonal and monoclonal antibody preparations.

[0023] As antibodies can be modified in a number of ways, the term "antibody" should be construed as covering antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain. The term “antibody” should also be construed as covering antibody mimetics, such as, but notlimited 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. 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.

[0024] 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. Unlike polyclonal antibodies, which include different antibodies directed against different epitopes, each monoclonal antibody of in a preparation is directed against a single epitope.

[0025] The antibody, or antigen-binding fragment thereof, described herein, "which binds" or is “capable of binding” an antigen, binds the antigen with sufficient affinity such that the antibody or antigen-binding fragment thereof may be useful as a therapeutic or diagnostic agent. 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.

[0026] “Specifically binds", "specific binding" or "selective binding" 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. The binding reaction may be shown with reference to a negative control test using an antibody of unrelated specificity.

[0027] The terms “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. Hence, “amino acid(s)” or “peptide residue(s)”, as used herein, denote both naturally occurring and synthetic amino acids. In some cases, the immunoglobulin proteins of the present invention may be synthesized using any in vivo or in vitro protein synthesis technique known in the art.

[0028] 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 chainvariable 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.

[0029] 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). 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.

[0030] Immunoglobulin molecules can be subdivided into various types class and subclass. In humans 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.

[0031] 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. Full-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).

[0032] 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. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1 , CDR2 and CDR3, for each of the variable regions. The term "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.

[0033] Heavy chain CDRs are designated HCDR1 , HCDR2 and HCDR3. Light chain CDRs are designated LCDR1 , LCDR2 and LCDR3.

[0034] 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.

[0035] The term "CDR" (complementarity-determining region) refers to the immunoglobulin hypervariable domains within an antibody or antigen binding fragment sequences. It is generallyaccepted that the CDRs determine the specific antibody binding. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1 , CDR2 and CDR3, for each of the variable regions. The term "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.

[0036] 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)). According to the IMGT numbering scheme, a CDR is a loop region of a variable domain, delimited according to the IMGT unique numbering for V domain. There are three CDR-IMGT in a variable domain: CDR1-IMGT (loop BC), CDR2-IMGT (loop C'C"), and CDR3-IMGT (loop FG).

[0037] The Kabat system as described above is used herein, unless otherwise stated. The terms "Kabat numbering", " Kabat definitions" and " Kabat labelling" are used interchangeably herein.

[0038] The term "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.

[0039] It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which generally retain the specificity of the original antibody. Such techniques may involve introducing the CDRs into a different immunoglobulin framework, or grafting variable regions onto a different immunoglobulin constant regions. Alternatively, a hybridoma or other cell producing an antibody molecule may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.

[0040] 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 orantigen-binding fragments are most desirable for use in antibody therapies, as such molecules would elicit little or no immune response in the human subject.

[0041] 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.

[0042] 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. Thus, the antigen-binding fragment may comprise or consist of any of these fragments.

[0043] 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.

[0044] 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).

[0045] The smallest antigen-binding fragment is the single variable fragment, namely the variable heavy (VH) or variable light (VL) chain domain. 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. named the isolated single VH domains "dAbs," for "domain antibodies." The term "dAb" or “sdAb” (for single domain antibody) generally refers to a single immunoglobulin variable domain (VH, VHH or VL) polypeptide that specifically binds antigen. For use in therapy, human singledomain antibodies are preferred over camelid derived VHH, primarily because they are not as likely to provoke an immune response when administered to a patient.

[0046] 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. In certain embodiments 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).

[0047] 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. For example 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.

[0048] 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. In an embodiment, the first antigen binding fragment may comprise a Fab, Fab’, F(ab)2, scFv, (scFv), heavy chain only antibody, single domain antibody, or nanobody. In an embodiment, 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. In an embodiment, the first and second antibody or antigen binding fragment may comprise a Fab or a Fab’. In an embodiment, 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.

[0049] As used herein, the term "single-chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain embodiments, one of the antigen binding moieties, e.g., antigen binding polypeptide construct, is a single-chain Fab molecule, i.e. a Fab molecule wherein the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain. In a particular such embodiment, 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. 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). In certain other embodiments, one of the antigen binding moieties is a single-chain Fv molecule (scFv).

[0050] 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. In one embodiment, 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. In one embodiment the scFv is a fusion protein comprising the heavy chain variable region and the light chain variable region linked via a peptide linker

[0051] 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.

[0052] The antibody or antigen-binding fragment may comprise a monoclonal antibody or antigen-binding fragment thereof.

[0053] 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.

[0054] 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. For example, the hinge region, or part thereof, may be an IgG 1 hinge region, or part thereof.

[0055] 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.

[0056] 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.

[0057] The antibody or antigen-binding fragment may comprise an Fc domain. For example, the first and / or second antibody or antigen-binding fragment may comprise an Fc domain. For example, one of the first or second antibody or antigen-binding fragment may comprise an Fc domain. For example, the first and / or second antibody or antigen-binding fragment maycomprise i) a Fab, F(ab')2, Fv, a single chain Fv fragment (scFv) and / or a single domain antibody and ii) an Fc domain. For example, 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.

[0058] The antibody or antigen-binding fragment may comprise an Fc region also referred to as an Fc domain. The term "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.

[0059] "Fc region", as used herein, 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.

[0060] The Fc sequence of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. By "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. In some embodiments, 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.

[0061] Thus, 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. In one embodiment, the hinge region, or part thereof, is a human lgG1 , lgG2, lgG3 or lgG4 hinge region, or part thereof. For example, the hinge region, or part thereof, is an lgG1 hinge region, or part thereof.

[0062] Thus, the binding molecule, antibody or antigen binding fragment thereof may comprise a CH3 domain. The sequence of the CH3 domain is not particularly limited. In one embodiment, 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.

[0063] Thus, 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.

[0064] 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.

[0065] 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.

[0066] Fc receptors (FcRs) 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).

[0067] "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.

[0068] "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.

[0069] "Antibody-dependent cellular phagocytosis and "ADCP" refer to the destruction of target cells via monocyte or macrophage-mediated phagocytosis.

[0070] The antibody or antigen-binding fragment may comprise a human IgG Fc with effector function.

[0071] In antibody ADCC, FcyRs on the surface of effector cells (natural killer cells, macrophages, monocytes and eosinophils) bind to the Fc region of an IgG which itself is bound to a target cell. Upon binding 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.

[0072] 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.

[0073] 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. In particular, the binding molecule, antibody or antigen binding fragment thereof may include a modified Fc region.

[0074] 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.

[0075] The Fc region may be modified to modulate the effect of the Fc region. For example 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.

[0076] 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).

[0077] 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. Alternatively, 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.

[0078] 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. Fc engineering approaches have been used to determine the key interaction sites for the lgG1 Fc domain with Fey receptors and C1 q and then mutate these positions to reduce or abolish binding. Through alanine scanning the binding site of C1q to a region covering the hinge and upper CH2 of the Fc domain was identified. 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 toFey 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.

[0079] Mutations to decrease or abrogate binding of the CH2 domain to one or more Fey receptors and / or complement are known in the art. 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"). Furthermore, 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). Additionally or alternatively, an antibody molecule of the invention may comprise an alanine, glutamine or glycine at EU position 297.

[0080] 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.

[0081] 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. An example of a modification that may be made to improve half-life is introduction of M252Y / S254T / T256E mutations in lgG1 CH2 domain, this modification is commonly referred to as a “YTE” variant.

[0082] 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. In such embodiments, 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.

[0083] The antibody or antigen-binding fragment may comprise an additional moiety. The additional moiety may provide further function to the molecule. For example the further moiety may be selected from a half-life extending moiety and / or a label.

[0084] 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.

[0085] 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. Thus, the binding may be detected and / or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance.

[0086] 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. The term "half-life" as used herein 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.

[0087] 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.

[0088] 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.

[0089] 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 VLdomain, 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. In an embodiment the antigen binding fragment that binds serum albumin may be a single domain antibody for example a VH. In an embodiment the antigen binding fragment that binds serum albumin may be an scFv.

[0090] 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. For example, 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).

[0091] 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.

[0092] There are several methods by which to produce recombinant antibodies or antigenbinding fragments thereof which are known in the art. One of these is production in an E. coli expression system. In the E. coli expression system, 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. For example, 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. coli, CHO or other host cell) comprising expressing T7 RNA polymerase in the cell which also includes a polynucleotide encoding an immunoglobulin chain that is operably linked to a T7 promoter. For example, 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).

[0093] 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.In addition, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art.

[0094] Thus, 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. When making 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.

[0095] A skilled person will know that there are different ways to identify, obtain and optimise antibodies and antigen-binding fragments that may be used herein, including in vitro and in vivo expression libraries. Optimisation techniques known in the art, such as display (e.g., ribosome and / or phage display) and I or mutagenesis (e.g., error-prone mutagenesis) can be used. The invention therefore also may comprise sequence optimised antibody variants or antigen-binding fragments thereof.

[0096] 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.

[0097] Preferably, the first and second antibody or antigen-binding fragment may each independently comprise an antigen-binding fragment.

[0098] 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. Preferably, the first and second antibody or antigen-binding fragment may each bind to different antigens. In embodiments where the first and second antibody or antigen-binding fragments each bind to different antigens, it will be appreciated that the compound is bispecific. By the term “bispecific”, and like terms as used herein, is meant that the compound is able to simultaneously bind to two different types of antigen or two different epitopes on the same antigen.

[0099] 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 orantigen-binding fragment may each independently comprise an antigen-binding fragment. For example, 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.

[0100] The first and second antibody or antigen-binding fragment may each independently bind to any suitable antigen. For example, the first and second antibody or antigen-binding fragment may each independently bind to CD33, CD7, B7H4, and / or CD56.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] As discussed herein, preferably, the first and second antibody or antigen-binding fragment may each bind to different antigens. For example, 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.

[0105] 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.

[0106] 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.

[0107] 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 fragmentmay 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.

[0108] 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.

[0109] 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. Thus, 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.

[0110] 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.

[0111] 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.

[0112] 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. It will be appreciated by the skilled person that the 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.

[0113] 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.

[0114] 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.

[0115] For the avoidance of doubt, by “derived from”, and like terms as used herein, in relation to the sulphur atoms is meant that said sulphur atom is that present in the side chain of a cysteine residue, (i.e.: -N-CH(CH3SH)-C(=O)-).Linker

[0116] 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.

[0117] The linker may be any suitable linker.

[0118] The linker may comprise a succinimide group. For example, the linker may comprise a succinimide group attached to the first and / or second sulphur atom. Preferably, 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.

[0119] The linker may comprise one or more polyethylene glycol (PEG) groups. For example, 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. As such, 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. Preferably, 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.

[0120] The linker may comprise a polycyclic group. By “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. Preferably, 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. In an embodiment, 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.

[0121] As defined hereinbelow, the linker may be formed via a click chemistry reaction. The term “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. Thus, preferably, 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. For example, 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.Method

[0122] The first and second antibody or antigen-binding fragment are conjugated together via click chemistry. The terms “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.

[0123] As such, 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. As used herein, 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 usedinterchangeably herein). Preferably, 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.

[0124] A click handle may be attached to the first and / or second antibody or antigen binding fragment, as appropriate, in any suitable manner. For example, 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. Sci., 2021 , 12, 9060-9068). Suitably, before attachment, 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.

[0125] 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.

[0126] 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.

[0127] Preferably, the click chemistry may be an inverse electron demand Diels-Alder (iEDDA) reaction.

[0128] 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.Scheme I: thiol-ene reaction (maleimide)

[0129] In some embodiments, the reaction may be between a thiol group and a maleimide group. As such, the first and second antibody or antigen-binding fragment may be conjugated together via a thiol-maleimide reaction.

[0130] In some embodiments, 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. In such embodiments, 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. Preferably, the alkene group, such as cyclic alkene group, or even maleimide group, is terminal. 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.

[0131] In other embodiments, 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’-L2-X”, wherein L2is a linker and each of X’ are alkene groups, such as cyclic alkene groups, or even maleimide groups.

[0132] 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:Scheme II: CuAAC reaction

[0133] 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). Alternatively, a copper-freecatalyst may be used. For example, a pentamethylcyclopentadienyl ruthenium chloride complex may be used as a catalyst to catalyse the azide-alkyne cycloaddition.

[0134] 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). When DIFO is used, for example, 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. Substituents other than fluorines on the cyclooctyne, such as benzene rings for example, also produce this effect. SPAACs reactions are exemplified in schemes Illa and II lb.Scheme lllb: SPAAC reaction (DIBO)

[0135] 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. 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. Thus, three-membered and four-membered cycloalkenes or cycloalkynes, as well as trans-cycloalkenes, make ideal dienophile substrates due to their high ring strain. Electron-donating substituents on the dienophile and electronwithdrawing substituents on the diene accelerate the iEDDA reaction by further reducing the HOMO(Dienophiie)-LUMO(Diene) separation. An iEDDA reaction is exemplified in scheme IV:Scheme IV: iEDDA reaction

[0136] Preferably, the click chemistry may be an inverse electron demand Diels-Alder (iEDDA) reaction. As such, preferably, 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. In some embodiments, the functional groups to be conjugated together may be a tetrazine group and a cyclooctene group, such as transcyclooctene (TCO). In some embodiments, the functional groups to be conjugated together may be a tetrazine group and a cyclooctyne group, such as bicyclononyne (BCN). It will be appreciated by a person skilled in the art that 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).

[0137] In some embodiments, 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. Suitably, the first linker arm, L’, may be attached to the first antibody or antigen-binding fragment via the first sulphur atom, S’. Suitably, the second linker arm, L”, may be attached to the second antibody or antigen-binding fragment via the second sulphur atom, S”.

[0138] Before the first and second antibody or antigen-binding fragment are conjugated together, 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. Before the first and second antibody or antigen-binding fragment are conjugated together, 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.

[0139] 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. 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.

[0140] 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. 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 a cyclooctene group, such as trans-cyclooctene (TCO), or a cyclooctyne group, such as bicyclononyne (BCN).

[0141] For the avoidance of doubt, 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.

[0142] For the avoidance of doubt, 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

[0143] Thus, according to a second aspect of the invention there is provided 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: A1-S’-Y’, wherein A1represents 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: A2-S”-Y”, wherein A2represents 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 oraliphatic or cyclic alkyne group; and when Y’ is -L’-X’ and X’ is an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, then Y” is hydrogen or -L”-X” and X” is an azide, nitrone, tetrazine or tetrazole group.

[0144] In some embodiments, 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.

[0145] In an embodiment, Y’ may be hydrogen, Y” may be -L”-X” and X” may be an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, such a cyclic alkene group, such as maleimide. In such embodiments, 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.

[0146] In some embodiments, 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. Alternatively, 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. In such embodiments, 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.

[0147] In some embodiments, Y’ may be -L’-X’, wherein X’ is a tetrazine group, and Y” may be - L”-X”, wherein X” is a cycloalkene group. Alternatively, Y’ may be -L’-X’, wherein X’ is a cycloalkene, and Y” may be -L”-X”, wherein X” is a tetrazine group. In such embodiments, 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.

[0148] In an embodiment, Y’ may be -L’-X’, wherein X’ is a tetrazine group, and Y” may be -L”- X”, wherein X” is a cycloalkyne group. Alternatively, Y’ may be -L’-X’, wherein X’ is a cycloalkyne, and Y” may be -L”-X”, wherein X” is a tetrazine group. In such embodiments, 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.

[0149] In an embodiment, 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. Alternatively, 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. For example, Y’ may be -L’-X’, wherein X’ is a tetrazine group, and Y” may be -L”-X”, wherein X” is a cyclooctene. Alternatively, Y’ may be -L’-X’, wherein X’ is a cyclooctene group, and Y” may be -L”-X”, wherein X” is a tetrazine group. For example, Y’ may be -L’-X’, wherein X’ is a tetrazine group, and Y” may be -L”-X”, wherein X” is a cyclooctyne. Alternatively, Y’ may be -L’- X’, wherein X’ is a cyclooctyne group, and Y” may be -L”-X”, wherein X” is a tetrazine group. In such embodiments, the first and second antibody or antigen binding fragments may beconjugated together via an inverse electron demand Diels-Alder (iEDDA) reaction, as defined herein.

[0150] In an embodiment, 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. Alternatively, 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. In such embodiments, 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.

[0151] The cyclic alkene conjugating groups may comprise any suitable group. For example, 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. Preferably, the cyclic alkene conjugating group may comprise trans-cyclooctene (TCO).

[0152] The cyclic alkyne conjugating groups may comprise any suitable group. For example, the cyclic alkyne conjugating groups may comprise a dibenzocyclooctyne (DBCO), difluorinated cyclooctyne (DIFO), bicyclononyne (BCN) and / or dibenzocyclooctyne (DICO) group. Preferably, the cyclic alkyne conjugating group may comprise a bicyclononyne (BCN) group, such as bicyclo[6.1 ,0]non-4-yne. In one embodiment, 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.

[0153] In one embodiment, Y’ may be -L’-X’, wherein X’ is a tetrazine group and Y” may be -L”- X”, wherein X” is a cyclooctene group. Alternatively, Y’ may be -L’-X’, wherein X’ is a cyclooctene group, and Y” may be -L”-X”, wherein X” is a tetrazine group. In such embodiments, 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.

[0154] In one embodiment, Y’ may be -L’-X’, wherein X’ is a tetrazine group and Y” may be -L”- X”, wherein X” is a trans-cyclooctene group. Alternatively, Y’ may be -L’-X’, wherein X’ is a trans-cyclooctene group, and Y” may be -L”-X”, wherein X” is a tetrazine group. In such embodiments, 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.

[0155] In an embodiment, Y’ may be -L’-X’, wherein X’ is a tetrazine group, and Y” may be -L”- X”, wherein X” is a bicyclononyne (BCN) group. Alternatively, Y’ may be -L’-X’, wherein X’ is a bicyclononyne (BCN) group, and Y” may be -L”-X”, wherein X” is a tetrazine group. In such embodiments, 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.

[0156] In one embodiment, 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. Alternatively, 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. In suchembodiments, 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.

[0157] When Y’ is -L’-X’, the -L’-X’ group may be attached to the first sulphur atom, S’, via any suitable method. For example, -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.

[0158] When Y” is -L”-X”, the -L”-X” group may be attached to S” via any suitable method. For example, -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.

[0159] When is -L’-X’, 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. As such, 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.

[0160] When Y” is -L”-X”, 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. As such, 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.Further components

[0161] The compound may optionally comprise one or more further moieties.

[0162] In some embodiments, 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).

[0163] The half-life extension moiety may comprise an antibody and / or antigen-binding fragment, a protein, and or a polypeptide. For example, the half-life extension moiety may comprise a serum albumin protein or an antibody or antigen-binding fragment that binds human serum albumin. Preferably, the half-life extension moiety may comprise an antibody or antigen-binding fragment that binds human serum albumin, more preferably an antigen-binding fragment thatbinds 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.

[0164] In some embodiments, the compound may comprise one or more further antibodies and / or antigen-binding fragments. For example, the compound may comprise a total of 3, 4, 5, etc., antibodies and / or antigen-binding fragments. Preferably, 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. For example, 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. It will be appreciated by the skilled person that 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.

[0165] It will be appreciated by a person skilled in the art that in certain embodiments a further antibody or antigen-binding fragment may also be a half-life extension moiety and vice versa.

[0166] 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.

[0167] 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. Thus, the binding may be detected and / or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance.

[0168] The one or more further moieties may be attached to the compound at any suitable position and by any suitable means. For example, when 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, A1and / or A2. The term “recombinantly attached” indicates that a nucleic acid molecule has been designed which encodes the first and / or second antibody and / or antigen-binding fragment, A1and / or A2in 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.

[0169] Suitable recombinant techniques will be known to a person skilled in the art. For example, nucleic acids encoding the first or second antibody and / or antigen-binding fragment as described herein, A1and / or A2, and the further antibody and / or antigen-binding fragment, protein and / or polypeptide, suitably separated by a polypeptide spacer / linker sequence, may beinserted into a plasmid and expressed in a suitable expression system. For example, nucleic acids encoding A1 / 2-(peptide linker)n-M may be expressed in a suitable expression system, wherein A1 / 2represents either of A1or A2, 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 A1 / 2-(peptide linker)n-M that is operably linked to a T7 promoter. For example, 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 A1 / 2-(peptide linker)n-M chain is induced by incubation of the host cell with IPTG (isopropyl-beta-D-thiogalactopyranoside). Transformation can be by any known method for introducing polynucleotides into a host cell. Suitable transformation methods are as already described herein.

[0170] In some embodiments, 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, A1and / or A2, via click chemistry, such as via an inverse electron demand Diels-Alder (iEDDA) reaction. In such embodiments, the compound may suitably comprise the formula:(A3-L3-S’1-)m-A1-S’-L-S”-A2-(-S”2-L4-A4)nwherein each of A1, A2, S’, S” and L are as defined herein; A3and A4each independently represent a further antibody or antigen-binding fragment as defined herein; S’1represents 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”2represents a sulphur atom, other than S”, that is derived from a non-naturally occurring cysteine residue of the first antibody or antigen-binding fragment; L3and L4are 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 A1or A2, as appropriate, via click chemistry. Suitable click chemistries are as defined herein. Preferably, each further antibody or antigen-binding fragment is conjugated to A1or A2via an inverse electron demand Diels-Alder (iEDDA) reaction, as set out herein.

[0171] For example, when each further antibody or antigen-binding fragment is conjugated to A1or A2via an inverse electron demand Diels-Alder (iEDDA) reaction, each further antibody or antigen-binding fragment may have a tetrazine functional group and A1or A2(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).

[0172] Preferably, m is 1 and n is 0 or m is 0 and m is 1 .

[0173] L3and L4are each linkers. Suitable linkers are as defined herein in relation to L. the linkers, L3and L4, are suitably formed by the conjugation of two linking arms using clickchemistry, such as an inverse electron demand Diels-Alder (iEDDA) reaction. Suitable linking arms are as defined herein in relation to L’ and L”.

[0174] L3is attached to A1via a non-naturally occurring cysteine residue, S’1, present in A1. L4is attached to A2via a non-naturally occurring cysteine residue, S”2, present in A2. Said non- naturally 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.

[0175] L3may be attached to A3via any suitable residue. L3may be attached to A3via a naturally occurring amino acid residue, such as a naturally occurring lysine or cysteine residue, such as a naturally occurring cysteine residue. L4may be attached to A4via any suitable residue. L4may be attached to A4via a naturally occurring amino acid residue, such as a naturally occurring lysine or cysteine residue, such as a naturally occurring cysteine residue.

[0176] L3may be attached to A3via a non-naturally occurring cysteine residue. In such embodiments, A3may 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). L4may be attached to A4via a non-naturally occurring cysteine residue. In such embodiments, A4may 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). In such embodiments, the compound may be of the following formula:(A3-S’”-L3-S’1-)m-A1-S’-L-S”-A2-(-S”2-L4-S””-A4)n wherein each of A1, A2, S’, S”, S’1, S”2, L, L3, L4, m and n are as defined herein; A3represents 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); A4represents 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-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 third antibody or antigen-binding fragment; S”” is a sulphur atom derived from a non-naturally occurring cysteine residue of the fourth antibody or antigen-binding fragment; wherein the first and second antibody or antigenbinding fragment are conjugated together via click chemistry, such as via an inverse electron demand Diels-Alder (iEDDA) reaction; wherein the first and third antibody or antigen-binding fragment are conjugated together via click chemistry, such as via an inverse electron demandDiels-Alder (iEDDA) reaction; and wherein the second and fourth antibody or antigen-binding fragment are conjugated together via click chemistry, such as via an inverse electron demand Diels-Alder (iEDDA) reaction.

[0177] In some embodiments, the compound may further comprise a payload.

[0178] A skilled person would know that different payloads are known in the art (see e.g., Gingrich J. How the Next Generation Antibody Drug Conjugates Expands Beyond Cytotoxic Payloads for Cancer Therapy - J. ADC. April 7, 2020).

[0179] In one embodiment, the payload may be a cell killing agent.

[0180] In one embodiment, the payload may be a macrophage class switching agent.

[0181] In one embodiment, the payload may be an immune-modulating payload.

[0182] In one embodiment, the payload may be a light activatable payload.

[0183] In one embodiment, the payload may be a molecular label. By “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. Examples of suitable molecular labels include, but are not limited to, fluorescent molecules, p-galactosidase, luciferase molecules, chemical dyes, fluorophores and / or radioisotopes.

[0184] 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. Thus, 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).

[0185] 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. Suitably, 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 Cu dotatate, [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).

[0186] As used herein, 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. Thus, 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).

[0187] Examples of payloads include agonists for the stimulator of interferon genes protein (STING; transmembrane protein 173; TMEM173). 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. Alternatively, payloads that act on toll-like receptors (TLRs) may be used. For example, agonists that bind to TLR7 and / or TLR8 can be used.

[0188] Another example is a macrophage class switching agent.

[0189] 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.

[0190] The cell killing agent may be a cytotoxin and the skilled person will understand that a range of cytotoxins will be compatible. For example, 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.

[0191] Preferably 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.

[0192] The payload may be a cytotoxic payload or a therapeutic compound, peptide or polypeptide. In particular, the payload is preferably a cytotoxin. The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term is intended to include 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, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including synthetic analogs and derivatives thereof. A "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. Examples of 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), Astrazeneca), AG1478, AG1571 (SU 5271 ; Sugen), alkylating agents such as thiotepa and CYTOXAN(R) cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; TLK 286 (TELCYTA(TM)); acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL(R)); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN(R)), CPT-II (irinotecan, CAMPTOSAR(R)), acetylcamptothecin, scopolectin, and 9- aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; bisphosphonates, such as clodronate; antibiotics such as the enediyne antibiotics (e. g., 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, dactinomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN(R) doxorubicin (including morpholino- doxorubicin, cyanomo[phi]holino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, and deoxydoxorubicin), esorubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; folic acid analogues such as denopterin, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals such as aminoglutethimide, mitotane, and trilostane; folic acid replenisher such as folinic acid (leucovorin); aceglatone; anti-folate anti- neoplastic agents such as ALEMTA(R), LY231514 pemetrexed, dihydrofolate reductase inhibitors such as methotrexate, antimetabolites such as 5-fluorouracil (5-FU) and its prodrugs such as UFT, S-l and capecitabine, and thymidylate synthase inhibitors and glycinamideribonucleotide formyltransferase inhibitors such as raltitrexed (TOMUDEX<1>A, TDX); inhibitors of dihydropyrimidine dehydrogenase such as eniluracil; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK(R) polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISENE(R), FILDESIN (R)); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids and taxanes, e.g., TAXOL(R) paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ.), ABRAXANE(TM) Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and TAXOTERE(R) doxetaxel (Rh[delta]ne-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR(R)); 6-thioguanine; mercaptopurine; platinum; platinum analogs or platinum-based analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine (VELBAN(R)); etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine (ONCOVIN(R)); vinca alkaloid; vinorelbine (NAVELBINE(R)); novantrone; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; topoisomerase inhibitor RFS 2000; topoisomerase inhibitor Dxd, difluorometlhylornithine (DMFO); retinoids such as retinoic acid; pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATESf(TM)) combined with 5-FU and leucovorin.

[0193] Also encompassed by the term “cytotoxic agent” are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX(R) tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LYI 17018, onapristone, and 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 ARHVIIDEX(R) anastrozole; and anti-androgens such as fiutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1 ,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as gene therapy vaccines, for example, ALLOVECTIN(R) vaccine, LEUVECTIN(R) vaccine, and VAXID(R) vaccine;PROLEUKIN(R) rlL-2; LURTOTECAN(R) topoisomerase 1 inhibitor; ABARELIX(R) rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the above

[0194] Preferably 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.

[0195] However, additionally or alternatively, the cytotoxin could also be selected from other known cytotoxins including ricin subunits and other peptide based cytotoxic materials.

[0196] In some embodiments, 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. Such agents may impart their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, proteasome and / or topoisomerase inhibition.

[0197] 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.

[0198] As used herein, an “ADC” is an antibody drug conjugate.

[0199] In one embodiment 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.

[0200] In an embodiment the cytotoxic or cytostatic agent is a tubulin inhibitor. In an embodiment, the tubulin inhibitor is selected from the group consisting of: (a) an auristatin; and (b) a maytansine derivative. In an embodiment, 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. In addition, this class of natural product has numerous asymmetric centres defined by total synthesis studies by Pettit et al (US 4,978,744). It would appear from structure activity relationships that the Dolaisoleuine and Dolaproine residues appear necessary for antineoplastic activity (US 5,635,483 and US 5,780,588). In an embodiment, the auristatin is selected from the group consisting of: Auristatin E (AE); Monomethylauristatin E (MMAE); Auristatin F (MMAF); vcMMAE; vcMMAF; mcMMAE and mcMMAF. In an embodiment, the cytotoxic or cytostatic agent is a maytansine or a structural analogue of maytansine. In an embodiment, 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. In an embodiment the maytansine is selected from the group consisting of: Mertansine (DM1); and a structural analogue of maytansine such as DM3 or DM4. Preferably, the drug is MMAE, MMAF or auristatin MMAF. In an embodiment the cytotoxic or cytostatic agent is an anti-neoplastic agent such as irinotecan or metabolites thereof. Suitable metabolites of irinotecan include SN- 38.

[0201] In an embodiment, the cytotoxic or cytostatic agent is DNA interacting agent. In an embodiment, the DNA interacting agent is selected from the group consisting of: (a) calicheamicins, (b) duocarmycins and (c) pyrrolobenzodiazepines (PBDs). In an embodiment, 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. In an embodiment, the calicheamicin is calicheamicin gamma 1 . In an embodiment, 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). In an embodiment, 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. In an embodiment, 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. In an embodiment, the cytotoxic or cytostatic agent is a synthetic derivative of two pyrrolobenzodiazepines units joined together via a flexible polymethylene tether. In an embodiment, 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.

[0202] In an embodiment, 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. In an embodiment, the cytotoxic or cytostatic agent is a DNA targeted mustard. For example, 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.

[0203] In an embodiment, the cytotoxic or cytostatic agent is selected from the group consisting of: Netropsin; Distamycin; Lexitropsin; Tallimustine; Dibromotallimustine; PNU 157977; and MEN 10710.

[0204] In an embodiment, the cytotoxic or cytostatic agent is a Bis-(benzimidazole) carrier. Preferably, the drug is Hoechst 33258.

[0205] A guanine-specific alkylating agent is a highly regiospecific alkylating agents that reacts at specific nucleoside positions. In an embodiment, 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. In an embodiment, the mitomycin is selected from: Mitomycin A; Mitomycin C; Porfiromycin; and KW- 2149. In an embodiment, the a carmethizole analogue is selected from: Bis- (Hydroxymethyl)pyrrolizidine; and NSC 602668. In an embodiment, the ecteinascidin analogue is Ecteinascidin 743.

[0206] Adenine-specific alkylating agents are regiospecific and sequence-specific minor groove alkylators reacting at the N3 of adenines in polypyrimidines sequences.

[0207] Cyclopropaindolones and duocamycins may be defined as adenine-specific alkylators. In an embodiment, the cytotoxic or cytostatic agent is a cyclopropaindolone analogue. Preferably, the drug is selected from: adozelesin; and carzelesin.

[0208] In an embodiment, the cytotoxic or cytostatic agent is a benz[e]indolone. Preferably, the cytotoxic or cytostatic agent is selected from: CBI-TMI; and iso-CBI.

[0209] In an embodiment, the cytotoxic or cytostatic agent is bizelesin. In an embodiment, 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.

[0210] In an embodiment, the cytotoxic or cytostatic agent is selected from: Cytarabine, Ara-C; Trabectedin (ET-743); and EribulinMesylate. In an embodiment, 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 F; Squalamine; Dehydrodidemnin B; Didemnin B; Cemadotin; Soblidotin; E7389; NVP-LAQ824; Discodermolide; HTI-286; LAF-389; KRN-7000 (Agelasphin derivative); Curacin A; DMMC; Salinosporamide A; Laulimalide; Vitilevuamide; Diazonamide; Eleutherobin; Sarcodictyin; Peloruside A; Salicylihalimides A and B; Thiocoraline; Ascididemin; Variolins; Lamellarin D; Dictyodendrins; ES-285 (Spisulosine); and Halichondrin B.

[0211] The following cytotoxic or cytostatic agent are also encompassed by the present invention: Amatoxins (a-amanitin)-bicyclic octapeptides produced by basidiomycetes of thegenus 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. These moieties possess potent cytotoxic activity which is linked to the stabilisation of microtubules and results in mitotic arrest at the G2 / M transition. Epothilones have demonstrated potent cytotoxicity across a panel of cancer cell lines and has often exhibited greater potency than paclitaxel (X. Pivot et al, European Oncology, 2008;4(2), P42-45). 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 In an embodiment, the drug is amatoxin. In an embodiment, the drug is tubulysin. In an embodiment the drug is Pseudomonas exotoxin. In an embodiment, the drug is cytolysin. In an embodiment, the drug is dolabellanin. In an embodiment, the drug is epothilone.

[0212] The following cytotoxic or cytostatic agent are also encompassed by the present invention. In an embodiment, 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; Etoposide; Hairpin polyamides; Etoposide phosphate; Vinblastine; Vincristine; Vindesine; Taxotere retinoic acid; N8-acetyl spermidine; Camptothecin; Esperamicin; and Ene-diynes.

[0213] In one embodiment, the cell killing portion is a peptide toxin, for example an auristatin such as MMAE or MMAF. In one embodiment, 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.

[0214] In certain embodiments the payload may comprise a small molecule inhibitor with an anticancer activity. For example 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.

[0215] In certain embodiments the payload comprises 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 tumorspecificity, 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.

[0216] Techniques for conjugating cytotoxic or cytostatic agents to proteins, and in particular to antibodies, are well-known. (See, e.g., Alley et ah, Current Opinion in Chemical Biology 2010 14: 1-9; Senter, Cancer J., 2008, 14(3): 154-169.). In certain embodiments, 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. For example, 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).

[0217] The cleavable linker can be pH-sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, the pH- sensitive linker is hydrolysable under acidic conditions. For example, 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) can be used.

[0218] Other 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).

[0219] In some embodiments the linker can be a protease cleavable linker, for example a valinecitrulline, which may be cleaved by cathepsin B in the lysosome.

[0220] 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.

[0221] 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 orantigen-binding fragments, drug moieties, and linkers. Alternatively or additionally, the payload may be conjugated to the first and / or second antibody or antigen-binding fragment via click chemistry as set out herein. For example, 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.

[0222] The linkers may be attached via nucleophilic groups on the first and / or second antibody or antigen-binding fragment, as appropriate. Such 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.

[0223] 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.

[0224] 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. In one embodiment, 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. In another embodiment, 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.

[0225] Likewise, 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.

[0226] 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.

[0227] 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.

[0228] Where a canonical amino acid is used to attach the payload or linker, site directed mutagenesis may be used to introduce a suitable amino acid residue at a suitable position within the binding molecule. In an embodiment 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. 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. Where a 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.

[0229] 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.

[0230] Where the compounds of the invention are provided as an immunoconjugate or an ADC they 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.

[0231] The compound may comprise, consist essentially of, or consist of the formula: A1-S’-L-S”- A2, wherein each of A1, A2, S’, S” and L are as defined herein.Use

[0232] According to 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

[0233] The invention 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.

[0234] 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.

[0235] The 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. In an embodiment, delivery is of the nucleic acid encoding the drug, e.g., a nucleic acid encoding the compound of the invention is delivered.

[0236] Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical, intra-articular or subcutaneous administration. In an embodiment, the compositions are administered parenterally.

[0237] The pharmaceutically acceptable carrier or vehicle can be particulate, so that the compositions are, for example, in tablet or powder form. The term “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. In addition, auxiliary, stabilizing, thickening, lubricating and colouring agents can be used. When administered to a human or animal subject, such as a human subject, 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.

[0238] 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.

[0239] 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.

[0240] As a solid composition for oral administration, the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition typically contains one or more inert diluents. In addition, one or more of the following can be present: 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. When the composition is in the form of a capsule (e. g. a gelatin capsule), it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

[0241] 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. When intended for oral administration, a composition can comprise one or more of a sweetening agent, preservatives, dye / colorant and flavor enhancer. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included.

[0242] Compositions can take the form of one or more dosage units.

[0243] 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.Therapy

[0244] The compounds of the present invention are capable of targeting specific cell populations, such as those found within malignant cell populations, as such they have utility in therapy. As used herein "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 theinvention. In certain embodiments, the cell may be a tumour cell, for example a tumour cell from a haematological malignancy, such as an AML cell.

[0245] 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.

[0246] The term malignancy as used herein 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. In certain embodiments 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. 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.

[0247] 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. Examples of 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.

[0248] In an embodiment the malignancy is an Acute Myeloid Leukaemia (AML) or AML derived cancer. In one embodiment, the disease may be a CD7+CD33+ haematological malignancy. The term "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). It will be appreciated that when the disease is a CD7+CD33+ haematological malignancy, one of the firstand 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.

[0249] 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).

[0250] As used herein, "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. a Phosphatidylserine (PS) expos ure / ann exin V binding assay, a caspase activation assay, a DNA fragmentation assay, a GSH / GSSG determination, a LDH cytotoxicity assay, a live-dead cell assay, or a non-caspase protease activation assay).

[0251] As used herein "inhibit the cell growth” (e.g., referring to target cells) 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. an ATP and ADP determination assay, a cell cycle assay, a cell proliferation assay, a cell viability assay, an LHD cytotoxicity assay, or a live-dead cell assay). 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).

[0252] When the disease is a cancer, 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. Examples of treatments and therapies include, but are not limited to, chemotherapy, immunotherapy, surgery and / or radiation therapy. Such 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.

[0253] Chemotherapy agents include alkylating agents, plant alkaloids, antimetabolites, anthracyclines, topoisomerase inhibitors and corticosteroids. For example, the chemotherapy can include vinorelbine, cisplatin, carboplatin, gemcitabine, paclitaxel, topotecan, docetaxel, irinotecan, pemetrexed, etoposide, or any combination thereof.

[0254] 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, LAG-3 (e.g. Relatlimab), Tim-3 or CTLA4 (e.g. Ipilimumab). The small molecule therapy may be Pexidartinib.

[0255] 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.

[0256] 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 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.

[0257] As an example 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 60mg / kg, 0.1 mg / kg to 40mg / kg, 0.5 mg / kg to 40mg / kg, 1 mg / kg to 40mg / kg, 2 mg / kg to 40mg / kg, 5 mg / kg to 40mg / kg, 10 mg / kg to 40mg / kg, 20 mg / kg to 40mg / kg, 0.1 mg / kg to 30mg / kg, 0.5 mg / kg to 30mg / kg, 1 mg / kg to 30mg / kg, 2 mg / kg to 30mg / kg, 5 mg / kg to 30mg / kg, 10 mg / kg to 30mg / kg, 15 mg / kg to 30mg / kg, 20 mg / kg to 30mg / kg, 0.1 mg / kg to 20mg / kg, 0.5 mg / kg to 20mg / kg, 1 mg / kg to 20mg / kg, 2 mg / kg to 20mg / kg, 5 mg / kg to 20mg / kg, 10 mg / kg to 20mg / kg, or 15 mg / kg to 20mg / kg.

[0258] As an example, 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. Within the treatment cycle the compound, conjugate or composition may be administered to the subject. 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.

[0259] As will be appreciated by the skilled person, the terms “treating”, “treats” and “treatment” 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. As used herein, "treat", "treating" or "treatment" means inhibiting or relieving a disease or disease. For example, 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. Thus, 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.

[0260] The term "subject" or "patient" refers to an animal which is the object of treatment, observation, or experiment. By way of example only, 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.

[0261] Wherein the compound, immunoconjugate or antibody drug conjugate is used in therapy it will be administered to the subject at a therapeutically effective dose. The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of cancer, 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. To the extent the drug may prevent growth and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. For cancer therapy, efficacy can, for example, be measured by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

[0262] The therapeutically effective amount of the compound or composition may be administered orally, topically, by inhalation, insufflation or parenterally. As the skilled person will appreciate, the compound may be formulated, for example in a composition as described herein. The compound be formulated for a particular administration route. For example, 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 afine 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.

[0263] As will be appreciated by the skilled person, 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.

[0264] As used herein, 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. For example, in order to elicit a favourable response in a subject when treating a disorder or infection, 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.

[0265] 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 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.

[0266] Typically, 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.

[0267] For administration by injection, 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. In one embodiment, 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

[0268] The compounds may also be used in various methods, for example in non-therapeutic methods.

[0269] For example, 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.

[0270] The term "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.

[0271] 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.

[0272] 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.

[0273] 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.

[0274] 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

[0275] An aspect of the invention relates to a kit comprising the compound described herein and optionally instructions for use.

[0276] 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 withthe 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.Selected Definitions

[0277] The term "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. These radicals may be optionally substituted with a chloro, bromo, iodo, cyano, nitro, OR19, OC(O)R20, C(O)R21, C(O)OR22, NR23R24, C(O)NR25R26, SR27, C(O)SR27, C(S)NR25R26, aryl or Het, wherein R19to R27each independently represent hydrogen, aryl or alkyl, and / or be interrupted by oxygen or sulphur atoms, or by silano or dialkylsiloxane groups. Examples of such 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. The term “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.

[0278] The term “alkenyl”, as used herein, 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. These radicals may be optionally substituted with a hydroxyl, chloro, bromo, iodo, cyano, nitro, OR19, OC(O)R20, C(O)R21, C(O)OR22, NR23R24, C(O)NR25R2e, SR27, C(O)SR27, C(S)NR25R2e, or aryl, wherein R19to R27each independently represent hydrogen, aryl or alkyl, and / or be interrupted by oxygen or sulphur atoms, or by silano or dialkylsiloxane groups. Examples of such 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. The term “alkenylene”, as used herein, relates to a bivalent radical alkenyl group as defined above. Forexample, an alkenyl group such as ethenyl which would be represented as -CH=CH2, becomes ethenylene, -CH=CH-, when represented as an alkenylene. Other alkenylene groups should be understood accordingly.

[0279] The term "alkynyl", as used herein, 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. These radicals may be optionally substituted with a hydroxy, chloro, bromo, iodo, cyano, nitro, OR19, OC(O)R20, C(O)R21, C(O)OR22, NR23R24, C(O)NR25R2e, SR27, C(O)SR27, C(S)NR25R2e, or aryl, wherein R19to R27each independently represent hydrogen, aryl or lower alkyl, and / or be interrupted by oxygen or sulphur atoms, or by silano or dialkylsiloxane groups. Examples of such radicals may be independently selected from alkynyl radicals include ethynyl, propynyl, propargyl, butynyl, pentynyl, hexynyl and the like. The term “alkynylene”, as used herein, relates to a bivalent radical alkynyl group as defined above. For example, an alkynyl group such as ethynyl which would be represented as -CECH , becomes ethynylene, -CEC-, when represented as an alkynylene. Other alkynylene groups should be understood accordingly.

[0280] The term “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. These radicals may be optionally substituted with a hydroxy, chloro, bromo, iodo, cyano, nitro, OR19, OC(O)R20, C(O)R21, C(O)OR22, NR23R24, C(O)NR25R26, SR27, C(O)SR27, C(S)NR25R26, or aryl, wherein R19to R27each independently represent hydrogen, aryl or lower alkyl, and / or be interrupted by oxygen or sulphur atoms, or by silano or dialkylsilcon groups. Examples of such 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-naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl and the like. The term “arylene”, as used herein, relates to a bivalent radical aryl group as defined above. For example, 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.

[0281] The term “cycloalkyl”, as used herein, refers to any of the above mentioned alkyl groups which comprise one or more cyclic group(s). The term “cycloalkylene”, as used herein, relates to a bivalent radical cycloalkyl group as defined above. For example, 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. It will be understood that the bivalent radical may be attached to two (organic) groups via anysuitable atoms on the ring. For example, the groups may be attached on adjacent atoms or otherwise (for example, at the meta, ortho or para position, as appropriate).

[0282] The terms “het” or “hetero” refer to sulphur, phosphorus, oxygen and / or nitrogen atoms. As such, “heteroalkyl”, “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. Representative examples of 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)NH2, -C(O)NHR', -C(O)N(R')2 , -NHC(O)R', -S(O)2R', -S(O)R', -OH, -halogen, - N3 , -NH2, -NH(R'), -N(R')2and -CN; wherein alkyl is preferably Ci-Cs alkyl and each R' is independently selected from -H, -alkyl, such as Ci-Cs alkyl and aryl. A substituted heterocyclic group may also include one or more of: -NHC=NH)NH2, -NHCONH2, -S(O)2R' and -SR'.

[0283] For the avoidance of doubt, the reference to 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.

[0284] As used herein, unless otherwise expressly specified, all numbers such as those expressing values, ranges, amounts or percentages may be read as if prefaced by the word "about", even if the term does not expressly appear. Also, the recitation of numerical ranges by endpoints includes all integer numbers and, where appropriate, fractions subsumed within that range (e.g. 1 to 5 can include 1 , 2, 3, 4 when referring to, for example, a number of elements, and can also include 1.5, 2, 2.75 and 3.80, when referring to, for example, measurements). The recitation of end points also includes the end point values themselves (e.g. from 1.0 to 5.0 includes both 1.0 and 5.0). Any numerical range recited herein is intended to include all subranges subsumed therein.

[0285] The terms "comprising", "comprises" and "comprised of’ as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.

[0286] The term “consisting essentially of’ or “consists essentially of’ means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components and the like.

[0287] The term “consisting of’ or “consists of’ means including the components specified but excluding other components.

[0288] Whenever appropriate, depending upon the context, the use of the term “comprises” or “comprising” may also be taken to include the meaning “consists essentially of’ or “consistingessentially of’, and also may also be taken to include the meaning “consists of’ or “consisting of’.

[0289] The optional features set out herein may be used either individually or in combination with each other where appropriate and particularly in the combinations as set out in the accompanying claims. The optional features for each aspect or exemplary embodiment of the invention, as set out herein are also applicable to all other aspects or exemplary embodiments of the invention, where appropriate. In other words, the skilled person reading this specification should consider the optional features for each aspect or exemplary embodiment of the invention as interchangeable and combinable between different aspects and exemplary embodiments.

[0290] It should be understood that while the use of words such as “preferable”, “preferably”, “preferred” or “more preferred” in the description suggest that a feature so described may be desirable, it may nevertheless not be necessary and embodiments lacking such a feature may be contemplated as within the scope of the invention as defined in the appended claims. In relation to the claims, it is intended that when words such as “a,” “an,” or “at least one,” are used to preface a feature there is no intention to limit the claim to only one such feature unless specifically stated to the contrary in the claim.

[0291] As used herein, 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.

[0292] The invention is further defined in the following numbered aspects:

[0293] 1 . A compound comprising the formula: A1-S’-L-S”-A2, wherein A1represents 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);A2represents 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; andL is a linker; wherein the first and second antibody or antigen-binding fragment are conjugated together via click chemistry.

[0294] 2. The compound according to aspect 1 , wherein the first and second antibody or antigen-binding fragment each bind to different antigens.

[0295] 3. The compound according to any one of aspects 1 or 2, 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.

[0296] 4. The compound according to aspect 3, 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.

[0297] 5. The compound according to any one of aspects 1-4, wherein the first and second antibody or antigen-binding fragment each independently bind to CD33, CD7, B7H4, and / or CD56.

[0298] 6. The compound according to aspect 5, wherein the first antibody or antigen-binding fragment binds to CD33 and the second antibody or antigen-binding fragment binds to CD7, CD56 or B7H4.

[0299] 7. The compound according to any one of aspects 1-6, wherein 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.

[0300] 8. The compound according to any one of aspects 1-7, wherein the compound is formed via the conjugation of a linker unit of formula X’-L2-X”, wherein L2is a linker, X’ is a first conjugating group selected from an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group and X” is a second conjugating group selected from an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, and each of -S’-H and S”-H as present on the first and second antibody or antigen-binding fragment, respectively, before the first and second antibody or antigen-binding fragment are conjugated together.

[0301] 9. The compound according to aspect 8, wherein X’ and X” each comprise a cyclic alkene group, such as a maleimide group.

[0302] 10. The compound according to any one of aspects 8 or 9, wherein L2comprises a moiety of the formula -(-O-CH2-CH2-)n-, wherein n is from 1 to 20.

[0303] 11. The compound according to any one of aspects 1-7, wherein the 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”.

[0304] 12. The compound according to aspect 11 , wherein, before the first and second antibody or antigen-binding fragment are conjugated together, 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 orcyclic 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.

[0305] 13. The compound according to aspect 12, wherein, before the first and second antibody or antigen-binding fragment are conjugated together, 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 second linker arm, L”. is an azide or tetrazine group.

[0306] 14. The compound according to any one of aspects 12 or 13, wherein the cyclic alkene conjugating groups comprise a cyclooctene group, such as a trans-cyclooctene group; and / or wherein the cyclic alkyne conjugating groups comprise a cyclooctyne group, such as a bicyclononyne group.

[0307] 15. The compound according to aspect 14, wherein, before the first and second antibody or antigen-binding fragment are conjugated together, 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.

[0308] 16. The compound according to aspect 15, wherein, before the first and second antibody or antigen-binding fragment are conjugated together, the first linker arm, L’, comprises a tetrazine conjugating group and the second linker arm, L”, comprises a cyclooctene group, such as a trans-cyclooctene group.

[0309] 17. The compound according to aspect 16, 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.

[0310] 18. The compound according to aspect 17, wherein, before the first and second antibody or antigen-binding fragment are conjugated together, 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.

[0311] 19. The compound according to aspect 19, 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.

[0312] 20. The compound according to any one of aspects 11-19, wherein the first linker arm, L’, is attached to S’ via a succinimide group; and / or wherein the second linker arm, L’, is attached to S” via a succinimide group.

[0313] 21 . The compound according to any one of aspects 11-20, wherein the first linker arm, L’, comprises a moiety of the formula -(-O-CH2-CH2-)n-, wherein n is from 1 to 20; and / or wherein the second linker arm, L”, comprises a moiety of the formula -(-O-CH2-CH2-)n-, wherein n is from 1 to 20.

[0314] 22. The compound according to any one of aspects 1-21 , wherein the 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).

[0315] 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.

[0316] 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.

[0317] 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.

[0318] 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: A1-S’-Y’, wherein A1represents 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: A2-S”-Y”,wherein A2represents 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 cyclic alkene or aliphatic or cyclic alkyne group, then Y” is hydrogen or -L”-X” and X” is an azide, nitrone, tetrazine or tetrazole group.

[0319] 27. The method according to aspect 26, wherein the first and second antibody or antigenbinding fragment each bind to different antigens.

[0320] 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.

[0321] 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.

[0322] 30. The method according to any one of aspects 26-29, wherein the first and second antibody or antigen-binding fragment each independently bind to CD33, CD7, B7H4, and / or CD56.

[0323] 31. The method according to aspect 30, wherein the first antibody or antigen-binding fragment binds to CD33 and the second antibody or antigen-binding fragment binds to CD7, CD56 or B7H4.

[0324] 32. The method according to any one of aspects 26-31 , wherein 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.

[0325] 33. The method according to any one of aspects 26-32, wherein Y’ is -L’-X’ and X’ is an azide, tetrazine, aliphatic or cyclic alkene or aliphatic or cyclic alkyne group and Y” is -L”-X” and 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.

[0326] 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.

[0327] 35. The method according to any one of aspects 26-34, wherein the cyclic alkene conjugating groups comprise cyclooctene; and / or wherein the cyclic alkyne conjugating groups comprise cyclooctyne.

[0328] 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.

[0329] 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.

[0330] 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.

[0331] 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.

[0332] 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.

[0333] 41 . The method according to any one of aspects 26-40, wherein the first linker arm, L’, is attached to S’ via a succinimide group; and / or wherein the second linker arm, L’, is attached to S” via a succinimide group.

[0334] 42. The method according to any one of aspects 26-41 , wherein the first linker arm, L’, comprises a moiety of the formula -(-O-CH2-CH2-)n-, wherein n is from 1 to 20; and / or wherein the second linker arm, L”, comprises a moiety of the formula -(-O-CH2-CH2-)n-, wherein n is from 1 to 20.

[0335] 43. The method according to any one of aspects 26-42, wherein 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.

[0336] 44. The method according to any one of aspects 26-43, wherein 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.

[0337] 45. The method according to aspect 44, wherein 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.

[0338] 46. The method according to any one of aspects 44 or 45, 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.

[0339] 46. 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 A1-S’-Y’, wherein A1represents 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: A2-S”-Y”, wherein A2represents 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 cyclic alkene or aliphatic or cyclic alkyne group, then Y” is hydrogen or -L”-X” and X” is an azide, nitrone, tetrazine or tetrazole group.

[0340] 48. The use according to aspect 47, wherein the first and second antibody or antigenbinding fragment each bind to different antigens.

[0341] 49. The use according to any one of aspects 47 or 48, 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.

[0342] 50. The use according to aspect 49, wherein the 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.

[0343] 50. The use according to any one of aspects 47-49, wherein the first and second antibody or antigen-binding fragment each independently bind to CD33, CD7, B7H4, and / or CD56.

[0344] 51. The use according to aspects 50, wherein the first antibody or antigen-binding fragment binds to CD33 and the second antibody or antigen-binding fragment binds to CD7, CD56 or B7H4.

[0345] 52. The use according to any one of aspects 47-51 , wherein 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.

[0346] 53. The use according to any one of aspects 47-52, wherein Y’ is -L’-X’ and X’ is an azide, tetrazine, aliphatic or cyclic alkene or aliphatic or cyclic alkyne group and Y” is -L”-X” and 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.

[0347] 54. The use according to any one of aspects 47-53, 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.

[0348] 55. The use according to any one of aspects 47-54, wherein the cyclic alkene conjugating groups comprise cyclooctene; and / or wherein the cyclic alkyne conjugating groups comprise cyclooctyne.

[0349] 56. The use according to any one of aspects 47-55, 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.

[0350] 57. The use according to aspect 56, 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.

[0351] 58. The method according to aspect 57, 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.

[0352] 59. The method according to aspect 58, 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.

[0353] 60. The compound according to aspect 59, 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.

[0354] 61. The use according to any one of aspects 46-60, wherein the first linker arm, L’, is attached to S’ via a succinimide group; and / or wherein the second linker arm, L’, is attached to S” via a succinimide group.

[0355] 62. The use according to any one of aspects 46-61 , wherein the first linker arm, L’, comprises a moiety of the formula -(-O-CH2-CH2-)n-, wherein n is from 1 to 20; and / or wherein the second linker arm, L”, comprises a moiety of the formula -(-O-CH2-CH2-)n-, wherein n is from 1 to 20.

[0356] 63. The use according to any one of aspects 46-62, wherein 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.

[0357] 64. The use according to any one of aspects 46-63, wherein 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.

[0358] 65. The use according to aspect 64, wherein 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.

[0359] 66. The use according to any one of aspects 64 or 65, 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.ExamplesReagents:

[0360] The following reagents were used in the examples: 1 X PBS, pH 7.4, Gibco™ (#15374875) Fisher Scientific Tris(2-carboxyethyl)phosphine HCI (C4706) Sigma- Aid rich (L)-Dehydroascorbic acid (#261556) Sigma-Aldrich Mal-prop-NHS (#HY-129526) MedChemExpressNH2-PEG3-N3 (#BP-20580) BroadPharmMal-PEG3-Tz-me (#AP10734) AxisPharm Mal-PEG3-DBCO (#CP-2030) ConjuProbe Mal-PEG4-Alkyne (#764248) Sigma- Aid richMal-PEG3-BCN (#CP-6005) ConjuProbe Mal-PEG3-TCO (790443) Sigma-AldrichBis-Mal-PEGn (#BP-22151) BroadPharmC11SO4.5H2O (#07631) Sigma-AldrichSodium ascorbate (#A7631) Sigma-AldrichProtein Concentrators PES, 30KDa MWCO (#15451025) Fisher ScientificSterile syringe filter, PVDF, 4mm, 0.22 pm (#10400031) Fisher ScientificCell Lines (KASUMI-3, HNT-34, JUKRAT, MV4-11 , SHI-1 , DND-39) DSMZClear bottom 96-well plates CytoOne®, Non-Treated (#007672-7596) STARLABSDisposable PS Reservoirs-StarTub PS (#E2310-1010) STARLABSCell Proliferation Reagent WST-1 (#ab155902) Abeam2mL Polypropylene Deep Well Plate (#AXYPDW20CS) SLSRPMI-1640 medium (#21875059) Life TechnologiesFetal Bovine Serum, Heat inactivated (#11533387) Life TechnologiesExample 1Method:

[0361] Compounds comprising two antibodies conjugated by click chemistry were prepared in accordance with the reaction scheme shown in Figure 1. The click chemistries used were as follows:SPAAC (Jewett & Bertozzi, Chem. Soc. Rev. 2010, 39 (4): 1272-1279);CuAAC (Sharpless et al., Ang. Chemie. Int. edt, 2002, 41 (14): 2596-2599); iEDDA (Bernardes, Oliveria & Guo, Chem. Soc. Rev. 2017, 46, 4895-4950)Functionalisation of Fab with R1 / R2and Bi-Fab formation:

[0362] Fabs were modified to permit Bi-Fab formation by bioorthogonal reactive partners.

[0363] 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.

[0364] 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.

[0365] Step 3: After 1 hour one of the following functionalisation reagents was introduced (10 eq.@ 10 mg / mL in DMSO):Mal-PEG3-N3*Mal-PEG3-Tz-meMal-PEG3-DBCOMal-PEG4-AlkyneMal-PEG3-BCNMal-PEG3-TCOBis-Mal-PEGu*Mal-PEG3-N3was synthesised from Mal-prop-NHS and NH2-PEG3-N3immediately prior to use.

[0366] 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.

[0367] 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:Table 1 - Conjugation pairs

[0368] For the azide-alkyne reaction, CuSO4.HCI (5 eq.) and sodium ascorbate (25 eq.) were also included to promote the CuAAC click reaction. If the scale was appropriate, impurities (unreacted Fab) were removed by pSEC, fractions containing purified bi-Fab were then combined and sterile filtered (0.22 pm) prior to storage or conjugation with payload.Results:

[0369] Initial work used a SPAAC reaction between DBCO and N3to link two Fabs together. The reaction required relatively harsh conditions (37°C and high concentration) and resulted in a bi- Fab yield of <15%. The bi-Fab formation step (Step 4, Figure 1) was therefore investigated for further improvement.

[0370] A trial was conducted of 3 bioorthogonal click reaction mechanisms, SPAAC, CuAAC and iEDDA, that could be used to form bi-Fabs. Each step of Fab functionalisation was performed at 37°C (Steps 1-3, Figure 1). Bi-Fab formation was performed at room temperature (Step 4, Figure 1). Over 24 hours passed between the purification of functionalised Fabs andcommencement of the Bi-Fab formation reactions. Samples of the Bi-Fab formation reactions were taken after 4 and 22 hours for analysis by aSEC. Samples were also analysed using SDS- PAGE after 22 hours which showed bands at 100 kDa for all 3 reactions (Error! Reference source not found.). New peaks were observed with all samples when they were analysed by aSEC, the area of the peak compared to the remaining starting material peaks was used to determine product conversion. Results are shown in Table 2 below, wherein % conversions were calculated from aSEC spectra.Table 2 - ResultsSelecting iEDDA reaction partners:

[0371] 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-PEG3-Tz-me, Mal-PEG3-TCO and Mal-PEG3-BCN and used immediately for Bi-Fab formation. Samples were taken after 1 hour and 18 hours for analysis by aSEC. Results are shown in Table 3 below, wherein % conversions are calculated from aSEC spectra. Samples were also analysed using SDS-PAGE after 18 hours which showed bands at 100 kDa (data not shown).

[0372] Immediate use of the functionalised Fabs increased the conversion to Bi-Fab for the Tz- TCO reaction (57% conversion, Entry 1 , Table 3) compared to when the functionalised Fabs were stored for over 24 hours before use in the Bi-Fab formation reaction (20% conversion, Entry 3, Table 2). There was little difference between the conversion to Bi-Fab when using Tz- TCO or Tz-BCN based reactions (Table 3).Table 3 - ResultsImproving reaction conditions:

[0373] 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 alarger 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).

[0374] During the Fab functionalisation steps, insoluble aggregate was observed at the larger scale. The performance of these steps at 37°C was examined as the cause of the aggregation. Trial reduction, reoxidation and functionalisation reactions were performed at room temperature. The room temperature reactions successfully produced me-Tz and BCN functionalised Fabs. When the room temperature conditions were applied to a larger scale reaction (5 mg Fab starting material), no aggregate was observed which resulted in a small increase in Bi-Fab yield (36%).Example 2: (anti-CD33)-mal-PEG11-mal-(anti-CD33) homodimer conjugated to MMAF (‘(anti-CD33)-mal-PEG11 -mal-(anti-CD33) homodimer ADC’)

[0375] A homodimer of two anti-CD33 Fabs linked by a bis-mal-PEG11 linker was first prepared according to the following method.Method - Formation of Bi-Fabs:

[0376] An anti-CD33 Fab having an I207C mutation in accordance with SEQ ID NO. 1 was prepared. Fabs were modified to permit homodimer formation via bis-maleimide reactive linkers.

[0377] The desired 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 pH7.4 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) was then introduced, and the reaction mixture was gently shaken before being incubated at room temperature for 90 minutes under agitation. After 90 minutes (L)-Dehydroascorbic acid (DHAA; 20 eq. @ 20 mg I mL in DMSO) was introduced. The 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.

[0378] Purification of the resultant homodimer Bi-Fab was performed by preparative SEC. The appropriate fractions were then combined and concentrated using a centrifugal ultrafiltration tube (MWCO 50 kDa) - aiming for a concentration of 1- 2 mg / mL. Once concentrated, the solution was sterile filtered (0.22 pM) for storage.Method - Cell cytotoxicity:

[0379] Cell lines were harvested, counted and the volume required to seed 20,000 (KASUMI-3, MV4-11 , SHI-1), 10,000 (HNT-34) and 5,000 (JURKAT, DND-39) cells per well in 50 pl media calculated for a 96-well plate. A 9-point dose response of ADC was prepared in assay media (RPMI-1640, 10% FBS) at 2x the final concentration with a top final concentration of 30nM. 50pl of each dose was pipetted across duplicate wells in a 96 well plate and a separate plate wasprepared for each cell line tested. 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.Results:

[0380] As shown in Figure 1 , 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.

[0381] As such, it has been shown that the (anti-CD33)-mal-PEG11-mal-(anti-CD33) homodimer ADC induces cell kill in CD33 expressing cell lines.Example 3:Method - Functionalisation of Fab

[0382] 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. @ 10 mg / mL in water) was then introduced, and the reaction mixture was gently shaken before being incubated at room temperature for 90 minutes. After 90 minutes, (L)- dehydroascorbic acid (DHAA; 20 eq. @ 20 mg I mL in DMSO) was introduced, and the reaction mixture was gently shaken before being incubated at room temperature for 1 hour. After this time, the desired functionalisation reagent, Mal-PEGs-Tz-me or Mal-PEGs-BCN, was introduced (10 eq. @ 10 mg / mL in DMSO). The reaction mixture was gently shaken, then incubated at room temperature for 30 minutes. 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.Functionalisation of Fab-Fc

[0383] An anti-CD33 Fab-Fc (1 eq.) was functionalised with a desired functionalisation reagent, Mal-PEGs-Tz-me or Mal-PEGs-BCN (10 eq. @ 10 mg / mL in DMSO), using the same method described above.Bi-Fab-Fc formation

[0384] The appropriately functionalised Fab and Fab-Fc (e.g. Fab-BCN and Fab-Fc-Tz) were combined at a 1 :1 molar ratio and gently shaken, then incubated at room temperature overnight. Unreacted Fab / Fab-Fc were removed by preparative size exclusion chromatography (pSEC) or a combination of pSEC and preparative ion exchange chromatography (plEX). Fractions containing purified Bi-Fab-FC were then combined and sterile filtered (0.22 pm) prior to storage or conjugation with payload.Payload conjugation (MMAF)

[0385] 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.Example 17 - Manufacturability Assessment of Bi-Fab ADC at gram scale

[0386] Manufacturability of the Bi-Fab ADCs was assessed at multi-gram scale by a global contract development and manufacturing organisation (CDMO). Fab protein starting materials were generated using non-optimised CMC platform processes by an affinity capture and IEX polishing step. 1 .5 g of Fab protein per litre clarified cell culture from CHO stable pools was obtained at high purity.

[0387] The manufacturing process was optimised for production at scale, resulting in an overall final process yield of 34 % or 5.55 g final drug substance was achieved from 8 g scale relative to each Fab protein input. Drug substance had Critical Quality Attributes of an average DAR 3.98 and 98 % monomer purity as assessed by reverse phase - mass spectrometry (RP-MS) and analytical SEC, respectively.Sequences:Anti-CD33 VH (SEQ ID NO. 1)EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKF KNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSSVH CDR1 (SEQ ID NO. 2)GYTITDSNVH CDR2 (SEQ ID NO. 3)IYPYNGGTVH CDR3 (SEQ ID NO. 4)VNGNPWLAYAnti-CD33 VL (SEQ ID NO. 5)DIQLTQSPSTLSASVGDRVTI ICRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKLEIKVL CDR1 (SEQ ID NO. 6)ESLDNYGIRFVL CDR2 (SEQ ID NO. 7)AASNVL CDR3 (SEQ ID NO. 8)QQTKEVPWS

Claims

CLAIMS1 . A compound comprising the formula:A1-S’-L-S”-A2wherein A1represents 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);A2represents 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; andL is a linker; wherein the first and second antibody or antigen-binding fragment are conjugated together via click chemistry.

2. The compound according to claim 1 , wherein the 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”.

3. The compound according to claim 2, wherein, before the first and second antibody or antigen-binding fragment are conjugated together, 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’, 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; and / orwherein, before the first and second antibody or antigen-binding fragment are conjugated together, 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 second linker arm, L”. is an azide or tetrazine group.

4. The compound according to claim 3, wherein the cyclic alkene conjugating groups comprise a cyclooctene group; and / or wherein the cyclic alkyne conjugating groups comprise a cyclooctyne group.

5. The compound according to claim 4, wherein, before the first and second antibody or antigen-binding fragment are conjugated together, the first linker arm, L’, comprises a tetrazine conjugating group and the second linker arm, L”, comprises a conjugating group selected from a cyclic alkene or cyclic alkyne group, and, optionally, 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); and / or wherein, before the first and second antibody or antigen-binding fragment are conjugated together, 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, optionally, 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); 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, and. optionally, wherein the click chemistry used to conjugate together the first and second antibody or antigen-binding fragments is a copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction and / or a strained-promoted azide-alkyne click chemistry (SPAAC) reaction.

6. The compound according to claim 5, wherein, before the first and second antibody or antigen-binding fragment are conjugated together, the first linker arm, L’, comprises a tetrazine conjugating group and the second linker arm, L”, comprises a conjugating groupselected from a trans-cyclooctene (TCO) group or a bicyclononyne (BCN) group, and, optionally, 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).

7. The compound according to any one of claims 2-6, wherein the first linker arm, L’, is attached to S’ via a succinimide group; and / or wherein the second linker arm, L’, is attached to S” via a succinimide group.

8. The compound according to any one of claims 4-8, wherein the first linker arm, L’, comprises a moiety of the formula -(-O-CH2-CH2-)n-, wherein n is from 1 to 20; and / or wherein the second linker arm, L”, comprises a moiety of the formula -(-O-CH2-CH2-)n-, wherein n is from 1 to 20.

9. The compound according to claim 1 , wherein the compound is formed via the conjugation of a linker unit of formula X’-L2-X”, wherein L2is a linker, X’ is a first conjugating group selected from an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, for example a cyclic alkene group, for example a maleimide group, and X” is a second conjugating group selected from an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, for example a cyclic alkene group, for example a maleimide group, and each of -S’-H and S”- H as present on the first and second antibody or antigen-binding fragment, respectively, before the first and second antibody or antigen-binding fragment are conjugated together.

10. The compound according to claim 9, wherein L2comprises a moiety of the formula -(-O- CH2-CH2-)n-, wherein n is from 1 to 20.

11. The compound according to any one of claims 1-10, wherein the 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.

12. The compound according to any one of claims 1-11 , 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).

13. 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: A1-S’-Y’,wherein A1represents 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: A2-S”-Y”, wherein A2represents 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 cyclic alkene or aliphatic or cyclic alkyne group, then Y” is hydrogen or -L”-X” and X” is an azide, nitrone, tetrazine or tetrazole group.

14. 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 A1-S’-Y’, wherein A1represents 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: A2-S”-Y”,wherein A2represents 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 cyclic alkene or aliphatic or cyclic alkyne group, then Y” is hydrogen or -L”-X” and X” is an azide, nitrone, tetrazine or tetrazole group.

15. The method according to claim 13 or use according to claim 14, wherein Y’ is -L’-X’ and X’ is an azide, tetrazine, aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, and Y” is -L”-X” and 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; or 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.

16. The method according to any one of claims 13 or 15 or use according to any one of claims 14 or 15, wherein the cyclic alkene conjugating groups comprise cyclooctene; and / or wherein the cyclic alkyne conjugating groups comprise cyclooctyne.

17. The method according to any one of claims 13, 15 or 16 or use according to any one of claims 14-16, wherein Y’ is -L’-X’ and X’ is a tetrazine group and Y” is -L”-X” and X” is a cyclic alkene or cyclic alkyne group, and, optionally, 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) and / or 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, optionally, 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);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, for example an alkyne or cyclooctene group, and. optionally, wherein the click chemistry used to conjugate together the first and second antibody or antigen-binding fragments is a copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction and / or a strained- promoted azide-alkyne click chemistry (SPAAC) reaction.

18. The method or use according to claim 17, wherein Y’ is -L’-X’, wherein 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’, wherein X’ is a trans-cyclooctene (TCO) group, and Y” is -L”-X”, wherein X” is a tetrazine group, and, optionally, 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).

19. The method or use according to claim 17, wherein Y’ is -L’-X’, wherein X’ is a tetrazine group, and Y” is -L”-X”, wherein X” is a bicyclononyne (BCN) group; and / or wherein Y’ is -L’-X’, wherein X’ is a bicyclononyne (BCN) group, and Y” is -L”-X”, wherein X” is a tetrazine group, and, optionally, 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).

20. The method according to any one of claims 13 or 15-19 or use according to any one of claims 14-19, wherein the first linker arm, L’, is attached to S’ via a succinimide group; and / or wherein the second linker arm, L’, is attached to S” via a succinimide group.

21. The method according to any one of claims 13 or 15-20 or use according to any one of claims 14-20, wherein the first linker arm, L’, comprises a moiety of the formula -(-O-CH2- CH2-)n-, wherein n is from 1 to 20; and / or wherein the second linker arm, L”, comprises a moiety of the formula -(-O-CH2-CH2-)n-, wherein n is from 1 to 20.

22. The method according to any one of claims 13 or 15-21 or use according to any one of claims 14-21 , wherein 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.

23. The method or use according to claim 22, wherein the click chemistry used to conjugate together the first and second units is an inverse electron demand Diels-Alder (iEDDA) reaction.

24. The compound according to any one of claims 1-12, method according to any one of claims 13 or 15-23 or use according to any one of claims 14-23, wherein the first and second antibody or antigen-binding fragment each bind to different antigens.

25. The compound according to any one of claims 1-12 or 24, method according to any one of claims 13 or 15-24 or use according to any one of claims 14-24, 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, optionally 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.

26. The compound according to any one of claims 1-12, 24 or 25, method according to any one of claims 13 or 15-25 or use according to any one of claims 14-25, wherein the first and second antibody or antigen-binding fragment each independently bind to CD33, CD7, B7H4, and / or CD56, for example wherein the first antibody or antigen-binding fragment binds to CD33 and the second antibody or antigen-binding fragment binds to CD7, CD56 or B7H4.

27. The compound according to any one of claims 1-12 or 24-26, method according to any one of claims 13 or 15-26 or use according to any one of claims 14-26, wherein 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.

28. The compound according to any one of claims 1-12 or 24-27, method according to any one of claims 13 or 15-27 or use according to any one of claims 14-27, wherein 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.

29. The compound, method or use according to claim 28, wherein the half-life extension moiety comprises a serum albumin protein, an antibody or antigen-binding fragment thatbinds to a serum albumin protein, an Fc domain and / or a modified Fc domain, for example an Fc domain and / or a modified Fc domain.

30. The compound, method or use according to any one of claims 28 or 29, 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.

Images