Antibody conjugate and methods

Abstract

The present invention relates to antibodies or antigen binding fragments thereof comprising functionalized monosaccharides in the Fab region. The invention also provides an antibody or an antigen binding fragment thereof, comprising at least one N-linked glycan in one or more specific position in the Fab region of said antibody or antigen binding fragment thereof, related methods for producing such antibody and payload enhancement.

Description

[0001] ANTIBODY CONJUGATE AND METHODS

[0002] Field of the Invention

[0003] The present invention relates to engineered / modified antibodies or antigen binding fragments thereof, comprising a modified Fab domain comprising at least one functionalized monosaccharide. The modified antibodies of the invention may be used for the preparation of immunoconjugates and radioimmunoconjugates.

[0004] Background

[0005] Antibody-drug conjugates (ADCs) and radioligand therapy (RLT) represent an innovative and rapidly growing class of targeted therapeutics that combine the specificity of monoclonal antibodies (mAbs) with the potent cytotoxicity activity of small-molecule drugs or radionuclides. By delivering cytotoxic payloads directly to tumour cells, ADCs offer significant therapeutic potential, particularly in oncology, where ADCs have demonstrated impressive clinical activity (Riccardi et al, 2023). Indeed, combining a toxic payload to an antibody minimizes off-target effects, spares healthy tissues, and enhances therapeutic efficacy. Similarly, radioligand therapy is an emerging treatment strategy in oncology that delivers targeted radiation to cancer cells (Parakh et al, Cancers (Basel). 2022 Mar 11 ;14(6):1454).

[0006] To harness the therapeutic potential of ADCs, a cytotoxic payload is chemically conjugated to the antibody via a stable linker. Traditional ADC conjugation methods primarily rely on the modification of naturally occurring cysteine or lysine residues on the antibody using various linker-payload technologies (Fu et al, 2022). However, these methods result in heterogenous ADC products, as they lack precise control over the drug-to-antibody ratio (DAR). Specifically, cysteine-mediated conjugation requires, in a typical mAb, reduction of the four interchain disulfide bonds, but the extent of reduction is difficult to control, leading to DAR values that can range anywhere between 0 to 8 depending on the number of bonds reduced (van den Berg et al, 2023; Zhu et al, 2014). Lysine-mediated conjugation lacks specificity as lysine residues are unevenly distributed throughout the antibody and vary in their local environment, impacting DAR heterogeneity (Haque et al, 2021). Indeed, these variations in the DAR can impact pharmacokinetics (PK), pharmacodynamics (PD), and overall therapeutic index of ADCs (McCombs & Owen, 2015; Boswell et al, 2011). For example, a low DAR reduces potency of the ADC, while a high DAR can result in increased toxicity due to off-target effects (Wagh et al, 2018; Nguyen et al, 2023). Furthermore, cysteine and lysine conjugation at non-optimal sites can cause antibody instability and aggregation further complicating the development of effective ADCs (Johann et al, 2024 (1); Johann et al, 2024 (2)). Therefore, there is a need in the art to develop better methods to precisely control the DAR.Other methods leverage the Fc N-glycan for conjugation, however this strategy is inherently limited by structural constraints of the antibody. The Fc region contains only a single native N-glycan site, restricting the number and positioning of conjugation sites, which in turn limits control over DAR. Additionally, because conjugation is confined to a single N-glycan per heavy chain, ADCs generated using this approach can have unfavourable biophysical properties due to the localized concentration of linker-payload hydrophobicity at the N-glycan site, which can impact stability and PK (Yang & Liu, 2024). Furthermore, Fc N-glycan modifications can alter interactions with Fey receptors (FcyRs), potentially disrupting immune effector functions, such as ADCC, which can be critical for therapeutic efficacy (Reusch & Tejada, 2015).

[0007] To address these issues, the present invention focuses on a novel N-glycan-based conjugation strategy aimed at facilitating efficient and controlled pay load conjugation to the Fab region of the antibody, without disrupting Fc-mediated functions or antigen binding. Specifically, the invention uses targeted Fab N-glycan engineering to introduce defined glycosylation sites for precise and homogenous conjugation of the payload. By introducing additional N-glycan sites within the Fab region, this approach provides greater flexibility in controlling DAR, allowing for the generation of both low- and high-DAR ADCs or radioligand therapy while maintaining product stability and minimizing aggregation. This glycan-mediated conjugation strategy ensures control over DAR and significantly reduces heterogeneity of the ADC or radioligand product, resulting in more consistent therapeutic properties. The present invention represents a significant advancement in ADC or radioligand design, providing a method to precisely control DAR and improve conjugation efficiency.

[0008] Summary of the Invention

[0009] The present inventors have surprisingly shown that by introducing engineered glycosylation sites at specific positions within the Fab region functionalized, enhanced conjugation efficiency can be achieved compared to conjugation at wild-type glycosylation sites. In particular, such enhanced conjugation efficiency may be reflected by an increased proportion of antibody molecules successfully conjugated with a payload, improved uniformity of conjugation across the antibody population, and / or greater control over the number of payloads attached per molecule. As such these modified antibodies are particularly useful in the preparation of antibody-drug conjugates wherein efficient payload or radionuclide conjugation is required. Furthermore, the inventors have also demonstrated that it is possible to introduce one or more engineered glycosylation sites and achieve higher drug to antibody ratio (DAR) with excellent conjugation efficiency and control.

[0010] In one aspect, the invention relates to an antibody or antigen binding fragment thereof, comprising engineered glycosylation site at one or more of amino acid position 190 of the lightchain of said antibody or antigen binding fragment thereof, 159 and / or 186 of the heavy chain of said antibody or antigen binding fragment thereof.

[0011] In one aspect, the invention relates to an antibody or antigen binding fragment thereof, comprising at least one N-linked glycan functionalized in the Fab region of said antibody or antigen binding fragment thereof, wherein the at least one N-linked glycan functionalized is present at one or more of amino acid position 190 of the light chain of said antibody or antigen binding fragment thereof, 159 and / or 186 of the heavy chain of said antibody or antigen binding fragment thereof.

[0012] In one embodiment the at least one N-linked glycan comprises a functionalized monosaccharide. In one embodiment the functionalized monosaccharide is selected from a functionalized sialic acid, a functionalized galactose, a functionalized N-acetylglucosamine (GIcNAc), a functionalized N-acetylgalactosamine GalNAc.

[0013] In one embodiment the functionalized monosaccharide is selected from N-azidoacetylneuraminic acid, N-azido acetylglucosamine, N-azidogalactose, N-azidoacetylgalactosamine.

[0014] In one embodiment the antibody or antigen binding fragment thereof comprises a further moiety covalently attached to said functionalized monosaccharide.

[0015] In one embodiment the further moiety is selected from a therapeutic moiety, half-life extending moiety or label.

[0016] In one embodiment the label is a fluorophore, a fluorescer, a radiolabel, a chemiluminescer, a nuclear magnetic resonance active label, a photosensitizer or a biotin tag.

[0017] In one embodiment the therapeutic moiety is a cell killing agent, an immune-modulating payload, a macrophage class switching agent, an oligonucleotide or a light activatable payload.

[0018] In one embodiment the immune-modulating payload is a STING agonist or a toll-like receptor agonist.

[0019] In one embodiment the cell killing agent comprises a cytotoxin.

[0020] In one embodiment said cytotoxin is selected from:

[0021] i) a peptide toxin;

[0022] ii) a chemical toxin: or

[0023] iii) radio toxin.

[0024] In one embodiment the cytotoxin is selected from the group comprising auristatins, maytansinoids, tubulysins, RNA polymerase II inhibitors, transcription inhibitors, calicheamicins, duocarmycins, pyrrolobenzodiazepines, camptothecin analogues, topoisomerase inhibitors and doxorubicin.

[0025] In one embodiment the further moiety comprises a linker covalently which attaches to said functionalized monosaccharide.

[0026] In one embodiment the linker comprises one or more of polyethylene glycol, glutaryl, valinecitrulline (Val-Cit), p-aminobenzylalcohol (PABA), 6-aminohexanoyl (Ahx), or N,N'-dimethylethylene diamine (DMEDA).In one embodiment the antibody is selected from an IgG 1 , lgG2, lgG3, lgG4.

[0027] In one embodiment said fragment is selected from a F(ab')2, Fab, heavy chain, light chain, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies.

[0028] In one embodiment the antibody or antigen binding fragment comprises one or more further N-linked glycans

[0029] In one aspect, the invention relates to an immunoconjugate or radioimmunoconjugate comprising the antibody or antigen binding fragment thereof as described above and a further moiety or radionuclide.

[0030] In one aspect, the invention relates to a nucleic acid encoding an antibody, antigen binding fragment thereof, or immunoconjugate or radioimmunoconjugate as described above.

[0031] In one aspect, the invention relates to a vector comprising a nucleic acid as described above. In one aspect, the invention relates to a host cell comprising the nucleic acid as described above, or a vector as described above.

[0032] In one aspect, the invention relates to a pharmaceutical composition comprising an antibody or antigen binding fragment thereof as described above and a pharmaceutically acceptable excipient.

[0033] In one aspect, the invention relates to an antibody or antigen binding fragment thereof as described above, an immunoconjugate or radioimmunoconjugate as described above, or a pharmaceutical composition as described above for use in the treatment of cancer, inflammatory disease, autoimmune disease, haematology conditions, endocrine disorders, infectious diseases, pulmonary diseases, cardiovascular disease, neurological disorders, allergic diseases, metabolic disorders, ophthalmic diseases, bone and joint diseases, dermatological diseases or transplantation associated diseases.

[0034] In one aspect, the invention relates to a method of treating of cancer, inflammatory disease, autoimmune disease, hematology conditions, endocrine disorders, infectious diseases, pulmonary diseases, cardiovascular disease, neurological disorders, allergic diseases, metabolic disorders, ophthalmic diseases, bone and joint diseases, dermatological diseases or transplantation associated diseases comprising administering antibody or antigen binding fragment thereof as described above, an immunoconjugate or radioimmunoconjugate as described above, or a pharmaceutical composition as described above to a subject.

[0035] In one embodiment said antibody or antigen binding fragment thereof is administered together with another therapy.

[0036] In one aspect, the invention relates to a method of preparing an immunoconjugate comprising conjugating a payload to the antibody or antigen binding fragment thereof as described above. In one aspect, the invention relates to a method of producing an immunoconjugate or radioimmunoconjugate with enhanced payload attachment, comprising:

[0037] modifying the amino acid sequence of an antibody or antigen binding fragment thereof to comprise one or more engineered N-glycan site at position 190 of the light chain of said antibodyor antigen binding fragment thereof, 159 and / or 186 of the heavy chain of said antibody or antigen binding fragment thereof;

[0038] functionalized; and

[0039] attaching a payload to said antibody or antigen binding fragment thereof via said functionalized engineered N-glycan site.

[0040] In one embodiment the method comprises introducing a functionalized monosaccharide at the one or more engineered N-glycan site, wherein the functionalized monosaccharide provides an attachment point for said payload.

[0041] In one embodiment the engineered N-glycan site comprises N-X-S and / or N-X-T sequence motifs, wherein X is any amino acid other than proline.

[0042] In one embodiment, the radio toxin is selected from the group comprising actinium-225 (225Ac), astatine-211 (211At), bismuth-213 (213Bi), indium-111 (111ln), iodine-123 (1231), iodine-124 (1241), iodine-131 (1311), lead-212 (212Pb), lutetium-177 (177Lu), technetium-99m (99mTc), copper-64 (64Cu), gallium-68 (68Ga), yttrium-86 (86Y), yttrium-90 (90Y), and zirconium-89 (89Zr), lodine-125 (1251), Rhenium-186 (186Re) Rhenium-188 (188Re), Samarium-153 (153Sm), Phosphorus-32 (32P), Cobalt-60 (60C).

[0043] In one aspect, the invention relates to a method of producing an antibody or antigen binding fragment thereof comprising at least one engineered N-glycan site comprising a functionalized monosaccharide in the Fab region of said antibody or antigen binding fragment thereof, comprising:

[0044] modifying the amino acid sequence of an antibody or antigen binding fragment thereof to comprise one or more N-glycan site comprising N-X-S and / or N-X-T sequence motifs, wherein X is any amino acid other than proline, at position 190 of the light chain of said antibody or antigen binding fragment thereof, 159 and / or 186 of the heavy chain of said antibody or antigen binding fragment thereof;

[0045] contacting said antibody or antigen binding fragment with a glycosyltransferase capable of covalently attaching a functionalized monosaccharide at one or more N-glycan site, and a functionalized monosaccharide under conditions sufficient to covalently attach said functionalized monosaccharide at N-glycan site.

[0046] In one embodiment the functionalized monosaccharide is selected from a functionalized sialic acid, a functionalized galactose, a functionalized N-acetylglucosamine (GIcNAc), a functionalized N-acetylgalactosamine GalNAc.

[0047] In one embodiment the functionalized monosaccharide is selected from N-azidoacetylneuraminic acid, N-azido acetylglucosamine, N-azidogalactose, N-azidoacetylgalactosamine.

[0048] In one embodiment the glycosyltransferase is selected from sialyltransferase, galactosyltransferase, O-GIcNActransferase, galactosaminyltransferase.In one embodiment the method further comprises a step of producing an immunoconjugate or radioimmunoconjugate comprising covalently attaching a further moiety to said antibody or antigen binding fragment thereof via said functionalized monosaccharide.

[0049] In another aspect, the invention relates to a method of enhancing payload attachment of an antibody, radioimmunoconjugate or immunoconjugate, comprising:

[0050] a) modifying the amino acid sequence of an antibody or antigen binding fragment thereof to comprise an engineered N-glycan site at amino acid position 190 of the light chain of said antibody or antigen binding fragment thereof, 159 and / or 186 of the heavy chain of said antibody or antigen binding fragment thereof according to Kabat numbering wherein said engineered N-linked glycan comprises a functionalized monosaccharide; and

[0051] b) attaching a payload to said antibody or antigen binding fragment thereof via said functionalized monosaccharide.

[0052] In another aspect, the invention relates to a method of enhancing payload attachment of an antibody, radioimmunoconjugate or immunoconjugate, comprising at least one engineered N-glycan site comprising a functionalized monosaccharide in the Fab region of said antibody or antigen binding fragment thereof, comprising:

[0053] a) modifying the amino acid sequence of an antibody or antigen binding fragment thereof to comprise an N-glycan site comprising N-X-S and / or N-X-T sequence motifs, wherein X is any amino acid other than proline, at amino acid position 190 of the light chain of said antibody or antigen binding fragment thereof, 159 and / or 186 of the heavy chain of said antibody or antigen binding fragment thereof according to Kabat numbering; and

[0054] b) contacting said antibody or antigen binding fragment with a glycosyltransferase capable of covalently attaching a functionalized monosaccharide at the N-glycan site, and a functionalized monosaccharide under conditions sufficient to covalently attach said functionalized monosaccharide at the N-glycan site.

[0055] Figures

[0056] FIGURE 1. A glycovariant design of trastuzumab showing a number of amino acid sites that were mutated to an asparagine residue in order to create an N-X-S / T sequence motif in the Fab constant region of trastuzumab that could be subsequently used as sites for payload conjugation to generate a trastuzumab-based antibody-drug conjugate. The modified residues are D170N, L154N, and Q166N of the light chain, and L166N and Q178N, both in the constant region of the heavy chain. The amino acid numbering referenced in the Figures is provided according to the sequence of Trastuzumab heavy chain (SEQ ID NO: 1) and light chain (SEQ ID NO: 2). Thus, the linear sequence of Trastuzumab is used as the reference sequence for this numbering. D170N corresponds to position 190 according to Kabat numbering, L166N corresponds to position 159 according to Kabat numbering, Q178N corresponds to position 186 according to Kabat numbering.FIGURE 2. Expression of trastuzumab glycovariants in glycan engineered CHO cells that add a complex biantennary N-glycan terminally capped with galactose moieties. Bold mutants indicate N-glycan sites that were successfully glycosylated and were used for subsequent payload conjugation. Underlined mutants indicate N-glycan sites that were either partially or not glycosylated and thus could not be used for payload conjugation. A) KB cells. B) CHO cells. FIGURE 3. Western blot showing payload conjugation to novel N-glycan sites in trastuzumab glycovariants with either (A) one or (B) two novel N-glycan sites per antibody molecule. TRZ wt refers to wild-type trastuzumab.

[0057] FIGURE 4. Mass spectroscopy results showing site L166N of trastuzumab (A) glycosylated with a A2G2F N-glycan and (B) conjugated with MMAE on the N-glycan. Glycan-modified and conjugated variants were tested.

[0058] Detailed Description of the Embodiments

[0059] The embodiments of the invention will now be further described. In the following passages, different embodiments are described. 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.

[0060] 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, Kontermann and Duebel, eds., Springer-Verlag, Heidelberg (2010).

[0061] 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, andtreatment of patients. Suitable assays to measure the properties as set out above are also described in the examples.

[0062] The present inventors have demonstrated that antibodies with high conjugation efficiency can be produced by introducing engineered glycosylation sites at one or more specific position within the Fab region of an antibody. By introducing a functionalized monosaccharide at one or more of these engineered glycosylation sites various payloads can be conjugated with excellent efficiency, which is highly beneficial for the production of immunoconjugates or radioimmunoconjugate, in particular antibody drug conjugates (ADCs) and radioligand therapy (RLT).

[0063] In all aspects of the invention, “engineered” or “modified” refers to any amino acid modification of a target sequence, e.g. a reference or wild type sequence. Modification may be the substitution of a reference amino acid residue with another amino acid residue, insertion of one or more amino acid into a reference sequence or deletion of one or more amino acid. Sequence motifs as described herein may be introduced into the amino acid sequence of the antibody or antigen binding fragmentthereof by several routine methods known to those in the art. Sequence modifications that introduce glycosylation sites are further described below.

[0064] A first aspect of the invention relates to an antibody or antigen binding fragment thereof, comprising at least one N-linked glycan functionalized in the Fab region of said antibody or antigen binding fragment thereof, wherein the at least one functionalized N-linked glycan is present at one or more of amino acid position 190 of the light chain of said antibody or antigen binding fragment thereof, 159 and / or 186 of the heavy chain of said antibody or antigen binding fragment thereof, wherein these amino acid positions are provided according to Kabat numbering herein.

[0065] In particular, the invention relates to an antibody or antigen binding fragment thereof, comprising an N-linked glycan functionalized in the Fab region of said antibody or antigen binding fragment thereof, wherein the functionalized N-linked glycan is present at amino acid position 159 (numbering according to Kabat) of the heavy chain of said antibody or antigen binding fragment thereof. Kabat position 159 corresponds to position IMGT CH1 position 45.1 numbering. This residue is located in the CH1 constant domain of the heavy chain. Kabat position 159 (based on alignment of the variable region ending at Kabat 113), corresponding to residue 166 of the mature linear sequence and IMGT CH1 position 45.1 Unless otherwise indicated, Kabat numbering is extended beyond the variable domain by sequential continuation following Kabat position 113.In one embodiment, the antibody or antigen binding fragment thereof comprises one or more further functionalized N-linked glycans. In one embodiment, the antibody or antigen binding fragment thereof does not comprise one or more further functionalized N-linked glycans.

[0066] As explained further herein, the N-linked glycan is inserted at an engineered glycosylation site. Thus, the antibody or antigen binding fragment of the invention comprises a modified / V-linked glycosylation site at one or more of amino acid position 190 of the light chain of said antibody or antigen binding fragment thereof, 159 or 186 of the heavy chain. In one embodiment, the antibody or antigen binding fragment of the invention comprises a modified / V-linked glycosylation site at position 159 of the heavy chain.

[0067] The antibody or antigen binding fragment of the invention may include an amino acid modification at one or more of amino acid position 190 of the light chain of said antibody or antigen binding fragment thereof, 159 or 186 of the heavy chain. Thus, in embodiments, the antibody or antigen binding fragment of the invention may thus be genetically engineered to comprise an / V-linked glycosylation site at a specific position, that is by the introduction of a sequence motif that directs the insertion of an / V-linked glycosylation. Thus, in an embodiment the antibody or antigen binding fragment thereof has been engineered to contain one or more / V-linked glycosylation site at amino acid position 190 of the light chain of said antibody or antigen binding fragment thereof, 159 or 186 of the heavy chain of said antibody or antigen binding fragment thereof. In an embodiment the antibody or antigen binding fragment thereof has been engineered to contain an / V-linked glycosylation site at amino acid position 159 of the heavy chain of said antibody or antigen binding fragment thereof. Further embodiments of these modifications are explained below.

[0068] Antibody and fragments

[0069] The term "antibody" as used herein broadly includes, but is not limited to, any immunoglobulin (Ig) molecule, or antigen binding portion 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.

[0070] In a full-length antibody, each heavy chain is comprised of a heavy chain variable region or domain (abbreviated herein as HCVR) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3. Each light chain has a light chain variable region or domain (abbreviated herein as LCVR) and a light chain constant region. The light chain constant region is comprised of one domain, CL.The heavy chain and light chain variable regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each heavy chain and light chain variable region are 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.

[0071] Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., lgG1 , lgG2, IgG 3, lgG4, lgA1 and lgA2) or subclass.

[0072] The term "CDR" or “CDR region” refers to the complementarity-determining region within antibody variable sequences. 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.

[0073] The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (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. The IMGT numbering scheme is described in Lefranc et al., Dev. Comp. Immunol., 29, 185-203 (2005).

[0074] The system described by Kabat is used herein unless otherwise mentioned, but alternatively IMGT or other numbering systems can be used. The terms "Kabat numbering", "Kabat definitions" and "Kabat labelling" are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e. , hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion.

[0075] The antibody may be human, humanized or chimeric. In one embodiment, the antibody may be a non-human mammal e.g. canine, feline, murine, bovine.

[0076] A “chimeric antibody” is a recombinant protein that contains the variable domains including the complementarity determining regions (CDRs) of an antibody derived from one species,preferably a rodent antibody, while the constant domains of the antibody molecule are derived from those of a human antibody.

[0077] 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. In certain embodiments, a limited number of framework region amino acid residues from the parent (rodent) antibody may be substituted into the human antibody framework region sequences.

[0078] Humanisation is typically undertaken since non-human antibodies will evoke an undesirable immune reaction when administered to a human subject. Non-human sequences may be derived from, but are not limited to, murine-derived antibodies, porcine-derived antibodies, bovine-derived antibodies, ovine-derived antibodies, simian-derived antibodies, leporine-derived antibodies, feline-derived antibodies, canine-derived antibodies, ursine-derived antibodies, piscine-derived antibodies and so on. The process of humanising antibodies most often involves the use of recombinant DNA techniques with which the skilled person will be familiar.

[0079] As used herein, the term “monoclonal antibody” or “mAb” is given its usual meaning in the art and refers to a monospecific antibody that is produced from homogeneous cells that are each clones of the same origin parent cell or cell line. The resultant antibodies are identical in sequence and therefore bind to the same epitope. In contrast to monoclonal antibodies, polyclonal antibodies (pAbs) are made from several different immune cells and have affinity for the same antigen but different epitopes. Monoclonal antibodies require production in laboratories whereas polyclonal antibodies can be found naturally, for example within the human body. In one embodiment, the antibodies of the present invention may be monoclonal antibodies.

[0080] As used herein, the term “antigen binding region” as used herein refers to an antibody, antigen binding fragment thereof or antibody mimetic.

[0081] The term "antigen binding site" refers to the part of the antibody or antibody fragment that comprises the area that specifically binds to an antigen. An antigen binding site may be provided by one or more antibody variable domains. An antigen binding site is typically comprised within the associated VH and VL of an antibody or antibody fragment.The term antibody as used herein also includes antibody fragments. Specifically, the invention also extends to antibody fragments. Antibody fragments are functional fragments of a full-length antibody, that is they retain the target specificity of a full antibody.

[0082] An antibody fragment is a portion of an antibody, for example a Fab (Fragment, antibody), F(ab')2, Fv, scFv (single chain variable chain fragments), heavy chain, light chain, variable heavy (VH) chain, variable light (VL) chain, CDR region, single VH or single VL domain, single VHH domain (nanobody), 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.

[0083] Thus, in one embodiment, the fragment is selected from a F(ab')2, Fab, Fv, scFv, heavy chain, light chain, variable heavy (VH), variable light (VL) chain, CDR region, single VH, VHH 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.

[0084] An “Fv" is the minimum antibody fragment which contains a complete antigen- recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0085] "Single-chain Fv" also abbreviated as "sFv" or "scFv" fragments (~25kDa) comprise of the two variable domains, VH and VL connected into a single polypeptide chain. 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).

[0086] Adiabody is a non-covalent dimer of a scFv fragment comprising the heavy chain variable region and light chain variable region connected by a small peptide linker. A triabody is a trimer of ScFvs, and a tetrabody is a tetramer of ScFvs.

[0087] A minibody is composed a single pair of ScFvs which are linked via a disulphide bond in a constant region of the heavy chain (known as the CH3 domain). Minibodies may be monospecificor bispecific. Further, a minibody may be generated by the addition of a third binding domain onto, for example, the constant region of the minibody.

[0088] The smallest antigen binding fragment is the single variable fragment, namely the single variable heavy (VH) or single variable light (VL) chain domain. VH and VL domains respectively are capable of binding to an antigen. Binding to a light chain / heavy chain partner respectively or indeed the presence of other parts of the full antibody is not required for target binding. The antigen-binding entity of an antibody, reduced in size to one single domain (corresponding to the VH or VL domain), is generally referred to as a “single domain antibody” or “single immunoglobulin variable domain”. A single domain antibody (~12 to 15 kDa) thus consists of either the VH or VL domain, but it does not comprise other parts of a full-length antibody. Single domain antibodies derived from camelid heavy chain only antibodies that are naturally devoid of light chains as well as single domain antibodies that have a human heavy chain domain have been described (Muyldermans J Biotechnol. 2001 Jun;74(4):277-302; Holliger Nat Biotechnol. 2005 Sep;23(9):1126-362). 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” as used herein generally refers to a single immunoglobulin variable domain (VH, VHH or VL) polypeptide that specifically binds antigen. Such a molecule only has the VH or VL binding domain respectively but does not comprise other parts of a full-length antibody. Unless otherwise specified, as used herein, the term refers to a single domain antibody that has a VH domain. For use in therapy, human single domain antibodies are often preferred, primarily because they are not as likely to provoke an immune response when administered to a patient.

[0089] The terms “single domain antibody”, “sdAb”, “VH domain antibody”, “single VH domain antibody”, “VH single domain antibody”, “single variable domain", “single variable domain antibody", “single variable heavy chain domain antibody" or immunoglobulin single variable domain (ISV)” are thus all well known in the art and describe the single variable fragment of an antibody that binds to a target antigen. These terms are used interchangeably herein.

[0090] Antibodies can be engineered to certain formats well known in the art and can, for example be multispecific, e.g. bispecific or trispecific. Other well-known formats are dual affinity retargeting antibodies (DARTs), as well as bi- and tri-specific killer engager antibodies (BiTE, BiKEs and TriKEs) and Tandem diabodies (TandAbs). Such formats are within the scope of the invention.

[0091] A binding molecule as used herein comprises an antibody or antigen binding fragment thereof.The antibody or antigen binding fragment thereof which comprises an / V-linked glycan in the Fab domain may be any antibody or fragment thereof described herein. For example, the antibody may be of any class, such as an IgM, IgA, IgD, IgE, or IgG class, or antibody subclass (such as IgG 1 or lgA2) or a fragment thereof so long as the antibody or fragment thereof has at least one / V-linked glycan in the Fab region. In certain embodiments, the antibody is an lgG1 antibody. The antibodies may be a monoclonal or polyclonal antibody. The antibody or antigen binding fragment thereof may be isolated. The antibody or antigen binding fragment thereof may be produced by a hybridoma or a cell line which expresses the antibody.

[0092] The term "isolated" refers to a moiety that is isolated from its natural environment. For example, the term "isolated" refers to an antibody or antigen binding fragment thereof that is substantially free of other antibodies or antibody fragments. Moreover, an isolated antibody or antigen fragment thereof is substantially free of other cellular material and / or chemicals. Antibodies or antigen binding fragments thereof of the invention are preferably isolated.

[0093] 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 / 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.

[0094] The term “antigen” as used herein therefore refers to the protein or polypeptide which is specifically recognised by the antibody or antigen binding fragment thereof according to the invention, for example a tumour associated antigen such as, but not limited to HER2. The terms antigen and target antigen are used interchangeably herein. By target, e.g. therapeutic target, is therefore meant the protein or polypeptide the antibody or antigen binding fragment thereof according to the invention specifically binds, for example a tumour associated antigen such as, but not limited to HER2. Exemplary targets are provided herein.

[0095] The term “epitope” or “antigenic determinant” refers to a site on the surface of an antigen to which an immunoglobulin, antibody or antibody fragment 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.

[0096] 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 antibody fragment (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides are tested for reactivity with a given antibody or antibody fragment. One method is Pepscan, a procedure for mapping and characterizing epitopes involving the synthesis of overlapping peptides and analysis of the peptides in enzyme-linked immunosorbent assays (ELISAs), see Westwood and Hay, Epitope Mapping: A Practical Approach, Oxford University Press, 2001 . Competition assays can also be used to determine if a test antibody binds to the same epitope as a reference antibody.

[0097] In one embodiment, the epitope for binding antigen is linear or conformational.

[0098] The degree of competition can be expressed as a percentage of the reduction in binding. Such competition or indeed binding can be measured using a real time, label-free bio-layer interferometry assay, e.g., on an Octet RED384 biosensor (Pall ForteBio Corp.), ELISA (enzyme-linked immunosorbent assays) or SPR (surface plasmon resonance), HTRF; flow cytometry; fluorescent microvolume assay technology (FMAT) assay, Mirrorball, high content imaging based fluorescent immunoassays, radioligand binding assays, bio-layer interferometry (BLI), surface plasmon resonance (SPR) and thermal shift assays.

[0099] The skilled person will understand that the principle of producing an antibody or antigen binding fragment thereof comprising at least one N-linked glycan in the Fab region of said antibody or antigen binding fragment thereof, wherein the at least one N-linked glycan is present at one or more of amino acid position 190 of the light chain of said antibody or antigen binding fragment thereof, 159 or 186 of the heavy chain of said antibody or antigen binding fragment thereof, can be applied to any antibody in accordance with any aspect of the invention. Thus, the antibody or antigen binding fragment thereof according to the invention is not limited to the examples shown herein, but applies to any antibody.

[0100] Preferably, the antibody or antigen binding fragment thereof is a therapeutic antibody or antigen binding fragment thereof. The antibody or antigen binding fragment thereof may bind to any therapeutic target of interest, for example a target in the therapy of diseases. A skilled person would be familiar with such targets which are, for example reviewed, in Sharma et al, Molecules.

[0101] 2023 Sep 5;28(18):6438.

[0102] In one embodiment, the antigen is a tumour associated antigen.Examples of target antigens include, but are not limited to: a transmembrane molecule; a receptor; a ligand; a growth factor; a growth hormone; a clotting factor; an anti-clotting factor; a plasminogen activator; a serum albumin; a receptor for a hormone or a growth factor; a neurotrophic factor; a nerve growth factor; a fibroblast growth factor; an interferon; a colony stimulating factor (CSF); an interleukin (IL); a T-cell receptor; a T-cell co-stimulatory receptor, such as CD137; a surface membrane protein; a viral protein; a tumor associated antigen; an integrin or an interleukin; VEGF; a renin; a human growth hormone; a growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; a lipoprotein; alpha-1 -antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factor VIIIC; clotting factor IX; tissue factor (TP); von Willebrand’s factor; Protein C; atrial natriuretic factor; a lung surfactant; urokinase; human urine; tissue-type plasminogen activator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha or -beta; enkephalinase; RANTES (Regulated on Activation Normally T-cell Expressed and Secreted); human macrophage inflammatory protein (MlP-l)-alpha; Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbial protein, beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associated antigen (CTLA); CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); protein A or D; a rheumatoid factor; bone-derived neurotrophic factor (BDNF); neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6); NGF- beta; platelet-derived growth factor (PDGF); aFGF; bFGF; epidermal growth factor (EGF); insulin-like growth factor-l or -II (IGF-I or IGF-II); des(l-3)-IGF-l (brain IGF-I), an insulin-like growth factor binding protein, erythropoietin; an osteoinductive factor; an immunotoxin; a bone morphogenetic protein (BMP); interferon-alpha, -beta, or -gamma; M- CSF, GM-CSF or G-CSF; IL-1 to IL-10; CD3, CD4, CD8, CD11a, CD11b, CD11c, CD18, CD19, CD20, CD34, CD40, 7 or CD46, an ICAM, VLA-4 or VCAM; or HER2, HER3 or HER4 receptor; a member of the ErbB receptor family; an EGF receptor; HER2, HER3 or HER4 receptor; a cell adhesion molecule; LFA-1 , Mac1 , pl50.95, VLA-4, ICAM-1 , VCAM, alpha4 / beta7 integrin or alphav / beta3 integrin; an alpha or beta subunit of a cell adhesion molecule; antibodies); a growth factor, VEGF; tissue factor (TF); alpha interferon (alpha-IFN); IL-8; IgE; blood group antigens Apo2; PD-1 ; PD-L1 ; PDL-1 , LAG3, TIGIT, 0X40, TIM-3, flk2 / flt3 receptor; LIV-1 , mesothelin, nectin-4, CTLA4, FOLR1 , PSMA, Trop2 or TNF-alpha. In one embodiment, the target is not TNF-alpha.

[0103] In particular, suitable targets in cancer therapy include checkpoint inhibitors such as PD-1 , PDL-1 , PDL-2, and CTLA-4. Examples of checkpoint inhibitors include pembrolizumab (Keytruda), ipilimumab (Yervoy), nivolumab (Opdivo) and atezolizumab (Tecentriq). Other targets in cancer therapy include immunooncology targets and T cell engagers. Examples are TIGIT, CD40, Mesothelin, 0X40 and LAG-3.Examples of tumor associated antigen targets include, but are not limited to: ADRB3, AFP, ALK, BCMA, beta human chorionic gonadotropin, CA-125 (MLIC16), CAIX, CD123, CD133, CD135, CD135 (FLT3), CD138, CD171 , CD19, CD20, CD22, CD24, CD276, CD33, CD33, CD38, CD44v6, CD79b, CD97, CDH3 (cadherin 3), CEA, CEACAM6, CLDN6, CLEC12A (CLL1), CSPG4, CYP1 B1 , EGFR, EGFRvlll, EpCAM, EPHA2, Ephrin B2, ERBBs (e. g. ERBB2), FAP, FGFR1 , folate receptor alpha, folate receptor beta, Fos-related antigen, GA733, GD2, GD3, GFRalpha4, globoH, GPC3, GPR20, GPRC5D, HAVCR1 , Her2 / neu (HER2), HLA-A2, HMWMAA, HPV E6 or E7, human telomerase reverse transcriptase, IL-11 Ra, IL-13Ra2, intestinal carboxyl esterase, KIT, Legumain, LewisY, LMP2, Ly6k, MAD-CT-1 , MAD-CT-2, ML-IAP, MN-CA IX, MSLN, MUC1 , mut hsp 70-2, NA- 17, NCAM, neutrophil elastase, NY-BR-1 , NY-ESO-1 , o-acetyl-GD2, OR51E2, PANX3, PDGFR-beta, PLAC1 , Polysialic acid, PSCA, PSMA, RAGE1 , ROR1 , sperm protein 17, SSEA-4, SSTR2, TAG72, TARP, TEM1 / CD248, TEM7R, thyroglobulin, Tn antigen, Tn-O-Glycopeptides, TPBG (5T4), TRP-2, TSHR, LIPK2 and VEGFR2. Examples are: CD138, CD79b, TPBG (5T4), HER2, MSLN, MUC1 , CA- 125 (MUC16), PSMA, BCMA, CD19, EpCAM, CLEC12A (CLL1), CD20, CD22, CEA, CD33, EGFR, GPC3, CD123, CD38, CD33, CD276, CDH3 (cadherin 3), FGFR1 , SSTR2, CD133, EPHA2, HLA-A2, IL13RA2, ROR1 , CEACAM6, CD135, GD-2, GA733, CD135 (FLT3), CSPG4 and TAG-72. Particular examples are: CD138, CD79b, CD123, MSLN, PSMA, BCMA, CD19, CD20, CEA, CD38, CD33, CLEC12a, HER2 and ROR1.

[0104] The antibody may be targeted against a target useful in medical therapy of a disease. Such diseases are listed below.

[0105] Examples of antibodies according to the invention include but are not limited to those listed in Lyo et al Antib Ther. 2022 Oct; 5(4): 233-257.

[0106] In one embodiment, examples of antibodies according to the invention include but are not limited to Muromonab, Nebacumab, Abciximab, Edrecolomab, Daclizumab, Rituximab, Infliximab, Trastuzumab, Palivizumab, Basiliximab, Gemtuzumab ozogamicin, Alemtuzumab, Adalimumab, Ibritumomab tiuxetan, Omalizumab, Efalizumab, Alefacept, Iodine 131 tositumomab, Cetuximab, Bevacizumab, Natalizumab, Tocilizumab, Abatacept, Panitumumab, Iodine 131 derlotuximab biotin, Iodine 131 metuximab, Nimotuzumab, Ranibizumab, Eculizumab, Racotumomab, Rilonacept, Certolizumab pegol, Romiplostim, Canakinumab, Catumaxomab, Ustekinumab, Ofatumumab, Golimumab, Denosumab, Brentuximab vedotin, Belimumab, Ipilimumab, Pertuzumab, Raxibacumab, Mogamulizumab, Trastuzumab emtansine, Obinutuzumab, Conbercept, Itolizumab, Blinatumomab, Pembrolizumab, Vedolizumab, Secukinumab, Ramucirumab, Efmoroctocog alfa, Siltuximab, Nivolumab, Elotuzumab,Alirocumab, Mepolizumab, Necitumumab, Idarucizumab, Dinutuximab, Daratumumab, Evolocumab, Atezolizumab, Olaratumab, Bezlotoxumab, Brodalumab,Ixekizumab, Reslizumab, Obiltoxaximab, Inotuzumab ozogamicin, Sarilumab, Dupilumab, Durvalumab, Avelumab, Emicizumab, Benralizumab, Ocrelizumab, Guselkumab, Erenumab, Moxetumomab pasudotox, Ravulizumab, Fremanezumab, Sintilimab, Ibalizumab, Galcanezumab, Tildrakizumab, Emapalumab, Cemiplimab, Toripalimab, Caplacizumab, Lanadelumab, Burosumab, Tislelizumab, Risankizumab, Trastuzumab deruxtecan, Brolucizumab, Crizanlizumab, Enfortumab vedotin, Romosozumab, Efgartigimod alfa, Camrelizumab, Polatuzumab vedotin, Luspatercept, Rabimabs, Netakimab, Inebilizumab, Teprotumumab, Sacituzumab govitecan, Atoltivimab, Odesivimab, Maftivimab, Cetuximab sarotalocan, Isatuximab, Belantamab mafodotin, Levilimab, Margetuximab, Satralizumab, Tafasitamab, Naxitamab, Ansuvimab, Eptinezumab, Prolgolimab, Olokizumab, Penpulimab, Dostarlimab, Evinacumab, Sugemalimab, Envafolimab, Regdanvimab, Amubarvimab, Romlusevimab, Amivantamab, Zimberelimab, Bimekizumab, Loncastuximab tesirine, Tisotumab vedotin, Tralokinumab, Tezepelumab, Aducanumab, Disitamab vedotin, Sotrovimab, Anifrolumab, Ormutivimab, Faricimab, Sutimlimab, Mosunetuzumab, Cadonilimab, Nemolizumab, Serplulimab.

[0107] In some aspects, the antibody is an immune checkpoint inhibitor. In some embodiments, the antibody targets cytotoxic T- lymphocyte antigen-4 (CTLA-4). In some aspects, the immune checkpoint inhibitor that targets CTLA-4 is Ipilimumab or tremelimumab (ticilimumab, CP-675.206). In some aspects, the immune checkpoint inhibitor targets PD-1. In some aspects, the immune checkpoint inhibitor that targets PD-1 is nivolumab (ONO-4538 / BMS-936558, MDX1106, OPDIVO®), pembrolizumab (MK-3475, KEYTRUDA®), pidilizumab (CT-011), atezolizumab (MPDL328OA), cemiplimab (LIBTAYO™), Spartalizumab (PDR001), Camrelizumab (SHR1210), Sintilimab (IBI308), Tislelizumab (BGB-A317), Toripalimab (JS 001), AMP-224, AMP-514 or Spartalizumab (PDR001). In some aspects, the immune checkpoint inhibitor targets PD-L-1 In some aspects, the immune checkpoint inhibitor that targets PD-L-1 is Avelumab, Atezolizumab, Durvalumab, KN035, CK-301 , AUNP12, CA- 170, or BMS-986189. In some aspects, the immune checkpoint inhibitor targets T cell immunoglobulin and mucin-domain containing-3 (TIM-3) and / or Lymphocyte Activating 3 (LAG3) proteins. In some aspects, the immune checkpoint inhibitor targeting TIM3 is MBG453; TSR-022; or LY3321367. In some aspects, the immune checkpoint inhibitor targeting LAG3 is IMP321 (Eftilagimod alpha), BMS-986016 (Relatlimab), LAG525 (anti- LAG-3 mAb), REGN3767 (anti-LAG-3 mAb), TSR-033 (anti-LAG-3 mAb). MGD013 (a PD-l / LAG-3 bispecific DART® protein), or FS118 (a LAG-3 / PD-L1 bispecific antibody).

[0108] In one embodiment, the cancer is a solid tumor. In one embodiment, the cancer is a hematological malignancy.In one embodiment, the antibody or antigen binding fragment thereof targets HER2 (ERBB2): (e.g., Trastuzumab deruxtecan, Trastuzumab emtansine), TROP2: (e.g., Sacituzumab govitecan), Nectin-4: (e.g., Enfortumab vedotin), FOLR1 (Folate Receptor Alpha): (e.g., Mirvetuximab soravtansine), EGFR, B7-H3 (CD276) & B7-H4, c-MET: Prime Targets for Hematological Malignancies, CD33: (e.g., Gemtuzumab ozogamicin), CD30: (e.g., Brentuximab vedotin), CD22: B (e.g., Inotuzumab ozogamicin), CD79b: (e.g., Polatuzumab vedotin), BCMA (B-cell maturation antigen): (e.g., Belantamab mafodotin) or CD19.

[0109] Non limiting examples of antibodies are set out below.

[0110] Key for sequence information

[0111] Italics = Fab variable region

[0112] Bold = Fab constant region

[0113] No highlight = Fc region

[0114] Underline = engineered N-glycan site

[0115] Linear numbering is used below. For Trastuzumab , the Kabat numbering is identified elsewhere herein.

[0116] In one embodiment, the antibody is trastuzumab (wild type sequences SEQ ID NO. 1 and 2, L166N modified heavy chain SEQ ID NO. 1).

[0117] SEQ ID NO: 1 Trastuzumab heavy chain wild type EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYAD SVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPG

[0118] SEQ ID NO: 2 Trastuzumab light chain wild type DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFS GSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVF\FPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC

[0119] SEQ ID NO: 3 Trastuzumab with L166N modification (heavy chain)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYAD SVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGANTSGVHTFPAVLQSSGLYS LSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP

[0120] Underline = engineered N-glycan site

[0121] SEQ ID NO: 4 Trastuzumab with Q178N modification (heavy chain) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYAD SVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLNSSGLYS LSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP

[0122] Underline = engineered N-glycan site

[0123] SEQ ID NO: 5 Trastuzumab with D170N modification (light chain) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFS GSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVF\FPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKNSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGECUnderline = engineered N-glycan site

[0124] Trastuzumab with Q178N modification (heavy chain SEQ ID NO: 4) can be combined with a light chain with the modification at D170N (light chain, SEQ ID NO:5)

[0125] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYAD SVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLNSSGLYS LSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP SEQ ID NO: 4 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFS GSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVF\FPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKNSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 5

[0126] In one embodiment, the antibody is Sacituzumab (anti-Trop2).

[0127] Sacituzumab wild type heavy chain sequence is shown below (SEQ ID NO. 6).

[0128] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTY TDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0129] The modified Sacituzumab sequence with a modification at Kabat 159 is shown below (L167 according to linear numbering, this has been modified to L167N) (SEQ ID NO. 7). The glycosylation motif is underlined.

[0130] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTY TDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGANTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0131] Light chain wild type (SEQ ID NO. 8)

[0132] DIQL TQSPSSLSA S VGDR VSITCKASQDVSIA VA WYQ QKPGKAPKLLIYSASYRYTG VPD RFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRT\ / AAPS\ / F\FPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0133] In one embodiment, the antibody is Atezolizumab (anti PD-L1).

[0134] Atezolizumab wild type heavy chain sequence is shown below (SEQ ID NO. 9).

[0135] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYY ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK

[0136] The modified Atezolizumab sequence with a modification at Kabat 159 is shown below (L164 according to linear numbering, this has been modified to L164N) (SEQ ID NO. 10). The glycosylation motif is underlined.EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYY ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGANTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAST' YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK

[0137] Light chain wild type (SEQ ID NO. 11) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAM>SVF\FPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0138] In one embodiment, the antibody is Rovalpitzumab (anti DLL3).

[0139] Rovalpitzumab wild type heavy chain sequence is shown below (SEQ ID NO. 12).

[0140] Q VQL VQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMG WINTYTGEPTY ADDFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARIGDSSPSDYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG

[0141] The modified Rovalpitzumab sequence with a modification at Kabat 159 is shown below (L164 according to linear numbering, this has been modified to L164N) (SEQ ID NO. 13). The glycosylation motif is underlined.

[0142] Q VQL VQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMG WINTYTGEPTY ADDFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARIGDSSPSDYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGANTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0143] Light chain wild type (SEQ ID NO. 14) EIVMTQSPATLSVSPGERATLSCKASQSVSNDVVWYQQKPGQAPRLLIYYASNRYTGIPA RFSGSGSGTEFTLTISSLQSEDFAVYYCQQDYTSPWTFGQGTKLEIKRTVAM>SVF\FPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0144] In one embodiment, the antibody is Enoblituzumab (anti B7-H3).

[0145] Enoblituzumab wild type heavy chain sequence is shown below (SEQ ID NO. 15).

[0146] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYY ADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELV GGPSVFLLPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQ YNSTLRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0147] The modified Enoblituzumab sequence with a modification at Kabat 159 is shown below (L168 according to linear numbering, this has been modified to L168N) (SEQ ID NO. 16). The glycosylation motif is underlined.

[0148] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYY ADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGANTSGVHTFPAVLQ SSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELV GGPSVFLLPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQ YNSTLRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0149] Light chain wild type (SEQ ID NO.17) DIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKRT\ / AAPS\ / F\FPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGlycans

[0150] In one embodiment the / V-linked glycan is inserted at an engineered glycosylation site. As such the antibody or antigen binding fragment thereof may be genetically engineered to comprise an / V-linked glycosylation site at a specific position. The antibody or antigen binding fragment thereof may be engineered to introduce a sequence motifthat directs the insertion of an / V-linked glycosylation. In an embodiment the antibody or antigen binding fragment thereof has been engineered to contain one or more / V-linked glycosylation site at amino acid position 190 of the light chain of said antibody or antigen binding fragment thereof, 159 or 186 of the heavy chain of said antibody or antigen binding fragment thereof.

[0151] As used herein, the term “sequence motif’ is given its usual meaning in the art and refers to an amino acid sequence that is usually conserved and related to a discrete biological function. A sequence motif may also be referred to as a “consensus sequence” or a “sequon”.

[0152] In the context of glycosylation of target proteins, a monosaccharide may be transferred to selected residues within certain sequence motifs within polypeptide chains. In particular, glycosylation sites are characterized by the N-X-S / T sequon, which acts as an acceptor sequence for / V-linked glycosylation. An N-X-S / T sequence motif is a sequence of amino acids having an asparagine residue upstream of an ‘X’ amino acid, where X can be any amino acid other than proline, followed by a serine or threonine residue.

[0153] The ‘X’ amino acid is not to be proline, as proline is known in the art to disrupt protein structures for example by breaking alpha helices within the secondary protein structure. Studies have indicated a lack of glycosyl-acceptor capabilities of sequons where ‘X’ is proline, potentially due to their inability to adopt a turn or loop conformation which permits the interaction between the hydroxy amino acid and the asparagine residue. In some embodiments, ‘X’ may be a canonical or a non-canonical amino acid. Where ‘X’ is a canonical amino acid, ‘X’ may be any of the following amino acids: alanine (A, Ala), arginine (R, Arg), asparagine (N, Asn), aspartate (D, Asp), cysteine (C, Cys), glutamate (E, Glu), glutamine (Q, Gin), (G, Gly), histidine (H, His), isoleucine (I, lie), leucine (L, Leu), lysine (K, Lys), methionine (M, Met), phenylalanine (F, Phe), proline (P, Pro), serine (S, Ser), threonine (T, Thr), tryptophan (W, Trp), tyrosine (Y, Tyr), or valine (V, Vai).

[0154] A / -glycosylation is thus a post-translational modification (PTM) attached to asparagine (Asn or N) amino acid residues within the N-(X)-S / T sequon, where X is any amino acid residue except proline, while S is serine and T is threonine. An A / -glycan is formed. This PTM in antibodies can take place naturally or be introduced through engineering at both fragment crystallizable region(Fc) and fragment antigen-binding (Fab) variable region. This most frequent and complex PTM plays an important role in antibody functions and properties. N-glycosylation in the constant Fc region is well characterized in all human antibody isotypes and modulates the Fc-mediated immune response through Fc receptors and complement system protein interaction.

[0155] The skilled person will be aware of suitable genetic engineering techniques to introduce an N-linked glycosylation site into an antibody sequence. Depending on the wild-type sequence of the antibody or fragment the N-linked glycosylation sequence motif may be introduced in a variety of ways. For example, in one embodiment, where there is a suitable X-S / T sequence downstream of the site at which the glycosylation is to be introduced, the sequence motif may be created by engineering into the amino acid sequence of an antibody or fragment thereof an asparagine residue at the desired position. However, in another embodiment, it is also possible that the N-X-S / T sequence motif is created by engineering into the amino acid sequence of an antibody or fragment thereof a serine or threonine residue downstream of any suitable N-X sequence. For example, engineering can be through an amino acid substitution of an existing residue with N, S or T or alternatively, a de novo residue may be inserted. The skilled person will be able to select the appropriate modification to make the glycosylation site at the desired position or positions, based on the wild-type sequence of the antibody or fragment thereof

[0156] The N-X-S and / or N-X-T sequence motifs is a motif for glycosylation. The antibody or antigen binding fragment of the invention comprises an N-linked monosaccharide at one or more of amino acid position 190 of the light chain of said antibody or antigen binding fragment thereof, 159 or 186 of the heavy chain of said antibody or antigen binding fragment thereof. As such the antibody or antigen fragment thereof comprise monosaccharides, such as glycans, at one or more of these positions. As described herein, glycans may be N-linked in that they are added to nitrogen atoms, for example on asparagine residues within the amino acid sequence of the antibody or antigen-binding fragment thereof. N-glycans may be incorporated onto existing asparagine residues within existing N-X-S and / or N-X-T sequence motifs, or onto N-X-S and / or N-X-T sequence motifs that have been engineered into the amino acid sequence. In one embodiment, one or more N-linked monosaccharides are engineered into the Fab region of the antibody or fragment.

[0157] As set out above, in order to introduce the N-linked glycan, the N-X-S and / or N-X-T sequence motif is required at the specified position.

[0158] In an embodiment the antibody or antigen binding fragment of the invention may comprise one or more of the following amino acid substitutions; X190N in the light chain of said antibody or antigen binding fragment, X159N in the heavy chain of said antibody or antigen binding fragment, and / or X186N in the heavy chain of said antibody or antigen binding fragment, whereinX refers to any amino acid and wherein amino acid positions are provided according to Kabat numbering. In particular, the X prior to the amino acid position denotes the wild type residue at that position. Thus, the antibody or antigen binding fragment thereof comprises a modified amino acid residue at one or more of position 190 in the light chain of said antibody or antigen binding fragment, 159 in the heavy chain and / or 186 in the heavy chain. In particular, the wild type amino acid residue at one or more of position 190 in the light chain of said antibody or antigen binding fragment, 159 in the heavy chain and / or 186 in the heavy chain is modified to N. Therefore, the antibody or antigen binding fragment thereof comprises amino acid residue N at one or more of position 190 in the light chain of said antibody or antigen binding fragment, 159 in the heavy chain and / or 186 in the heavy chain. In one embodiment, the antibody or antigen binding fragment of the invention may comprises the following modification X159N.

[0159] As an example the antibody according to the invention may comprise an anti-HER2 antibody for example Trastuzumab, or an antibody comprising a heavy chain comprising at least 80%, 85%, 90%, 95% sequence identity to SEQ ID NO: 1 and a light chain comprising at least 80%, 85%, 90%, 95% sequence identity to SEQ ID NO: 2 but with one or more modification as set out herein. In an embodiment the anti-HER2 antibody comprises at least one N-linked glycan in the Fab region of said antibody, wherein the at least one N-linked glycan is present at one or more of amino acid position 170 of the light chain of said antibody, amino acid position 166 and / or 178 of the heavy chain of said antibody, wherein the numbering of the heavy chain is provided according to SEQ ID NO: 1 and the numbering of the light chain is provided according to SEQ ID NO: 2. In embodiment wherein the antibody comprises an anti-HER2 antibody, for example Trastuzumab, the antibody may comprise one or more of the following amino acid substitutions in the Fab region of the antibody: D170N in the light chain, L166N in the heavy chain and / or Q178N in the heavy chain, wherein the numbering of the heavy chain is provided according to SEQ ID NO: 1 and the numbering of the light chain is provided according to SEQ ID NO: 2. In a specific embodiment the antibody may comprise one or more of SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5. Where an anti-HER2 antibody is provided as an example antibody of the invention, the numbering of the amino acid positions is provided according to the heavy and light chain sequences provided in SEQ ID NO:1 and SEQ ID NO:2 and also as Kabat numbering.

[0160] In another example, the antibody according to the invention may comprise an anti-Trop2 antibody, for example Sacituzumab, antibody comprising a heavy chain comprising at least 80%, 85%, 90%, 95% sequence identity to SEQ ID NO: 6 and a light chain comprising at least 80%, 85%, 90%, 95% sequence identity to SEQ ID NO: 8, but with one or more modification as set out herein. In an embodiment the anti-Trop2 antibody comprises a modification at position 159 according to Kabat. In an embodiment the anti-Trop2 antibody comprises the heavy chain sequence of SEQ ID NO: 8 or a variant having at least 80%, 85%, 90%, 95% sequence identity to SEQ ID NO: 8, but with the modification position at 159.In another example, the antibody according to the invention may comprise an anti-PD-L1 antibody, for example Atezolizumab, antibody comprising a heavy chain comprising at least 80%, 85%, 90%, 95% sequence identity to SEQ ID NO: 9 and a light chain comprising at least 80%, 85%, 90%, 95% sequence identity to SEQ ID NO: 11, but with one or more modification as set out herein. In an embodiment, the anti-PD-L1 antibody comprises a modification at position 159 according to Kabat. In an embodimentthe anti-PD-L1 antibody comprises the heavy chain sequence of SEQ ID NO: 10 or a variant having at least 80%, 85%, 90%, 95% sequence identity to SEQ ID NO: 10, but with the modification position at 159.

[0161] In another example, the antibody according to the invention may comprise an anti-DLL3 antibody, for example Rovalpitzumab, antibody comprising a heavy chain comprising at least 80%, 85%, 90%, 95% sequence identity to SEQ ID NO: 12 and a light chain comprising at least 80%, 85%, 90%, 95% sequence identity to SEQ ID NO: 14, but with one or more modification as set out herein. In an embodiment, the anti-DLL3 antibody comprises a modification at position 159 according to Kabat. In an embodiment the anti-DLL3 antibody comprises the heavy chain sequence of SEQ ID NO: 13 or a variant having at least 80%, 85%, 90%, 95% sequence identity to SEQ ID NO: 13, but with the modification position at 159.

[0162] In another example, the antibody according to the invention may comprise an anti-B7-H3 antibody, for example Enoblituzumab, antibody comprising a heavy chain comprising at least 80%, 85%, 90%, 95% sequence identity to SEQ ID NO: 15 and a light chain comprising at least 80%, 85%, 90%, 95% sequence identity to SEQ ID NO: 17, but with one or more modification as set out herein. In an embodiment, the anti- B7-H3 antibody comprises a modification at position 159 according to Kabat. In an embodiment the anti- B7-H3 antibody comprises the heavy chain sequence of SEQ ID NO: 16 or a variant having at least 80%, 85%, 90%, 95% sequence identity to SEQ ID NO: 16, but with the modification position at 159.

[0163] The N-linked glycan that is present at one or more of amino acid position 190 of the light chain of said antibody or antigen binding fragment thereof, 159 and / or 186 of the heavy chain of said antibody or antigen binding fragment thereof, may be any suitable glycan. N-glycans can be classified into three types. Firstly, oligomannose N-glycans, in which only mannose residues are present. Secondly, complex N-glycans, in which “antennae” are initiated by GIcNAc, extend the core. Thirdly, hybrid N-glycans, in which mannose extends the Mana1-6 arm of the core and one or two GIcNAc-initiated antennae extend the Mana1-3 arm. The preference of the present invention is for the N-glycans to be complex N-glycans.

[0164] In one embodiment, the complex N-glycan may be bi-antennary, in that it comprises two antennas. In another embodiment, the complex N-glycan may be tri-antennary, in that itcomprises three antennas. In another embodiment, the complex N-glycan may be tetra-antennary, in that it comprises four antennas.

[0165] In one embodiment the N-glycan comprises a galactose moiety, the galactose moiety may serve at the attachment point for a functionalized monosaccharide. In one embodiment the N-glycan is a complex biantennary N-glycan comprising a galactose moiety. In one embodiment the N-glycan is a complex biantennary N-glycan comprising a galactose moiety, wherein the galactose moiety is attached to a functionalized monosaccharide.

[0166] Depending on the N-glycan that is desired at a specific position there are various methods known in the art to control the glycosylation that occurs, this can include using specific cell lines for expression (for example engineered cell lines), addition of media supplements during fermentation and / or manipulation of metabolic pathways involved in glycosylation. The skilled person will be aware of suitable cell lines and methods to introduce specific glycans (Edwards et al,. 2022). In particular the skilled person will be aware of cell lines which are capable of incorporating complex biantennary N-glycan comprising a galactose moiety.

[0167] Functionalized monosaccharides

[0168] The glycans which are introduced at the one or more N-linked glycan sites in the Fab domain of the antibody or antigen binding fragment thereof provide an attachment point for a further moiety. Glycan specific conjugation techniques are known in the art, for example functionalization of glycans through oxidation of adjacent diols of terminal monosaccharide, production of sialylated glycans which are mildly oxidized by sodium periodate to form aldehyde groups and then conjugated via oxime ligation, introduction of functionalized monosaccharide groups. The present invention extends to use of any glycan specific technique to attach a further moiety via said one or more glycan in the Fab region.

[0169] In a preferred embodiment, the glycans which are introduced at the one or more N-linked glycan sites comprise at least one functionalized monosaccharide at one or more of the positions disclosed herein. In an embodiment there are one or more further non-functionalized monosaccharides at the N-linked glycan site. The functionalized monosaccharide may be present at the terminal end of the N-linked glycan.

[0170] The term “functionalized monosaccharide” as used herein refers to any monosaccharide comprising a functional group which can participate in a covalent linkage with a functionalized further moiety e.g. a payload or cargo molecule. The functionalized group present on the functionalized monosaccharide serves at the attachment point for the further moiety.In an embodiment the monosaccharide portion of the functionalized monosaccharide may be selected from sialic acid, galactose, N-acetylglucosamine (GIcNAc), N-acetylgalactosamine (GalNAc), mannose, or any suitable glycan. As used herein the term “sialic acid” includes N- or O-substituted derivatives of neuraminic acid, 2- keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulosonic acid (Neu5Ac), and 2-keto-3- deoxy-D-glycero-D-galactonononic acid (KDN). For example, sialic acid and its derivatives can include and are sometimes referred to as A / -acetylneuraminic acid, NANA, NeuAc, Neu5Ac, or Neu5Gc.

[0171] The functional group may be any functional group suitable for forming a covalent linkage with a further functional group. In some embodiments the functionalized monosaccharide comprises a functional group capable of participating in a click chemistry reaction i.e. a click reactive group. 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, non-aldol carbonyl chemistry and carbon multiple bond additions.

[0172] Any suitable click reactive group may be present on the functionalized monosaccharide. The click reactive group may typically be selected based on the functional group that the functionalized monosaccharide is to be conjugated with and the click chemistry that is utilised for the conjugation. The skilled person if aware of suitable click reactive groups and will be able to select the appropriate click reactive group. The click chemistry may be selected from a thiolene 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. Preferably, the click chemistry may be copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strained-promoted azide-alkyne click chemistry (SPAAC) reaction.

[0173] 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. 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-free catalyst may be used. For example, a pentamethylcyclopentadienyl ruthenium chloride complex may be used as a catalyst to catalyse the azide-alkyne cycloaddition. In some embodiments the functionalized monosaccharide may comprise an azide group or an alkyne group. An azide group may be conjugated with an appropriate alkyne group. An alkyne group may be conjugated with an appropriate azide group.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. In some embodiments the functionalized monosaccharide may comprise an azide group or a strained alkyne group. An azide group may be conjugated with an appropriate strained alkyne group. A strained alkyne group may be conjugated with an appropriate azide group.

[0174] 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 catalysed 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 catalysed by either a base or a nucleophile, resulting in a similar anti-Markovnikov addition product as the free-radical addition reactions. In some embodiments the functionalized monosaccharide may comprise a thiol group, a maleimide group, an alkene group. A thiol group may be conjugated with an appropriate maleimide group. A maleimide group may be conjugated with an appropriate thiol group. An alkene group may be conjugated with an appropriate thiol group.

[0175] 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 HOMO(Dienophiie) and LUMO(Diene) 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 electron-withdrawing substituents on the diene accelerate the iEDDA reaction by further reducing the HOMO(Dienophiie)-LUMO(Diene) separation. In some embodiments the functionalized monosaccharide may comprise a diene group, or a dienophile group. A diene group may be conjugated with an appropriate dienophile group. A dienophile group may be conjugated with an appropriate diene group.In one embodiment, the functional group, or click reactive group is positioned at any suitable position in the glycan. In one embodiment the functional group, or click reactive group is positioned at C-9 of the sialic acid. In another embodiment, the functional group is positioned at C-5 of the sialic acid. Functionalized sialic acid can contain one, two or more functional groups capable of covalent linkage with a cargo molecule, positioned at C-9, C-5 or any other suitable position on the sialic acid such as, without limitation, C-1 , C-2, C-4, C-7, or C-8. A functionalized sialic acid containing more than one functional group may contain the same or different functional groups. In one embodiment the functional group, or click reactive group is positioned at any suitable position of GIcNAc, GalNAc, mannose, or galactose

[0176] Exemplary functional groups include but are not limited to azide, nitrone, nitrile oxide, azoxy, diazo, acyl diazo, and trans-cyclooctene. In certain embodiments, the modified antibody comprising at least one N-linked glycan that has an azido-modified sialic acid in the Fab region. Exemplary functionalized monosaccharides may be selected from A / -azidoacetylneuraminic acid, A / -azido acetylglucosamine, A / -azidogalactose, A / -azidoacetylgalactosamine. In specific embodiments, the functionalized sialic acid is A / -azidoacetylneuraminic acid (Neu5NAz).

[0177] Methods are known in the art how to introduce functionalized monosaccharides. For example, a transferase enzyme may be used to enzymatically attach a suitable functionalized monosaccharide. Examples of suitable transferase enzyme include sialyltransferase, galactosyltransferase, O-GIcNActransferase, galactosaminyltransferase.

[0178] Typically, the transferase enzyme will recognise a nucleotide associated monosaccharide and catalyse the attachment of the monosaccharide to the end of the glycan chain. For example, the functionalized sialic acid substrate is typically a nucleotide associated sialic acid, such as a CMP-sialic acid. In certain embodiments, the functionalized CMP-sialic acid is a CMP- azidomodified sialic acid. In specific embodiments, the functionalized sialic acid substrate can be CMP-Neu5Ac9N3 or alternatively CMP-Neu5NAz. The functionalized CMP-sialic acid may be produced by contacting cytidine triphosphate (CTP) and CMP-sialic acid synthetase for a time and under conditions sufficient to produce a functionalized CMP-sialic acid.

[0179] The sialyltransferase, ST6Gal1 , has been previously found to catalyze the sialy lation of N- linked glycans in both the Fc and Fab regions of antibodies using functionalized CMP-sialic acid derivative as a substrate. Surprisingly, it was found that certain bacterial sialyltransferases catalyze sialylation of N-linked glycans in the Fab region of antibodies but not N-linked glycans in the Fc region of antibodies. Accordingly, these sialyltransferases may be used to specifically modify N-linked glycans in the Fab region of antibody with a functionalized sialic acid.The bacterial sialyltransferase includes sialyltransferase derived from Gram negative bacterium such as bacterium from the Pasteurellaceae family and Photobacterium spp. from the family Vibrionaceae. In certain embodiments, the bacterial sialyltransferase is derived from an Actinobacillus sp., including but not limited to Actinobacillus suis bacterium (GenBank Accession # AFU19871). In certain other embodiments, the bacterial sialyltransferase is derived from CAZy glycosyltransferase family 80. In certain embodiments, the bacterial sialyltransferase comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, or 100% identity to the amino acid sequence as set forth in GenBank Accession # AFU19871 or an active fragment thereof.

[0180] Galactosyltransferases may be used to incorporate the functionalized galactose. O-GIcNActransferase may be used to incorporate functionalized GIcNAc. Galactosaminyltransferase may be used to incorporate functionalized GalNAc.

[0181] Conjugates

[0182] The present inventors have demonstrated that by introducing engineered / V-linked glycan sites within the Fab region of an antibody or antigen binding fragment thereof, attachment of payload to the antibody or fragment thereof can be enhanced. Methods are known in the art to attach further moieties such as payloads via glycans. In certain embodiments the attachment of the payload occurs via a functionalized monosaccharide present in the N-linked glycan. As such in an embodiment the antibody or antigen binding fragment thereof comprises a further moiety or pay load.

[0183] The antibody or fragment thereof may be conjugated to a further moiety. The further moiety may be a half-life extender, 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). 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).

[0184] The antibody or fragment of the invention may be conjugated to a payload i.e. a therapeutic moiety. A “payload” may be selected from but not limited to a cell killing agent, an immune-modulating payload, a macrophage class switching agent, a detectable label or a light activatable payload. In certain embodiments where the antibody or fragment thereof is conjugated to a further moiety such as a payload, this is referred to as an immunoconjugate. The antibody or fragment of the invention may be conjugated to a radionuclide. In certain embodiments where the antibody or fragment thereof is conjugated to a radionuclide, this is referred to as a radioimmunoconjugate.

[0185] In one embodiment, the payload may be a cell killing agent. In one embodiment, the payload may be a macrophage class switching agent. In one embodiment, the pay load may be an immune-modulating payload. In one embodiment, the payload may be a light activatable payload. 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.

[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. Another example is a macrophage class switching agent.The payload may be a cytotoxic payload or a therapeutic compound, peptide or polypeptide. In particular, the payload is preferably a cytotoxin.

[0188] 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), camptothecin analogues, topoisomerase inhibitors and doxorubicin.

[0189] However, additionally or alternatively, the cytotoxin could also be selected from other known cytotoxins including ricin subunits and other peptide based cytotoxic materials.

[0190] In some embodiments the payload may be a cytotoxic or cytostatic agent, i.e., a compound that kills or inhibits tumour cells. Such agents may impart their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, proteasome and / or topoisomerase inhibition. 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 "cytotoxic agent" as used herein also includes radioactive isotopes (also termed radio toxins, radionuclides, radioligand therapy (RLT), and targeted radionuclide therapy (TRT) herein) (chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including synthetic analogues and derivatives thereof.

[0191] Examples of radioactive isotopes include, but are not limited to, actinium-225 (225Ac), astatine-211 (211At), bismuth-213 (213Bi), indium-111 (111ln), iodine-123 (123l), iodine-124 (124l), iodine-131 (131l), lead-212 (212Pb), lutetium-177 (177Lu), technetium-99m (99mTc), copper-64 (64Cu), gallium-68 (68Ga), yttrium-86 (86Y), yttrium-90 (90Y), and zirconium-89 (89Zr), lodine-125 (125l), Rhenium-186 (186Re) Rhenium-188 (188Re), Samarium-153 (153Sm) ,Phosphorus-32 (32P), Cobalt-60 (60C), and radioactive isotopes of Lu. Suitable radionuclides and tehcniques for conjugating antibodies to these agents are reviewed in Parakh et al, Cancers (Basel). 2022 Mar 11 ;14(6):1454.

[0192] Thus, the present invention relates to the generation of radioligand therapies (RLTs), or radioconjugates, through site-specific modification of antibody N-glycans to introduce bioorthogonal functional groups, including azides, at defined positions. A chelator comprising a complementary reactive moiety, such as a strained alkyne (e.g., dibenzocyclooctyne, DBCO), is conjugated to the modified glycan via strain-promoted azide-alkyne cycloaddition (SPAAC) or other click chemistry methods. The chelator is configured to coordinate with radioactive payload, including but not limited to radiometals or radioisotopes, and may be radiolabeled prior to or subsequent to conjugation. Such conjugation techniques are well known in the art.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.

[0193] Examples of chemotherapeutic agents include, but are not limited to, 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, andzorubicin; 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 glycinamide ribonucleotide 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.

[0194] 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, andFARESTON(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); siRNA, miRNA, small activating RNA, 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

[0195] 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.

[0196] 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.

[0197] In an embodiment the cytotoxic or cytostatic agent is a tubulin inhibitor. In an embodiment, the tubulin inhibitor is selected from the group comprising 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 / 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 (AF), monomethylauristatin 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 cytostaticagent is an anti-neoplastic agent such as irinotecan or metabolites thereof. Suitable metabolites of irinotecan include SN-38.

[0198] 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 doublestrand 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. 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.

[0199] 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 targetedmustard. 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.

[0200] 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.

[0201] In an embodiment, the cytotoxic or cytostatic agent is a Bis-(benzimidazole) carrier. Preferably, the drug is Hoechst 33258.

[0202] 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 guaninespecific 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.

[0203] Adenine-specific alkylating agents are regiospecific and sequence-specific minor groove alkylators reacting at the N3 of adenines in polypyrimidines sequences.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] The following cytotoxic or cytostatic agent are also encompassed by the present invention: Amatoxins (a-amanitin)- bicyclic octapeptides produced by basidiomycetes of the genus Amanita, e.g., the Green Deathcap mushroom; Tubulysins; Pseudomonas exotoxin; Cytolysins; dolabellanins; Epothilone A, B, C, D, E, F. Epothilones - constitute a class of non-taxane tubulin polymerisation agents and are obtained by natural fermentation of the myxobacterium Sorangiumcellulosum. 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. 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 or payload is amatoxin. In an embodiment, the drug or payload is tubulysin. In an embodiment the drug or payload is Pseudomonas exotoxin. In an embodiment, the drug or payload is cytolysin. In an embodiment, the drug or payload is dolabellanin. In an embodiment, the drug or payload is epothilone.

[0209] 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.

[0210] 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 an anti-CD33 anti-CD7 bispecificantibody or binding portion thereof 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.

[0211] In certain embodiments the payload comprises a small molecule inhibitor with an anti-cancer 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.

[0212] 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 tumour specificity, while using infrared light to activate the biophysical mechanism of the drug to accurately induce rapid death of cancer cells without harming the surrounding normal tissues. Suitable light-activatable payloads include IRDye700DX. In one embodiment 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.

[0213] Immunoconjugates comprising the further moieties e.g. payloads as described herein may be prepared by in vitro methods known to one of ordinary skill in the art. Techniques for conjugating cytotoxic or cytostatic agent 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.)

[0214] The further moieties or payloads, described herein, may be modified to comprise a functional group to allow attachment to the antibody or fragment thereof via the N-linked glycan or specifically the functionalized monosaccharide. The functional group may be any functional group suitable for forming a covalent linkage with a further functional group. In some embodiments the further moiety or payload comprises a functional group capable of participating in a click chemistry reaction i.e. a click reactive group. The click reactive group may be suitable to form a covalent linkage via cycloaddition, nucleophilic ring opening, non-aldol carbonyl chemistry and carbon multiple bond additions.Any suitable click reactive group may be present on the further moiety or payload. The click reactive group may typically be selected based on the corresponding functional group present on the functionalized monosaccharide, and the click chemistry that is utilised for the conjugation. The skilled person if aware of suitable click reactive groups and will be able to select the appropriate click reactive group. The click chemistry may be selected from a thiol-ene reaction, a copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strained-promoted azidealkyne click chemistry (SPAAC) reaction, a 1 ,3-dipolar cycloaddition reaction and / or an inverse electron demand Diels-Alder (iEDDA) reaction. Preferably, the click chemistry may be copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strained-promoted azide-alkyne click chemistry (SPAAC) reaction.

[0215] In some embodiments the further moiety or payload may comprise an azide group or an alkyne group. An azide group may be conjugated with an appropriate alkyne group. An alkyne group may be conjugated with an appropriate azide group. Suitable azide groups include but are not limited to arylazide groups.

[0216] In some embodiments the further moiety or payload may comprise an azide group or a strained alkyne group. An azide group may be conjugated with an appropriate strained alkyne group. A strained alkyne group may be conjugated with an appropriate azide group. Suitable strained alkyne groups may include but are not limited to cyclooctyne groups such as difluorooctyne (DIFO), dibenzylcyclooctyne (DIBO) and biarylazacyclooctynone (BARAC).

[0217] In some embodiments the further moiety or payload may comprise a sydnone group or an alkyne group or an alkene group. A sydnone group may be conjugated with an appropriate alkyne group or alkene group. An alkyne group or alkene group may be conjugated with an appropriate sydnone group.

[0218] In some embodiments the further moiety or payload may comprise a thiol group, a maleimide group, an alkene group. A thiol group may be conjugated with an appropriate maleimide group. A maleimide group may be conjugated with an appropriate thiol group. An alkene group may be conjugated with an appropriate thiol group.

[0219] In some embodiments the further moiety or payload may comprise a diene group, or a dienophile group. A diene group may be conjugated with an appropriate dienophile group. A dienophile group may be conjugated with an appropriate diene group. Suitable diene groups include but are not limited to tetrazine groups. Suitable dienophile groups include but are not limited to cycloalkene or cycloalkyne group, for example a cyclooctene or bicyclononyne group.In one embodiment, the functional group, or click reactive group is positioned at any suitable position on the further moiety or payload. In some embodiments the further moiety or payload is modified to comprise a linker which comprises the functional group or click reactive group. Suitable linkers include but are not limited to polyethylene glycol, glutary I, valine-citrulline (Val-Cit), p-aminobenzylalcohol (PABA), 6-aminohexanoyl (Ahx), or N,N'-dimethylethylene diamine (DM EDA).

[0220] An aspect of the invention relates to an immunoconjugate comprising an antibody or antigen binding fragment as described herein and a further moiety or payload, in particular at position 159 according to Kabat. The further moiety and payload may comprise any of the features as set out herein.

[0221] Immunoconjugates / radioimmunoconjugates

[0222] An aspect of the invention relates to a radioimmunoconjugate comprising an antibody or antigen binding fragment as described herein conjugated to a radionuclide, in particular at position 159 according to Kabat.

[0223] In an embodiment the immunoconjugate may comprise a DAR of 1 , 2, 3, 4, 5 or 6.

[0224] Nucleic acids, vectors, hosts

[0225] An aspect of the invention relates to a nucleic acid encoding an antibody, antigen binding fragment thereof, radioimmunoconjugate or immunoconjugate as described herein.

[0226] The term "nucleic acid," "polynucleotide," or "nucleic acid molecule" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination of a DNA or RNA. RNA includes in vitro transcribed RNA, synthetic RNA or mRNA sequence. The nucleic acid construct may further comprise a suicide gene. The construct may be in the form of a plasmid, vector, transcription or expression cassette.

[0227] In another aspect, the invention relates to an isolated nucleic acid construct comprising a nucleic acid as defined above. The construct may be in the form of a plasmid, vector, transcription or expression cassette.

[0228] The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. The vectors can be suitable for replication andintegration eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0229] In one embodiment, the vector is an in vitro transcribed vector, e.g., a vector that transcribes RNA of a nucleic acid molecule described herein. The expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2013). A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses such as adenovirus vectors can be used. In one embodiment, a lentivirus vector is used.

[0230] To produce the antibodies, monoclonal antibodies or antigen-binding fragments thereof, the aforementioned expression vectors are used for stable transfection or transient transfection of a host cell, such as a mammalian cell. A stable transfection refers to any method in which genes are integrated into the host genome of a cell that allows for stable expression of the genes under the control of a constitutive or inducible promoter. A stable transfection allows for integration into the host genome thus the gene is replicated, and the expression of the genes are sustained long-term. A transient transfection refers to any method in which genes are introduced and expressed under the control of a constitutive or inducible promoter. A transient transfection does not allow for integration into the host genome thus the gene is not inherited during cell division, and the expression of the genes is therefore for a finite period of time. The expression vector may be a plasmid.

[0231] In a further aspect, the invention also relates to an isolated cell or cell population comprising one or more nucleic acid construct or vector as described above. In one embodiment, the cell is an isolated recombinant host cell comprising one or more nucleic acid construct as described above. The host cell may be a bacterial, viral, plant, mammalian or other suitable host cell.

[0232] Such host cells are well known in the art and many are available from the American Type Culture Collection (ATCC). These host cells include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and a number of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Other cell lines that may be used are insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, such as E. coli, plant cells and fungal cells. Fungal cells include yeast and filamentous fungus cells including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichiathermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella. Derivatives of known cells lines, e.g. genetically modified cell lines can also be used.

[0233] In one embodiment, a method of making the antibody or antigen binding fragment thereof as described herein is provided, wherein the method comprises culturing the host cell under conditions suitable for expression of the polynucleotide encoding an antibody or antigen binding fragment thereof as described herein and isolating the antibody or antigen binding fragment thereof.

[0234] Pharmaceutical Composition

[0235] In another aspect, there is provided a pharmaceutical composition comprising an antibody or antigen binding fragment thereof of the invention, a radioimmunoconjugate or immunoconjugate of the invention and optionally a pharmaceutically acceptable carrier. The terms composition and formulation are used interchangeably herein. An antibody or antigen binding fragment thereof or the pharmaceutical composition of the invention can be administered by any convenient route, including but not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitreal, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, by inhalation, or topical, particularly to the ears, nose, eyes, or skin or by inhalation.

[0236] Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. Preferably, the compositions are administered parenterally.

[0237] Delivery or the antibody or antigen binding fragment may be by any suitable route. In one embodiment delivery may be via gene therapy, e.g. in using a recombinant adeno-associated virus (rAAV) vector having: a viral capsid and comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding an antibody according to the invention or an antigen binding fragment thereof.

[0238] 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 an antibody or antigen binding fragment thereof of the present invention is administered. Such pharmaceutical carriers can be liquids, such as water and oils, includingthose 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 coloring agents can be used. In one embodiment, when administered to an animal, the antibody or antigen binding fragment thereof of the present invention or compositions and pharmaceutically acceptable carriers are sterile. Water is a preferred carrier when the binding molecule or an antibody or antigen binding fragment thereof of the present invention is 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.

[0239] The pharmaceutical composition of the invention 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 subcutaneously.

[0240] When intended for oral administration, the composition is preferably 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.

[0241] 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.

[0242] 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.

[0243] Compositions can take the form of one or more dosage units.

[0244] In specific embodiments, it can be desirable to administer the composition locally to the area in need of treatment, or by intravenous injection or infusion.

[0245] The amount of the therapeutic that is effective / active in the treatment of a particular disorder or condition will depend on the nature of the disorder 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 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.

[0246] Typically, the amount is at least about 0.01 % of an antibody or antigen binding fragment thereof 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. Oral compositions can comprise from about 4% to about 50% of an antibody or antigen binding fragment thereof of the present invention by weight of the composition.

[0247] 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 an antibody or antigen binding fragment thereof of the present invention. The invention also relates to a device, such as a pre-filled syringe which comprises an antibody or antigen binding fragment thereof of the invention.

[0248] For administration by injection, the composition can comprise from about typically about 0.1 mg / kg to about 250 mg / kg of the subject’s body weight, preferably, between about 0.1 mg / kg and about 20 mg / kg of the subject’s body weight, and more preferably about 1 mg / kg to about 10 mg / kg of the subject’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.

[0249] As used herein, “treat”, “treating” or “treatment” means inhibiting or relieving a disease or disorder. For example, treatment can include a postponement of development of the symptomsassociated with a disease or disorder, 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., human patients, being treated. Many medical treatments are effective for some, but not all, patients that undergo the treatment.

[0250] 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.

[0251] As used herein, the term “effective amount” means an amount of the antibody or antigen binding fragment thereof as described herein, 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.

[0252] Therapy

[0253] A skilled person will understand that the antibody, antigen binding fragment thereof, radioimmunoconjugate or immunoconjugate of the invention can be used to treat a multitude of disease, depending on the therapeutic target of the antibody. Therapeutic antibodies and conjugates are well known and for example discussed in Sharma et al Molecules. 2023 Sep 5;28(18):6438.

[0254] In another aspect, the invention relates to an antibody, antigen binding fragment thereof, radioimmunoconjugate, immunoconjugate or a pharmaceutical composition according to the invention for use in the treatment of cancer, inflammatory disease, autoimmune disease, hematology conditions, endocrine disorders, infectious diseases, pulmonary diseases, cardiovascular disease, neurological disorders, allergic diseases, metabolic disorders, ophthalmic diseases, bone and joint diseases, dermatological diseases or transplantation associated diseases.

[0255] In another aspect, the invention relates to a method of treating cancer, inflammatory disease, autoimmune disease, hematology conditions, endocrine disorders, infectious diseases, pulmonary diseases, cardiovascular disease, neurological disorders, allergic diseases, metabolic disorders, ophthalmic diseases, bone and joint diseases, dermatological diseases or transplantation associated diseases comprising administering antibody, antigen bindingfragment thereof, radioimmunoconjugate, immunoconjugate or a pharmaceutical composition according to the invention.

[0256] The invention also relates to the use of an antibody, antigen binding fragment thereof, radioimmunoconjugate or immunoconjugate according to the invention in the manufacture of a medicament for the treatment of cancer, inflammatory disease, autoimmune disease, hematology conditions, endocrine disorders, infectious diseases, pulmonary diseases, cardiovascular disease, neurological disorders, allergic diseases, metabolic disorders, ophthalmic diseases, bone and joint diseases, dermatological conditions or transplantation associated diseases

[0257] An inflammatory disease may be rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, dermatitis, Crohn's disease, plaque psoriasis or ulcerative colitis.

[0258] A hematology disease may be hemophilia A or sickle cell disease.

[0259] A pulmonary disease may be asthma, lung cancer, and respiratory infections, COPD, respiratory syncytial virus (RSV) or IPF.

[0260] An infectious disease may be HIV, tuberculosis or malaria.

[0261] A neurological disorder may be multiple sclerosis (MS), Alzheimers or Parkinsons.

[0262] A metabolic disorder may be Pompe disease.

[0263] An ophthalmic disease may be age-related macular degeneration.

[0264] A bone and joint disease may be osteoporosis.

[0265] A dermatological condition may be skin diseases such as psoriasis or atopic dermatitis.

[0266] In one embodiment, the disease is selected from psoriasis, systemic lupus erythematosis, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis, idiopathic inflammatory myopathies, Sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, thyroiditis, diabetes mellitus, immune-mediated renal disease, demyelinating diseases of the central and peripheral nervous systems such as multiple sclerosis, idiopathic demyelinating polyneuropathy or Guillain Barre syndrome, chronic inflammatory demyelinating polyneuropathy, hepatobiliary diseases such as infectious, autoimmune chronic active hepatitis, primary biliary cirrhosis,granulomatous hepatitis, and sclerosing cholangitis, inflammatory bowel disease, glutensensitive enteropathy, and Whipple's disease, autoimmune or immune-mediated skin diseases including bullous skin diseases, erythema multiforme and contact dermatitis, allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity and urticaria, immunologic diseases of the lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, autoimmune haematological disorders (including e.g. hemolytic anaemia, aplastic anaemia, pure red cell anaemia and idiopathic thrombocytopenia), autoimmune inflammatory bowel disease (including e.g. ulcerative colitis, Crohn's disease and Irritable Bowel Syndrome), transplantation associated diseases including graft rejection and graft-versus-host-disease.

[0267] In one embodiment, the disease is cancer and the invention thus relates to methods for the prevention and / or treatment of cancer, comprising administering to a subject a cell or cell population comprising an antibody or antigen binding fragment thereof as described herein, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an antibody or antigen binding fragment thereof or pharmaceutical composition of the invention. The invention also relates to an antibody or antigen binding fragment thereof or pharmaceutical composition according to the invention for use in therapy. The invention also relates to an antibody or antigen binding fragment thereof or pharmaceutical composition according to the invention as described herein for use in the treatment of cancer. The invention also relates to the use of an antibody or antigen binding fragment thereof or pharmaceutical composition according to the invention in the manufacture of a medicament for the treatment of cancer.

[0268] The term "cancer" refers to a disease characterized by the uncontrolled growth of aberrant cells. Cancer includes all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs irrespective of the histopathologic type or stage of invasiveness. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. The cancer may be a primary or a secondary cancer. Thus, a molecule as described herein may be for use in a method of treating cancer in an individual, wherein the cancer is a primary tumour and / or a tumour metastasis.

[0269] In one embodiment, the cancer is selected from a haematological cancer or malignancy or a solid tumor. Hematologic cancers are cancers of the blood or bone marrow. Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas.

[0270] The cancer to be treated using an antibody or antigen binding fragment thereof or pharmaceutical composition of the invention may be a solid cancer. Examples of various cancers are described further herein and include, but are not limited to, mesothelioma, breast cancer,ovarian cancer, cervical cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, lung cancer, and prostate cancer.

[0271] In one embodiment, the cancer is metastatic.

[0272] In particular, cancers that may be treated by methods, uses, an antibody or antigen binding fragment thereof and compositions described herein include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant;choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0273] In reference to cancer, an effective amount may comprise an amount sufficient to cause a tumour to shrink and / or to decrease the growth rate of the tumour (such as to suppress tumour growth) or to prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay development or prolong survival or induce stabilisation of the cancer or tumour. In some embodiments, a therapeutically effective amount is an amount sufficient to prevent or delay recurrence. For example, a "therapeutically effective dosage" may induce tumour shrinkage by at least about 5% relative to baseline measurement, such as at least about 10%, or about 20%, or about 60% or more. The baseline measurement may be derived from untreated subjects.

[0274] In reference to an autoimmune disease, an effective amount may comprise an amount sufficient to partially or fully prevent disease activity, or to prevent or delay new disease activity or flare ups.

[0275] In some embodiments, an effective amount is an amount sufficient to delay the development of symptoms or prolong symptom-free periods or induce stabilisation of the disease.

[0276] In some embodiments, a therapeutically effective amount is an amount sufficient to prevent or delay recurrence of the disease, flare ups of the disease or incidences of new disease activity.A treatment may not take effect immediately. For example, treatment may be followed by an increase in disease activity, but over time eventual stabilization or reduction in disease activity in a given subject may subsequently occur.

[0277] Additional adverse symptoms and complications associated with neoplasia and autoimmune disease that can be inhibited, reduced, decreased, delayed, or prevented include, for example, nausea, swollen glands, lack of appetite, lethargy, pain, swelling, and discomfort. Thus, a partial or complete decrease or reduction in the severity, duration or frequency of an adverse symptom or complication associated with or caused by a cellular hyperproliferative disorder, an improvement in the subject’s quality of life and / or well-being, such as increased energy, appetite, psychological well-being, are all particular non-limiting examples of therapeutic benefit.

[0278] A therapeutic benefit or improvement therefore can also include a subjective improvement in the quality of life of a treated subject. In an additional embodiment, the invention prolongs or extends lifespan (survival) of the subject. In a further embodiment, a method improves the quality of life of the subject.

[0279] Exemplary combinations with other agents

[0280] An antibody or antigen binding fragment thereof or pharmaceutical composition of the invention may be administered as the sole active ingredient or in combination with one or more other therapeutic agent, e.g. an anti-cancer therapy. A therapeutic agent is a compound or molecule which is useful in the treatment of a disease. Examples of therapeutic agents include antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, pro-apoptotic agents, anti-angiogenic agents, boron compounds, photoactive agents or dyes and radioisotopes. An antibody molecule includes a full antibody or fragment thereof (e.g., a Fab, F(ab')2, Fv, a single chain Fv fragment (scFv) or a single domain antibody, for example a VH domain, antibody mimetic protein.

[0281] The anti-cancer therapy may include a therapeutic agent or radiation therapy and includes gene therapy, viral therapy, RNA therapy bone marrow transplantation, nanotherapy, targeted anticancer therapies or oncolytic drugs. Examples of other therapeutic agents include checkpoint inhibitors, antineoplastic agents, immunogenic agents, attenuated cancerous cells, tumor antigens, antigen presenting cells such as dendritic cells pulsed with tumor-derived antigen or nucleic acids, immune stimulating cytokines (e.g., IL-2, IFNa2, GM-CSF), targeted small molecules and biological molecules (such as components of signal transduction pathways, e.g. modulators of tyrosine kinases and inhibitors of receptor tyrosine kinases, and agents that bind to tumor- specific antigens, including EGFR antagonists), an anti-inflammatory agent, a cytotoxic agent, a radiotoxic agent, or an immunosuppressive agent and cells transfected with agene encoding an immune stimulating cytokine (e.g., GM-CSF) or chemotherapy. In another specific embodiment, the chemotherapeutic agent or radiation therapy is administered prior or subsequent to administration of the composition of the present invention, preferably at least an hour, five hours, 12 hours, a day, a week, a month, more preferably several months (e. g. up to three months), prior or subsequent to administration of composition of the present invention.

[0282] With reference to checkpoint inhibitors, checkpoint molecules include PD-1 , PDL-1 , PDL-2, LAG-3, CTLA-4, TIGIT and TIM-3. Checkpoint inhibitors include PD-1 inhibitors (Nivolumab, Pembrolizumab, and Cemiplimab), PDL-1 inhibitors (Atezolimumab, Durvalumab and Avelumab), and CTLA-4 inhibitor (Ipilimumab).

[0283] In one embodiment, administration is in combination with surgery. An antibody or antigen binding fragment thereof or pharmaceutical composition of the invention may be administered at the same time or at a different time as the other therapy, e.g., simultaneously, separately or sequentially.

[0284] Methods

[0285] The present invention also relates to method of making the antibodies, antigen binding fragment and / or immunoconjugates or radioimmunoconjugate as described herein.

[0286] An aspect of the invention relates to a method of preparing an immunoconjugate comprising conjugating a payload to the antibody or antigen binding fragment thereof as disclosed herein. An aspect of the invention relates to a method of preparing a radioimmunoconjugate comprising conjugating a radionuclide to the antibody or antigen binding fragment thereof as disclosed herein.

[0287] Conjugation of the payload may be performed by attaching the payload via the functionalized monosaccharide present on the antibody or antigen binding fragment thereof. In a preferred embodiment the conjugation comprises conjugation via a click reaction. Suitable click reactive groups which may be present on the functionalized monosaccharide and the payload are described herein above.

[0288] An aspect of the invention relates to a method of producing an immunoconjugate with enhanced payload attachment, comprising:

[0289] modifying the amino acid sequence of an antibody or antigen binding fragment thereof to comprise one or more engineered N-glycan site at position 190 of the light chain of said antibody or antigen binding fragment thereof, 159 and / or 186 of the heavy chain of said antibody or antigen binding fragment thereof;

[0290] andattaching a payload to said antibody or antigen binding fragment thereof via said functionalized monosaccharide.

[0291] The invention also relates to a method of enhancing payload attachment of an antibody, radio immunoconjugate, or immunoconjugate, comprising:

[0292] a) modifying the amino acid sequence of an antibody or antigen binding fragment thereof to comprise an engineered N-glycan site at amino acid position 159 of the heavy chain of said antibody or antigen binding fragment thereof according to Kabat numbering wherein said engineered N-linked glycan comprises a functionalized monosaccharide; and

[0293] b) attaching a payload to said antibody or antigen binding fragment thereof via said functionalized monosaccharide.

[0294] The invention also relates to a method of enhancing payload attachment of an antibody, radioimmunoconjugate, or immunoconjugate, comprising at least one engineered N-glycan site comprising a functionalized monosaccharide in the Fab region of said antibody or antigen binding fragment thereof, comprising:

[0295] a) modifying the amino acid sequence of an antibody or antigen binding fragment thereof to comprise an N-glycan site comprising N-X-S and / or N-X-T sequence motifs, wherein X is any amino acid other than proline, at amino acid position 159 of the heavy chain of said antibody or antigen binding fragment thereof according to Kabat numbering; and

[0296] b) contacting said antibody or antigen binding fragment with a glycosyltransferase capable of covalently attaching a functionalized monosaccharide at the N-glycan site, and a functionalized monosaccharide under conditions sufficient to covalently attach said functionalized monosaccharide at the N-glycan site.

[0297] In an embodiment the method of producing an immunoconjugate or radioimmunoconjugate and enhancing payload attachment of an antibody, radioimmunoconjugate, or immunoconjugate comprises a step of introducing a functionalized monosaccharide at the one or more engineered N-glycan site. In an embodiment the functionalized monosaccharide provides an attachment point for the payload.

[0298] In an embodiment the engineered N-glycan site comprises N-X-S and / or N-X-T sequence motifs, wherein X is any amino acid other than proline. The skilled person will be aware of suitable genetic engineering techniques to introduce an N-linked glycosylation site into an antibody sequence. Depending on the wild-type sequence of the antibody or fragment the N-linked glycosylation sequence motif may be introduced in a variety of ways. For example, in one embodiment, where there is a suitable X-S / T sequence downstream of the site at which the glycosylation is to be introduced, the sequence motif may be created by engineering into theamino acid sequence of an antibody or fragment thereof an asparagine residue at the desired position. However, in another embodiment, it is also possible that the N-X-S / T sequence motif is created by engineering into the amino acid sequence of an antibody or fragment thereof a serine or threonine residue downstream of any suitable N-X sequence. For example, engineering can be through an amino acid substitution of an existing residue with N, S or T or alternatively, a de novo residue may be inserted. The skilled person will be able to select the appropriate modification to make the glycosylation site at the desired position or positions, based on the wild-type sequence of the antibody or fragment thereof

[0299] An aspect of the invention relates to a method of producing an antibody or antigen binding fragment thereof comprising at least one engineered N-glycan site comprising a functionalized monosaccharide in the Fab region of said antibody or antigen binding fragment thereof, comprising:

[0300] modifying the amino acid sequence of an antibody or antigen binding fragment thereof to comprise one or more N-glycan site comprising N-X-S and / or N-X-T sequence motifs, wherein X is any amino acid other than proline, at position 190 of the light chain of said antibody or antigen binding fragment thereof, 159 and / or 186 of the heavy chain of said antibody or antigen binding fragment thereof;

[0301] contacting said antibody or antigen binding fragment with a glycosyltransferase capable of covalently attaching a functionalized monosaccharide at one or more N-glycan site, and a functionalized monosaccharide under conditions sufficient to covalently attach said functionalized monosaccharide at N-glycan site.

[0302] Modifying the amino acid sequence of an antibody or antigen binding fragment thereof to comprise one or more N-glycan site may comprise the substitution of a reference amino acid residue with another amino acid residue, insertion of one or more amino acid into a reference sequence or deletion of one or more amino acid. The modification of the amino acid sequence may be performed at the nucleic acid level by altering of modifying nucleotides to encode a different amino acid at specific positions. The method of the invention may comprise a step of expressing the antibody or antigen binding fragment. During the step of expressing the antibody or antigen binding fragment an N-linked glycan may be introduced at the engineered position. The N-linked glycan may comprise an oligomannose N-glycan, a complex N-glycan, or a hybrid N-glycan. In one embodiment, the complex N-glycan may be bi-antennary, in that it comprises two antennas. In another embodiment, the complex N-glycan may be tri-antennary, in that it comprises three antennas. In another embodiment, the complex N-glycan may be tetra-antennary, in that it comprises four antennas.

[0303] In one embodiment the N-glycan comprises a galactose moiety, the galactose moiety may serve at the attachment point for the functionalized monosaccharide. In one embodiment the N-glycanis a complex biantennary N-glycan comprising a galactose moiety. In one embodiment the N-glycan is a complex biantennary N-glycan comprising a galactose moiety, wherein the galactose moiety is attached to the functionalized monosaccharide. As described herein the skilled person will be aware of methods suitable to introduce specific N-glycans.

[0304] In an embodiment the step of contacting said antibody or antigen binding fragment with a glycosyltransferase capable of covalently attaching a functionalized monosaccharide at one or more N-glycan site, and a functionalized monosaccharide under conditions sufficient to covalently attach said functionalized monosaccharide at N-glycan site, results in covalent attachment to a galactose moiety present on the N-glycan.

[0305] The functionalized monosaccharide may comprise any of the features of the functionalized monosaccharides described herein above. In particular the functionalized monosaccharide may be selected from a functionalized sialic acid, a functionalized galactose, a functionalized N-acetylglucosamine (GIcNAc), a functionalized N-acetylgalactosamine GalNAc. In an embodiment the functionalized monosaccharide may be selected from N-azidoacetylneuraminic acid, N-azido acetylglucosamine, N-azidogalactose, N-azidoacetylgalactosamine.

[0306] According to the methods of the invention a transferase enzyme may be used to attach the functionalized glycan at the engineered N-linked glycosylation site. Suitable glycosyltransferases include but are not limited to sialyltransferase, galactosyltransferase, O-GIcNActransferase, galactosaminyltransferase.

[0307] The methods of the invention may further comprise a step of producing an immunoconjugate or radioimmunoconjugate comprising covalently attaching a further moiety to said antibody or antigen binding fragment thereof via said functionalized monosaccharide.

[0308] The antibodies and methods of the present invention comprise functionalized glycans to attach payloads or further moieties to said antibody, by using this approach the present inventors have demonstrated that conjugation can be achieved with excellent efficiency leading to more reliable DAR. A DAR or 2-4 is generally considered as optimal to obtain sufficient potency while preserving good physico-chemical properties for the. The inventors have demonstrated that conjugation can be achieved at multiple sites within the Fab region allowing for higher DAR ratios.

[0309] Another aspect of the invention relates to the use of an engineered antibody or antigen binding fragment thereof (comprising a modification at Kabat position 159) for use in enhancing payload conjugation.Exemplary Kits

[0310] In another aspect, the invention provides a kit comprising antibody or antigen binding fragment thereof, radioimmunoconjugate or immunoconjugate of the invention. The kit may also comprise instructions for use. The kits may include a labeled antibody or antigen binding fragment thereof of the invention as described above and one or more compounds for detecting the label. Also provided is an antibody or antigen binding fragment thereof of the invention packaged in lyophilized form or packaged in an aqueous medium. The kits may include a reagent, (e.g. for reconstituting) and / or instructions for use and / or a device for administration.

[0311] The kit may contain materials useful for the treatment of the disorders described above is provided. The kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds the bispecific binding molecule, radioimmunoconjugate, 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 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.

[0312] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present disclosure, including methods, as well as the best mode thereof, of making and using this disclosure, the following examples are provided to further enable those skilled in the art to practice this disclosure. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present disclosure will be apparent to those skilled in the art in view of the present disclosure.

[0313] All documents mentioned in this specification are incorporated herein by reference in their entirety, including references to gene accession numbers, scientific publications and references to patent publications."and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and / or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[0314] The term “comprising” or “comprises” where used herein means including the component(s) specified but not to the exclusion of the presence of other components. 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.

[0315] The term “consisting of’ or “consists of’ means including the components specified but excluding other components.

[0316] 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 “consisting essentially of’, and also may also be taken to include the meaning “consists of’ or “consisting of’.

[0317] 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.

[0318] 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.

[0319] Numbered Embodiments1. An antibody or antigen binding fragment thereof, comprising at least one N-linked glycan in the Fab region of said antibody or antigen binding fragment thereof, wherein the at least one N-linked glycan is present at one or more of amino acid position 190 of the light chain of said antibody or antigen binding fragment thereof, 159 and / or 186 of the heavy chain of said antibody or antigen binding fragment thereof.

[0320] 2. The antibody or antigen binding fragment thereof according to embodiment 1 , wherein the at least one N-linked glycan comprises a functionalized monosaccharide.

[0321] 3. The antibody or antigen binding fragment thereof according to embodiment 2, wherein the functionalized monosaccharide is selected from a functionalized sialic acid, a functionalized galactose, a functionalized N-acetylglucosamine (GIcNAc), a functionalized N-acetylgalactosamine GalNAc.

[0322] 4. The antibody or antigen binding fragment thereof according to any preceding embodiment, wherein the functionalized monosaccharide is selected from N-azidoacetylneuraminic acid, N-azido acetylglucosamine, N-azidogalactose, N-azidoacetylgalactosamine.

[0323] 5. The antibody or antigen binding fragment thereof according to any preceding embodiment comprising a further moiety covalently attached to said at least one N-linked glycan in the Fab region of said antibody or antigen binding fragment thereof.

[0324] 6. The antibody or antigen binding fragment thereof according to embodiment 5, wherein the further moiety is selected from a therapeutic moiety, half-life extending moiety or label.

[0325] 7. The antibody or antigen binding fragment thereof according to embodiment 6, wherein the label is a fluorophore, a fluorescer, a radiolabel, a chemiluminescer, a nuclear magnetic resonance active label, a photosensitizer or a biotin tag.

[0326] 8. The antibody or antigen binding fragment thereof according to embodiment 5, wherein the therapeutic moiety is a cell killing agent, an immune-modulating payload, a macrophage class switching agent, an oligonucleotide or a light activatable payload.

[0327] 9. The antibody or antigen binding fragment thereof according to embodiment 8, wherein the immune-modulating payload is a STING agonist or a toll-like receptor agonist.

[0328] 10. The antibody or antigen binding fragment thereof according to embodiment 8, wherein the cell killing agent comprises a cytotoxin.

[0329] 11. The antibody or antigen binding fragment thereof according to embodiment 10, wherein said cytotoxin is selected from:

[0330] i) a peptide toxin;

[0331] ii) a chemical toxin: or

[0332] iii) radio toxin.

[0333] 12. The antibody or antigen binding fragment thereof according to embodiment 10, wherein the cytoxin is selected from the group comprising auristatins, maytansinoids, tubulysins, RNA polymerase II inhibitors, transcription inhibitors, calicheamicins, duocarmycins, pyrrolobenzodiazepines, camptothecin analogues, topoisomerase inhibitors and doxorubicin.13. The antibody or antigen binding fragment thereof according to any one of embodiments 5 to 12, wherein the further moiety comprises a linker covalently which attaches to said functionalized monosaccharide.

[0334] 14. The antibody or antigen binding fragment thereof according to embodiment 13, wherein the linker comprises one or more of polyethylene glycol, glutaryl, valine-citrulline (Val-Cit), p-aminobenzylalcohol (PABA), 6-aminohexanoyl (Ahx), or N,N'-dimethylethylene diamine (DM EDA).

[0335] 15. The antibody or antigen binding fragment thereof according to any preceding embodiment wherein the antibody is selected from an lgG1, lgG2, lgG3, lgG4.

[0336] 16. The antibody or antigen binding fragment thereof according to according to any preceding embodiment wherein said fragment is selected from a F(ab')2, Fab, heavy chain, light chain, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies.

[0337] 17. The antibody or antigen binding fragment thereof according to any preceding embodiment, wherein the antibody or antigen binding fragment comprises one or more further N-linked glycans

[0338] 18. An immunoconjugate comprising the antibody or antigen binding fragment thereof according to any preceding embodiment and a further moiety.

[0339] 19. A nucleic acid encoding an antibody, antigen binding fragment thereof, or immunoconjugate according to any preceding embodiment.

[0340] 20. A vector comprising a nucleic acid according to embodiment 19.

[0341] 21. A host cell comprising the nucleic acid according to embodiment 19, or a vector according to embodiment 20.

[0342] 22. A pharmaceutical composition comprising an antibody or antigen binding fragment thereof according to any of embodiments 1 to 17 and a pharmaceutically acceptable excipient.

[0343] 23. An antibody or antigen binding fragment thereof according to any one of embodiments 1 to 17, an immunoconjugate according to embodiment 18, or a pharmaceutical composition according to embodiment 22 for use in the treatment of cancer, inflammatory disease, autoimmune disease, haematology conditions, endocrine disorders, infectious diseases, pulmonary diseases, cardiovascular disease, neurological disorders, allergic diseases, metabolic disorders, ophthalmic diseases, bone and joint diseases, dermatological diseases or transplantation associated diseases.

[0344] 24. A method of treating of cancer, inflammatory disease, autoimmune disease, hematology conditions, endocrine disorders, infectious diseases, pulmonary diseases, cardiovascular disease, neurological disorders, allergic diseases, metabolic disorders, ophthalmic diseases, bone and joint diseases, dermatological diseases or transplantation associated diseases comprising administering antibody or antigen binding fragment thereof according to any one of embodiments 1 to 17, an immunoconjugate according to embodiment 18, or a pharmaceutical composition according to embodiment 22 to a subject.25. The antibody or antigen binding fragment thereof for use according to embodiment 23 or the method according to embodiment 24 wherein said antibody or antigen binding fragment thereof is administered together with another therapy.

[0345] 26. A method of preparing an immunoconjugate comprising conjugating a payload to the antibody or antigen binding fragment thereof according to any one or embodiments 1 to 17. 27. A method of producing an immunoconjugate with enhanced payload attachment, comprising:

[0346] modifying the amino acid sequence of an antibody or antigen binding fragment thereof to comprise one or more engineered N-glycan site at position 190 of the light chain of said antibody or antigen binding fragment thereof, 159 and / or 186 of the heavy chain of said antibody or antigen binding fragment thereof;

[0347] and

[0348] attaching a payload to said antibody or antigen binding fragment thereof via said functionalized monosaccharide.

[0349] 28. The method of embodiment 27, further comprising a step of introducing a functionalized monosaccharide at the one or more engineered N-glycan site.

[0350] 29. The method of embodiment 27 or 28, wherein the engineered N-glycan site comprises N-X-S and / or N-X-T sequence motifs, wherein X is any amino acid other than proline.

[0351] 30. A method of producing an antibody or antigen binding fragment thereof comprising at least one engineered N-glycan site comprising a functionalized monosaccharide in the Fab region of said antibody or antigen binding fragment thereof, comprising:

[0352] modifying the amino acid sequence of an antibody or antigen binding fragment thereof to comprise one or more N-glycan site comprising N-X-S and / or N-X-T sequence motifs, wherein X is any amino acid other than proline, at position 190 of the light chain of said antibody or antigen binding fragment thereof, 159 and / or 186 of the heavy chain of said antibody or antigen binding fragment thereof;

[0353] contacting said antibody or antigen binding fragment with a glycosyltransferase capable of covalently attaching a functionalized monosaccharide at one or more N-glycan site, and a functionalized monosaccharide under conditions sufficient to covalently attach said functionalized monosaccharide at N-glycan site.

[0354] 31. The method of embodiment 30, wherein the functionalized monosaccharide is selected from a functionalized sialic acid, a functionalized galactose, a functionalized N-acetylglucosamine (GIcNAc), a functionalized N-acetylgalactosamine GalNAc.

[0355] 32. The method of embodiment 31 , wherein the functionalized monosaccharide is selected from N-azidoacetylneuraminic acid, N-azido acetylglucosamine, N-azidogalactose, N-azidoacetylgalactosamine.

[0356] 33. The method according to any one of embodiments 30 to 32 wherein the glycosyltransferase is selected from sialyltransferase, galactosyltransferase, O-GIcNActransferase, galactosaminyltransferase.34. The method according to any one of embodiments 30 to 33, further comprising a step of producing an immunoconjugate comprising covalently attaching a further moiety to said antibody or antigen binding fragment thereof via said functionalized monosaccharide.

[0357] EXAMPLES

[0358] Example 1 - Method for identifying novel N-glycan sites for payload conjugation Candidate N-glycan sites D17, L166 and Q178 within the Fab region of trastuzumab were selected using a structure-guided approaching. Selection criteria included:

[0359] 1. Structural context: Novel N-glycan sites were positioned within linker or disordered regions of the Fab constant domain to minimize disruption to protein folding to ensure that glycosylated at selected sites did not introduce steric hindrance or alter domain stability.

[0360] 2. Solvent accessibility: Asparagine residues (native or introduced) predicted to be solventexposes were identified through in silica analysis. Solvent accessibility was quantified to ensure residue exposure sufficient for glycosylation and subsequent payload conjugation

[0361] 3. Functional considerations: Candidate sites were selected to avoid antigen-binding regions and known protein-protein interaction domains.

[0362] Figure 1 shows a glycovariant design of trastuzumab showing a number of amino acid sites that may be mutated to an asparagine residue in order to create an N-X-S / T sequence motif. In Trastuzumab, the modified residues are D170N, L154N, and Q166N of the light chain (with reference to SEQ ID NO: 2), and L166N and Q178N of the heavy chain (with reference to SEQ ID NO: 1). The amino acid numbering referenced in the Examples is provided according to the sequence of Trastuzumab heavy chain (SEQ ID NO: 1) and light chain (SEQ ID NO: 2). D170N corresponds to position 190 according to Kabat numbering, L166N corresponds to position 159 according to Kabat numbering, Q178N corresponds to position 186 according to Kabat numbering. For other exemplary antibody sequences, the position at 159 Kabat is also highlighted herein.

[0363] Example 2 - Expression of trastuzumab glycovariants

[0364] Once the novel N-glycan sites on trastuzumab were identified, the genes encoding trastuzumab were cloned into mammalian expression vectors - pFUSE2ss-CLIg-hK and pFUSEss-CHIg-hG1 - using standard cloning techniques. Glycovariants of trastuzumab were generated through sitespecific mutagenesis to introduce the following mutations: D170N, L154N, and Q166N in the light chain, and L166N and Q178N in the heavy chain. The plasmids were transfected into glycan-engineered CHO cells and wild-type CHO-S cells using PEI-mediated transfection, and transient expression of each trastuzumab glycovariant was carried out for 7 days at 37 °C. Following cell culture, supernatants were collected by centrifugation. Protein expression in the supernatant was analyzed by SDS-PAGE. An increase in molecular weight was observed forthree out of the five glycovariants, consistent with the presence of additional N-glycans. Figure 2 shows the expression of D170N, L154N, Q166N, L166N, and Q178N in genetically engineered CHO cells and wild-type CHO-S cells.

[0365] Example 3 - Conjugation of payload to trastuzumab glycovariants

[0366] Trastuzumab glycovariants were harvested from cell culture supernatants and purified using Protein A affinity chromatography. Purified antibodies were enzymatically modified by incorporating a functionalized sialic acid moiety, N-azidoacetylneuraminic acid (NeuNAz), at the terminal position of each N-glycan using a sialyltransferase. Following an additional purification step to remove excess reagents, click chemistry was employed to conjugate a linker-payload system containing monomethyl auristatin E (MMAE), a microtubule-disrupting cytotoxic agent commonly used in antibody-drug conjugates (ADCs).

[0367] Conjugation efficiency was assessed by Western blot using an anti-MMAE antibody. Results demonstrated that N-glycans engineered in the Fab region showed superior conjugation efficiency relative to the native Fc N-glycan. As shown in Figure 3, this trend was observed across three single mutants (D170N, L166N, and Q178N) and a double mutant (D170N / Q178N), highlighting the enhanced payload attachment at engineered Fab N-glycan sites.

[0368] Example 4 - Mass spectroscopy of L166N non-conjugated and conjugated to payload Antibody sample trastuzumab with the modification at L166N, including glycan-modified and conjugated variants, were subjected to liquid chromatography-mass spectrometry (LC-MS) analysis to assess N-glycosylation and MMAE conjugation. Prior to analysis, antibodies were enzymatically digested to generate defined subunits and, where appropriate, further treated under reducing and / or denaturing conditions to obtain additional fragment species.

[0369] The resulting samples were separated by liquid chromatography and analyzed by mass spectrometry to acquire intact mass data for the different species present. Chromatographic and mass spectral data were processed to determine the molecular weights of the observed species and compared to theoretical values.

[0370] This enabled identification of glycan-modified fragments, discrimination between conjugated and unconjugated species, and detection of mass shifts associated with linker cleavage or payload attachment.

[0371] Relative abundances of the different species were used to assess conjugation efficiency and stability of the conjugated constructs.

[0372] References

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[0374] Zhu Z, Ramakrishnan B, Li J, Wang Y, Feng Y, Prabakaran P, Colantonio S, Dyba MA, Qasba PK, Dimitrov DS. Site-specific antibody-drug conjugation through an engineered glycotransferase and a chemically reactive sugar. MAbs. 2014;6(5):1190-200. doi: 10.4161 / mabs.29889.

[0375] Haque M, Forte N, Baker JR. Site-selective lysine conjugation methods and applications towards antibody-drug conjugates. Chem Commun (Camb). 2021 Oct 14;57(82):10689-10702. doi: 10.1039 / d1cc03976h.

[0376] McCombs JR, Owen SC. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. AAPS J. 2015 Mar;17(2):339-51. doi: 10.1208 / s12248-014-9710-8. Boswell CA, Mundo EE, Zhang C, Bumbaca D, Valle NR, Kozak KR, Fourie A, Chuh J, Koppada N, Saad O, Gill H, Shen BQ, Rubinfeld B, Tibbitts J, Kaur S, Theil FP, Fielder PJ, Khawli LA, Lin K. Impact of drug conjugation on pharmacokinetics and tissue distribution of anti-STEAP1 antibody-drug conjugates in rats. Bioconjug Chem. 2011 Oct 19;22(10):1994-2004. doi: 10.1021 / bc200212a.

[0377] Wagh A, Song H, Zeng M, Tao L, Das TK. Challenges and new frontiers in analytical characterization of antibody-drug conjugates. MAbs. 2018 Feb / Mar;10(2):222-243. doi: 10.1080 / 19420862.2017.1412025.

[0378] Nguyen TD, Bordeau BM, Balthasar JP. Mechanisms of ADC Toxicity and Strategies to Increase ADC Tolerability. Cancers (Basel). 2023 Jan 24; 15(3):713. doi: 10.3390 / cancersl 5030713. Johann F, W6II S, Winzer M, Gieseler H. Agitation-Induced Aggregation of Lysine- And Interchain Cysteine-Linked Antibody-Drug Conjugates. J Pharm Sci. 2024 May ;113(5): 1265-1274. doi: 10.1016 / j.xphs.2023.12.003.

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Claims

66CLAIMS1. An antibody or antigen binding fragment thereof, comprising at least one N-linked glycan in the Fab region of said antibody or antigen binding fragment thereof, wherein the at least one N-linked glycan is present at amino acid position 159 of the heavy chain of said antibody or antigen binding fragment thereof according to Kabat numbering wherein the at least one N-linked glycan comprises a functionalized monosaccharide.

2. The antibody or antigen binding fragment thereof according to claim 1 , wherein the functionalized monosaccharide is selected from a functionalized sialic acid, a functionalized galactose, a functionalized N-acetylglucosamine (GIcNAc), a functionalized N-acetylgalactosamine GalNAc.

3. The antibody or antigen binding fragment thereof according to any preceding claim, wherein the functionalized monosaccharide is selected from N-azidoacetylneuraminic acid, N-azido acetylglucosamine, N-azidogalactose, N-azidoacetylgalactosamine.

4. The antibody or antigen binding fragment thereof according to any preceding claim comprising a further moiety covalently attached to said at least one N-linked glycan in the Fab region of said antibody or antigen binding fragment thereof.

5. The antibody or antigen binding fragment thereof according to claim 4, wherein the further moiety is selected from a therapeutic moiety, half-life extending moiety or label.

6. The antibody or antigen binding fragment thereof according to claim 5, wherein the label is a fluorophore, a fluorescer, a radiolabel, a chemiluminescer, a nuclear magnetic resonance active label, a photosensitizer or a biotin tag.

7. The antibody or antigen binding fragment thereof according to claim 5, wherein the therapeutic moiety is a cell killing agent, an immune-modulating payload, a macrophage class switching agent, an oligonucleotide or a light activatable payload.

8. The antibody or antigen binding fragment thereof according to claim 7, wherein the immune-modulating payload is a STING agonist or a toll-like receptor agonist.

9. The antibody or antigen binding fragment thereof according to claim 7, wherein the cell killing agent comprises a cytotoxin.

10. The antibody or antigen binding fragment thereof according to claim 9, wherein said cytotoxin is selected from:i) a peptide toxin;ii) a chemical toxin: oriii) radio toxin.

11. The antibody or antigen binding fragment thereof according to claim 9, wherein the cytoxin is selected from the group comprising auristatins, maytansinoids, tubulysins, RNA polymerase II inhibitors, transcription inhibitors, calicheamicins, duocarmycins,67pyrrolobenzodiazepines, camptothecin analogues, topoisomerase inhibitors and doxorubicin.

12. The antibody or antigen binding fragment thereof according to claim 10, wherein the radio toxin is selected from the group comprising actinium-225 (225Ac), astatine-211 (211At), bismuth-213 (213Bi), indium-111 (111ln), iodine-123 (1231), iodine-124 (1241), iodine-131 (1311), lead-212 (212Pb), lutetium-177 (177Lu), technetium-99m (99mTc), copper-64 (64Cu), gallium-68 (68Ga), yttrium-86 (86Y), yttrium-90 (90Y), and zirconium-89 (89Zr), lodine-125 (1251), Rhenium-186 (186Re) Rhenium-188 (188Re), Samarium-153 (153Sm) , Phosphorus-32 (32P), Cobalt-60 (60C).

13. The antibody or antigen binding fragment thereof according to any one of claims 4 to 12, wherein the further moiety comprises a linker covalently which attaches to said functionalized monosaccharide.

14. The antibody or antigen binding fragment thereof according to claim 13, wherein the linker comprises one or more of polyethylene glycol, glutaryl, vali n e-citru Hi ne (Val-Cit), p-aminobenzylalcohol (PABA), 6-aminohexanoyl (Ahx), or N,N'-dimethylethylene diamine (DMEDA).

15. The antibody or antigen binding fragment thereof according to any preceding claim, wherein the antibody is selected from an lgG1 , lgG2, lgG3, lgG4.

16. The antibody or antigen binding fragment thereof according to according to any preceding claim, wherein said fragment is selected from a F(ab')2, Fab, heavy chain, light chain, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies.

17. The antibody or antigen binding fragment thereof according to any preceding claim, wherein the antibody or antigen binding fragment comprises one or more further N- linked glycans.

18. The antibody or antigen binding fragment thereof according to any preceding claim, wherein the antibody or antigen binding fragment binds to a tumour associated antigen, a checkpoint inhibitor or an antigen expressed in a hematological malignancy.

19. The antibody or antigen binding fragment thereof according to any preceding claim, wherein the antibody or antigen binding fragment binds to HER2, Trop2, B7-H3, DLL3 or PD-L1 .

20. The antibody or antigen binding fragment thereof according to preceding claim 19, wherein the antibody is Trastuzumab, Enoblituzumab Sacituzumab, Rovalpitzumab or Atezolizumab or an or antigen binding fragment thereof.

21. An immunoconjugate comprising the antibody or antigen binding fragment thereof according to any preceding claim and a further moiety.

22. A radioimmunoconjugate comprising the antibody or antigen binding fragment thereof according to any preceding claim and a radionuclide.6823. A nucleic acid encoding an antibody, antigen binding fragment thereof, radioimmunoconjugate or immunoconjugate according to any preceding claim.

24. A vector comprising a nucleic acid according to claim 23.

25. A host cell comprising the nucleic acid according to claim 23, or a vector according to claim 24.

26. A pharmaceutical composition comprising an antibody or antigen binding fragment thereof according to any of claims 1 to 20, an immunoconjugate according to claim 21 or a radioimmunoconjugate according to claim 22 and a pharmaceutically acceptable excipient.

27. An antibody or antigen binding fragment thereof according to any one of claims 1 to 20, an immunoconjugate according to claim 21 , a radioimmunoconjugate according to claim 22, or a pharmaceutical composition according to claim 26 for use in the treatment of cancer, inflammatory disease, autoimmune disease, haematology conditions, endocrine disorders, infectious diseases, pulmonary diseases, cardiovascular disease, neurological disorders, allergic diseases, metabolic disorders, ophthalmic diseases, bone and joint diseases, dermatological diseases or transplantation associated diseases.

28. A method of treating of cancer, inflammatory disease, autoimmune disease, hematology conditions, endocrine disorders, infectious diseases, pulmonary diseases, cardiovascular disease, neurological disorders, allergic diseases, metabolic disorders, ophthalmic diseases, bone and joint diseases, dermatological diseases or transplantation associated diseases comprising administering antibody or antigen binding fragment thereof according to any one of claims 1 to 20, an immunoconjugate according to claim 21 , a radioimmunoconjugate according to claim 22, or a pharmaceutical composition according to claim 26 to a subject.

29. The antibody or antigen binding fragment thereof for use according to claim 27 or the method according to claim 28 wherein said antibody or antigen binding fragment thereof is administered together with another therapy.

30. A method of preparing an immunoconjugate comprising conjugating a payload to the antibody or antigen binding fragment thereof according to any one or claims 1 to 20.

31. A method of preparing a radioimmunoconjugate comprising conjugating a radionuclide to the antibody or antigen binding fragment thereof according to any one or claims 1 to 20.

32. A method of producing an immunoconjugate with enhanced payload attachment, comprising:a) modifying the amino acid sequence of an antibody or antigen binding fragment thereof to comprise an engineered N-glycan site at amino acid position 159 of the heavy chain of said antibody or antigen binding fragment thereof according to Kabat numbering69wherein said engineered N-linked glycan comprises a functionalized monosaccharide; andb) attaching a payload to said antibody or antigen binding fragment thereof via said functionalized monosaccharide.

33. The method of claim 32, further comprising a step of introducing a functionalized monosaccharide at amino acid position 159.

34. The method of claim 32 or 33, wherein the engineered N-glycan site comprises N-X- S and / or N-X-T sequence motifs, wherein X is any amino acid other than proline.

35. A method of producing an antibody or antigen binding fragment thereof comprising at least one engineered N-glycan site comprising a functionalized monosaccharide in the Fab region of said antibody or antigen binding fragment thereof, comprising:a) modifying the amino acid sequence of an antibody or antigen binding fragment thereof to comprise an N-glycan site comprising N-X-S and / or N-X-T sequence motifs, wherein X is any amino acid other than proline, at amino acid position 159 of the heavy chain of said antibody or antigen binding fragment thereof according to Kabat numbering; andb) contacting said antibody or antigen binding fragment with a glycosyltransferase capable of covalently attaching a functionalized monosaccharide at the N-glycan site, and a functionalized monosaccharide under conditions sufficient to covalently attach said functionalized monosaccharide at the N- glycan site.

36. The method of claim 35, wherein the functionalized monosaccharide is selected from a functionalized sialic acid, a functionalized galactose, a functionalized N- acetylglucosamine (GIcNAc), a functionalized N-acetylgalactosamine GalNAc.

37. The method of claim 35, wherein the functionalized monosaccharide is selected from N-azidoacetylneuraminic acid, N-azido acetylglucosamine, N-azidogalactose, N- azidoacetylgalactosamine.

38. The method according to any one of claims 35 to 37 wherein the glycosyltransferase is selected from sialyltransferase, galactosyltransferase, O-GIcNActransferase, galactosaminyltransferase.

39. The method according to any one of claims 35 to 38, further comprising a step of producing an immunoconjugate comprising covalently attaching a further moiety to said antibody or antigen binding fragment thereof via said functionalized monosaccharide.

40. A method of enhancing payload attachment of an antibody, radio immunoconjugate or immunoconjugate, comprising:70a) modifying the amino acid sequence of an antibody or antigen binding fragment thereof to comprise an engineered N-glycan site at amino acid position 159 of the heavy chain of said antibody or antigen binding fragment thereof according to Kabat numbering wherein said engineered N-linked glycan comprises a functionalized monosaccharide; andb) attaching a payload to said antibody or antigen binding fragment thereof via said functionalized monosaccharide.

41. A method of enhancing payload attachment of an antibody, radioimmunoconjugate or immunoconjugate, comprising at least one engineered N-glycan site comprising a functionalized monosaccharide in the Fab region of said antibody or antigen binding fragment thereof, comprising:a) modifying the amino acid sequence of an antibody or antigen binding fragment thereof to comprise an N-glycan site comprising N-X-S and / or N-X-T sequence motifs, wherein X is any amino acid other than proline, at amino acid position 159 of the heavy chain of said antibody or antigen binding fragment thereof according to Kabat numbering; andb) contacting said antibody or antigen binding fragment with a glycosyltransferase capable of covalently attaching a functionalized monosaccharide at the N-glycan site, and a functionalized monosaccharide under conditions sufficient to covalently attach said functionalized monosaccharide at the N- glycan site.

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