Antibody-drug conjugates having a tailor-made drug-to-antibody ratio

Abstract

The present invention provides an antibody-drug conjugate (ADC), wherein the antibody is conjugated to the drug by cysteine-based site-specific conjugation, wherein the antibody comprises a first polypeptide comprising an immunoglobulin fragment crystallisable (Fc) region, at least one variable domain N-terminal of the Fc region and an engineered IgG hinge region between the Fc region and the at least one variable domain, and wherein the engineered IgG hinge region is heterologous to the Fc region and / or is mutated to provide a predetermined number of cysteine residues.

Description

[0001] ANTIBODY-DRUG CONJUGATES HAVING A TAILOR-MADE DRUG-TO-ANTIBODY RATIO

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to antibody-drug conjugates (ADCs) having a tailor-made drug- to-antibody ratio (DAR). In particular, the ADCs of the invention comprise an engineered IgG hinge region that provides a predetermined number of cysteine residues. This enables the production of ADCs having the desired DAR and a homogenous ADC product using sitespecific cysteine conjugation methods. The present invention also relates to methods of producing the ADCs and the therapeutic use of the ADCs.

[0004] BACKGROUND TO THE INVENTION

[0005] Antibody-based therapies have proven revolutionary in the treatment of diseases such as cancer. Traditional use of monoclonal antibodies (mAbs) has certain limitations, including a lack of efficacy in certain indications or low tumour expression of the antigen the mAb is targeting. This has led to the development of next-generation antibody-based therapies, including bispecific antibodies (bsAbs), mRNA-encoded antibodies and antibody drug conjugates (ADCs).

[0006] ADCs are conjugates of mAbs, bsAbs or antigen-binding antibody fragments that may be specific for a cancer related antigen, with a chemical linker sequence and a drug (often referred to as a “payload”). The payload is generally a molecule with cytotoxic activity, including small molecules, peptide toxins and radionuclides. By generating an ADC, the payload benefits from a long in vivo half-life and directed tumour targeting, avoiding off-target activity, due to the conjugated antibody or antigen-binding fragment. Additionally, the chemical linker between the antibody and payload may be designed in a cleavable manner, thus upon internalization of the ADC, tumour or endosome-specific proteases may free the payload enabling cell-specific activity. The payload is then free to mediate toxicity via different pathways, including the inhibition of topoisomerases, the inhibition of polymerization of tubulin or DNA damage.

[0007] The DAR is a key property used to measure the quality of ADCs because it can significantly affect ADC efficacy. It has been demonstrated that higher DARs mediate a higher potency of an ADC. It is essential to measure the DAR because a low DAR value may indicate decreased efficacy, while a relatively high DAR value may negatively impact safety. The optimal DAR value for a given ADC varies depending upon several factors such as the payload and target tissue. Various strategies exist for the conjugation of antibodies to linker-payload molecules, including random conjugation to primary amines present on surface exposed lysine side chains, enzyme-mediated conjugation to specific peptide tags (e.g. transglutaminase or sortase tags) or the utilization of introduced non-natural amino acids or direct conjugation to the N297 glycan structure. However, coupling via lysine side chains yields heterogenic products with varying drug-to-antibody ratios (DAR). Enzyme-mediated conjugation approaches also come with limitations, including the additional manufacturing steps to produce the enzymes and remove the enzymes after the conjugation steps since any residual bacterial enzymes would be immunogenic. Moreover, for sortase a peptide tag must be added to the antibody, which can change the physicochemical properties of the protein, and which might be immunogenic as well.

[0008] Despite the success of some first-generation ADCs, their complex heterogeneity has been associated with several problems, including suboptimal therapeutic index, higher clearance rates for high-DAR species, narrower therapeutic windows, and poor stability. To overcome these challenges, second-generation, site-specific ADCs have been designed and developed. The production of site-specific ADCs precisely controls not only the average DARs but also the number of unique conjugation sites. Compared with the production of first-generation ADCs, which yields thousands to millions of different structures, the production of second- generation ADCs yields only a few unique structures, which can be optimized, developed, and manufactured more easily.

[0009] One of the main obstacles in the ADC field lies in creating ADCs exhibiting exactly the anticipated DAR. A standard IgG 1 antibody exhibits two inter-heavy chain disulfide bonds and two inter-heavy-light-chain disulfide bonds. This consequently results in a maximal DAR 8 upon reducing these disulfides to two cysteine residues each. However, when lower DARs are anticipated, the resulting ADC will exhibit a very heterogeneous DAR distribution. A good example is given by Fabre et al. 2020, (Fig 1b DOI: 10.1158 / 1078-0432. CCR-19-2238). Here an IgG 1 antibody was conjugated to an ADC exhibiting a DAR of 3.5. However, as lgG1s have eight free cysteine residues upon reduction, the authors yielded a mixture of DAR 1 , DAR 2, DAR 3, DAR 4, DAR 5, DAR 6, DAR 7 and DAR 8 species. While their relative quantity to one another yielded a theoretical average DAR of 3.5, this ADC is very heterogeneous as actually a mixture of different ADCs. Since different DAR species can display different PK profiles or cytotoxic effects, a more homogeneous product would be favourable. The results presented by Jones et al., 2020 (DOI: 10.1080 / 19420862.2019.1682895) underlines the heterogeneity in state-of-the-art ADCs and clearly demonstrates that ADCs with DARs significantly lower than the maximal achievable. Furthermore, the prior art has been fraught with other issues of heterogeneity in antibody products. Glaser et al., 2005 described a hinge engineering approach for CH2-deleted antibodies which can be present in two isoforms: the desired isoform A where the hinge cysteine from inter-chain disulfide bonds and the undesired isoform B where intra-chain disulfide bonds are formed and the two antibody chains are not covalently linked (Glaser et al. Journal of biological chemistry 2005 280(50):41494-41503, doi: 10.1074 / j be. M 508739200). Glaser and colleagues aimed to solve this problem by insertion of an lgG3 hinge motif into the lgG1 hinge of this antibody. This sequence was termed G1 / G3:PAP. While this isotype mixed hinge boosted the presence of the isoform A to over 98%, “mass spectrometry analysis suggest that at least two, and as many as five, inter-heavy chain disulfide linkages may be present’. That means Glaser and colleagues failed in utilizing an lgG3 hinge motif to create a homogeneous antibody product, in which all five inter-chain disulfides form in the desired interchain manner but yielded a heterogeneous product containing a mixture of different hinge species. As for the formation of isoform A only one of the five inter-chain disulfides need to be present, Glaser et al., achieved the primary goal of their research. However, this clearly demonstrated that hinge sequences cannot be used in a simple plug-and-play manner to create well-behaving and homogeneous antibody products.

[0010] Consequently, in standard lgG1 antibodies, scientists need to choose between high homogeneity and a DAR of approximately 8, or a lower DAR which might better fit the target biology but is resulting in a more heterogeneous ADC product. Further, for certain antigens and payloads DARs of over 8 might be envisioned, but this is not achievable with unmodified lgG1 antibodies and cysteine-targeting conjugation methods.

[0011] Numerous approaches have been developed to generate site-specific ADCs. Conjugation strategies include site-specific coupling via cysteines, which is based on the incorporation of a thiol-reactive group on the linker-payload molecule. One such approach, the Thiomab approach, relies on artificially introducing cysteine residues in the heavy chain (HC) and / or light chain (LC) of the antibody, wherein a toxin-linker comprising maleimide, a moiety capable of covalently conjugating to thiol groups of cysteines, conjugates to the antibody of interest. However, free (unpaired) cysteines and the complicated conjugation strategy can negatively impact the chemistry, manufacturing and controls (CMC) development process of the antibody (Kostova V et al., Pharmaceuticals, 2021 ; 14: 442). These CMC challenges include, but are not limited to: antibody heterogeneity, low stability, aggregation, low solubility and reduced potency. Furthermore, artificial introduction of cysteine residues may occur at immunogenic positions within the antibody (for example, in conserved regions of the antibody backbone) and alter the immunogenicity profile of the antibody. Moreover, with the ThioMab technology, introduced cysteines are always coupled with glutathione or the amino acid cysteine and require activation before conjugation. This is achieved by reduction of the antibody. This in turn results in the opening of naturally occurring inter-chain disulfides, which must subsequently be closed again in an oxidation reaction. Only after reduction and oxidation, can the conjugation to the linker-payload can be performed. This additional oxidation step leads to a more complicated process for the generation of the ADC (e.g. Fig 1a of Juntula et al. Nature Biotechnology 2008 26:925-32; https: / / doi.org / 10.1038 / nbt.1480). Further, unpaired cysteines are highly undesirable in antibodies as they can mediate a number of obstacles and CMC challenges. These CMC challenges include, but are not limited to: antibody heterogeneity, low stability, aggregation, low solubility and reduced potency (Xu et al., 2019; DOI: 10.1080 / 19420862.2018.1553476, Metcalfe et al., 2022; DOI: 10.3389 / fmolb.2022.886417, Zhang et al., 2023; DOI: 10.1093 / abt / tbac029). Furthermore, if the cysteine is introduced in conserved regions of the antibody backbone, immunogenicity issues might arise.

[0012] In view of the limitations associated with introducing cysteine residues into the HC and / or LC of an antibody, an alternative conjugation strategy relies on the cysteine residues that are naturally occurring within the antibody. For example, an lgG1 antibody displays four interchain disulphide bonds, two between the HCs and one between each HC with its respective LC. It is therefore possible to reduce the inter-chain disulphides to expose eight free cysteines that can be subsequently conjugated to the linker-payloads. Using such an approach, a homogenous product with a DAR of eight can be generated. Antibody fragments such as VHHs, however, are limited in their number of disulphide bridges. A conventional VHH-Fc, for example, exhibits two inter-chain disulphide bridges between the hinge regions of the two HCs. Upon reduction and subsequent conjugation to a linker-payload conjugate via thiolreactive coupling strategy, such as a maleimide-based conjugation, a homogenous product with a DAR of four may therefore be generated. However, relying upon the naturally occurring cysteine residues within the antibody limits the DAR of the ADC product to the number of naturally occurring inter-chain disulphide-forming cysteine residues within the antibody.

[0013] Single domain antibodies (sdAb) like VHHs gained interest in the scientific community due to their small size and high versatility as fusion partners. Due to their small size (~15 KDa) they allow for deep tissue and tumour penetration. However, they generally also exhibit very short half-lives in the range of minutes. As such, VHHs are often fused to half-life extending fusion partners such as serum albumin binding moieties (e.g. peptides or VHHs) or lgG1-derived fragment crystallizable (Fc) portions. VHH-Fc fusions are able to mediate effector functions in a similar fashion to conventional IgGs, while being smaller in size, being less complex in architecture, but still having similar half-lives to conventional IgGs. However, the utilization of VHHs in the context of ADCs is still in an early phase. Moreover, these antibody formats have intrinsic limitations in their use as ADCs.

[0014] In particular, for VHH-based ADCs, the DAR problem comes in a slightly different form. To increase half-life, these molecules most of the time exhibit an lgG1 Fc, which by binding to FcRn elongates the half-life. The fusion of the VHH to the Fc is achieved by utilizing an lgG1 - derived hinge. Examples for these molecules are Rimteravimab, Simridarlimab, Ozekibart, Erfonrilimab, Letolizumab, Envafolimab and Porustobart. As all exhibit only two inter-heavy chain disulfides, a VHH-Fc based ADC following this state-of-the-art protein design could only exhibit a maximal DAR of 4.

[0015] Since the sixties, antibody hinge regions have been considered liable for proteolytic cleavage. (Turner & Bennich Biochem J 1968 107(2):171-8 doi: 10.1042 / bj1070171 ). The cleavage can be mediated by a number of proteases (Ryan et al. Mol Immunol 2008 45(7):1837-46 doi: 10.1016 / j.molimm.2007.10.043) and especially lgG3 hinges are considered to be prone for proteolytic cleavage (Baici et al. Scan J Immunol 1980 12(1):41-50 doi: 10.1111 / j.1365- 3083.1980. tb00039.x; Turner & Natvig Nature 225(5235):853-5 doi: 10.1038 / 225853b0; Virella & Parkhouse Immunochemistry 1971 8(3):243-50 doi: 10.1016 / 0019-2791(71)90478- 2). One report even mentioned the cleavage of antibody hinge regions in a non-enzymatic manner (Cordoba et al. Journal of Chromatography B 2005 818(2):115-121 doi: 10.1016 / j.jchromb.2004.12.033).

[0016] Further, scientists refrain especially from lgG3 antibodies for ADC development, partly because of the lgG3 hinge region. Hoffmann et al. states that lgG3 “has so far been avoided for the development of ADCs because of its low half-life in serum compared to the other classes (e.g., 7 days instead of 21 days for lgG1, 2 and 4), its long hinge region that is subject to proteolysis and also evidence of potential immunogenicity’ (Hoffmann et al. Oncoimmunology 2018 7(3):e1395127 doi: 10.1080 / 2162402X.2017.1395127).

[0017] In general, scientists refrain from engineering hinge regions in antibodies. This is emphasized by the fact that many papers describe the engineering of variable and constant domains, while the limited number of papers describing hinge engineering experiments investigate unrelated concerns such as fragmentation resistance and recognition by pre-existing anti-hinge antibodies, or describe the removing the hinge sequence from the antibody completely. While there are reports in which the cysteines are removed in lgG1 hinges, in combination with homologous lgG1 Fes, to lower the DAR (Cho et al. Large molecule therapeutics 2018 17(10):2176-2186 doi: 10.1158 / 1535-7163. MCT-17-0982), there are no hinge engineering techniques described in the art to elevate the DAR above the threshold of 8 for IgGs or the threshold of 4 for VHH-Fcs by hinge engineering. Using heterologous hinge regions is further taught away from in the art due to the intrinsic instabilities of IgG hinges. Moritz and Stracke states that “the hinge region is prone to cleavage and is involved in pathways that lead to thioether bond formation, cysteine racemization, and iso -Asp (Asp, aspartic acid) formation. Disulfide or sulfhydryl groups were found to be prone to reductive cleavage, trisulfide formation, cysteinylation, glutathionylation, disulfide bridging to further light chains, and disulfide scrambling. With regard to potency, disulfide cleavage, hinge cleavage, disulfide bridging to further light chains, and cysteinylation were found to influence antigen binding and fragment crystallizable (Fc) effector functionalities. Renal clearance of small fragments may be faster, whereas clearance of larger fragments appears to depend on their neonatal Fc receptor (FcRn) functionality, which in turn may be impeded by disulfide bond cleavage" (Moritz & Stricke Electrophoresis 2017 38(6):769-785 doi: 10.1002 / elps.201600425).

[0018] US 2016 / 129129 A1 discloses an antibody-drug conjugate comprising an lgG4 Fc domain and homologous lgG4 hinge region. In the antibody, a sequence comprising cysteine has been deleted. Avoiding N-glycosylation is also disclosed. This document does not disclose more substantive engineering such as via heterologous Fc region-hinge region pairings.

[0019] US 2009 / 117100 A1 discloses an antibody-drug conjugate engineered to include unpaired / free cysteines for drug conjugation. Similar technology is described in US 7855275 B2, US 7723485 B2, US 2019 / 300559 and CA 3178093 A1. The engineering in these documents does not involve substantively engineering the hinge regions or drug conjugation to paired cysteines that form inter-chain disulphide bonds between the hinge regions.

[0020] US 2016 / 176964 A1 discloses similar technology wherein one of a pair of cysteine residues is mutated, leaving an unpaired cysteine residue for conjugation. This document does not disclose substantively engineering the hinge region or heterologous Fc -hinge region pairing.

[0021] EP 4001303 A1 discloses antibody-drug conjugate engineering in the context of am / ne-linked conjugation.

[0022] WO 2015 / 095972 A1 discloses antibody-drug conjugate engineering in which a linker extension comprising a cysteine conjugation site is introduced onto the C-terminus of the antibody light chain. This engineering does not involve substantive engineering of the hinge regions.

[0023] There is a demand for new and effective ADC therapeutics, and methods of producing said therapeutics. A barrier to this is the inherent unpredictability of making novel modifications to the polypeptide sequences of antibody constant domains, which may result in unforeseeable disadvantageous / deleterious effects on protein structure. Current approaches in the art have explored specific point mutations or deletions, but have not yet considered the possibility of engineering entire regions of the polypeptide sequence for ADC purposes.

[0024] SUMMARY OF THE INVENTION

[0025] The present invention is based, at least in part, on the inventors’ development of antibodydrug conjugates (ADCs) which have particularly advantageous and surprising properties.

[0026] It is desirable to be able to control the DAR of an ADC, for example to produce an ADC which is optimised for a specific therapeutic use by balancing efficacy and safety of the product. The present inventors have developed engineered IgG-derived hinge regions that connect an Fc region with a variable domain N-terminal of the Fc region, wherein the hinge region advantageously comprises a predetermined number of cysteine residues. This enables the production of a homogenous ADC product with the desired DAR via site-specific cysteine- based conjugation strategies. Moreover, by utilising an engineered hinge region that is based upon a naturally occurring IgG hinge region, immunogenicity is reduced. The present inventors have also demonstrated that modifications to avoid O-glycosylation are tolerated within the engineered IgG hinge regions. This is advantageous, since O-glycosylation may lead to CMC challenges of the later ADC, for example by increasing antibody heterogeneity.

[0027] The present inventors have surprisingly shown that an ADC comprising an engineered IgG hinge region demonstrates preserved or elevated ADC properties, including stability, cell binding, internalization, antibody-dependent cellular cytotoxicity (ADCC) and complementdependent cytotoxicity (CDC). In particular, the present inventors have achieved this using heterologous-isotype pairings between Fc regions and hinge regions, i.e. engineering the antibody polypeptide sequence to comprise a hinge region that is derived from a different isotype (e.g. lgG1 , 2, 3 or 4) to the isotype of the Fc region. Herein, the present inventors surprisingly demonstrate that this approach leads to the provision of stable and effective ADC constructs, wherein the DAR can be controlled via the choice of the heterologous hinge region sequences that are used. Furthermore, the present inventors have surprisingly found that, prior to conjugation, all of the cysteine residues within the engineered IgG hinge regions form stable inter-chain disulphide bonds, i.e. between the corresponding cysteine residues of the engineered IgG hinge regions of the first and second polypeptides of the antibody.

[0028] Accordingly, in a first aspect, the present invention provides an antibody-drug conjugate (ADC), wherein the antibody is conjugated to the drug by cysteine-based site-specific conjugation, wherein the antibody comprises a first polypeptide comprising an immunoglobulin fragment crystallisable (Fc) region, at least one variable domain N-terminal of the Fc region and an engineered IgG hinge region between the Fc region and the at least one variable domain, and wherein the engineered IgG hinge region is heterologous to the Fc region and / or is mutated to provide a predetermined number of cysteine residues.

[0029] In a preferred embodiment, the engineered IgG hinge region is heterologous to the Fc region and optionally is mutated to provide a predetermined number of cysteine residues.

[0030] In a further aspect, the present invention provides a method of producing an antibody-drug conjugate (ADC) having a predetermined drug-to-antibody ratio (DAR), comprising the steps of:

[0031] (i) (a) providing at least one polynucleotide sequence encoding an antibody comprising a first polypeptide comprising an immunoglobulin fragment crystallisable (Fc) region and at least one variable domain N-terminal of the Fc region;

[0032] (b) modifying the at least one polynucleotide sequence to encode an engineered IgG hinge region between the Fc region and the at least one variable domain, wherein the engineered IgG hinge region is heterologous to the Fc region and / or is mutated to provide a predetermined number of cysteine residues;

[0033] (c) introducing the at least one polynucleotide sequence into a cell;

[0034] (d) culturing the cell under conditions suitable for the expression of the at least one polynucleotide sequence;

[0035] (e) isolating the antibody expressed by the cell;

[0036] (ii) reducing the antibody with a reducing agent; and

[0037] (iii) conjugating the reduced antibody with the drug.

[0038] In some embodiments, the at least one variable domain is selected from the group consisting of a single-chain variable fragment (scFv); an Fab; an Fab’; an F(ab)’2; an Fv; a single domain antibody (sdAb); a VHH; a single chain variable domain; a designed ankyrin repeat protein (DARPin); and an aptamer.

[0039] In some embodiments, the antibody is selected from the group consisting of a full-length immunoglobulin, a scFv-Fc, a Fab-Fc, an Fv-Fc, a sdAb-Fc, or a VHH-Fc.

[0040] In some embodiments, the antibody is a VHH-Fc. In some embodiments, the antibody further comprises a second polypeptide comprising an Fc region, and step (i)(b) further comprises modifying the at least one polynucleotide sequence to encode an engineered IgG hinge region N-terminal of the Fc region of the second polypeptide, wherein the engineered IgG hinge region is heterologous to the Fc region of the second polypeptide and / or is mutated to provide a predetermined number of cysteine residues, and wherein the engineered IgG hinge region of the first polypeptide and the engineered IgG hinge region of the second polypeptide are complementary engineered IgG hinge regions.

[0041] In some embodiments, the second polypeptide further comprises at least one variable domain N-terminal of the engineered IgG hinge region.

[0042] In a further aspect, the present invention provides an antibody-drug conjugate (ADC), wherein the antibody is conjugated to the drug by cysteine-based site-specific conjugation, wherein the antibody comprises a first polypeptide and a second polypeptide, wherein each of the first polypeptide and the second polypeptide comprises an immunoglobulin fragment crystallisable (Fc) region, at least one variable domain which is a VHH N-terminal of the Fc region and an engineered IgG hinge region between the Fc region and the at least one VHH, wherein the engineered IgG hinge region of the first polypeptide and the engineered IgG hinge region of the second polypeptide are complementary engineered IgG hinge regions, and wherein the engineered IgG hinge region is heterologous to the Fc region and / or is mutated to provide a predetermined number of cysteine residues.

[0043] In a further aspect, the present invention provides a method of producing an antibody-drug conjugate (ADC) having a predetermined drug-to-antibody ratio (DAR), comprising the steps of:

[0044] (i) providing an antibody as defined herein;

[0045] (ii) reducing the antibody with a reducing agent; and

[0046] (iii) conjugating the reduced antibody with the drug.

[0047] In a further aspect, the present invention provides a method of producing an antibody-drug conjugate (ADC) having a predetermined drug-to-antibody ratio (DAR), comprising the steps of:

[0048] (i) (a) providing at least one polynucleotide sequence encoding an antibody wherein the antibody comprises a first polypeptide and a second polypeptide, wherein each of the first polypeptide and the second polypeptide comprises an immunoglobulin fragment crystallisable (Fc) region and at least one variable domain which is a VHH N-terminal of the Fc region;

[0049] (b) modifying the at least one polynucleotide sequence to encode an engineered IgG hinge region between the Fc region and the at least one variable domain of each of the first polypeptide and the second polypeptide, wherein the engineered IgG hinge region of the first polypeptide and the engineered IgG hinge region of the second polypeptide are complementary engineered IgG hinge regions, and wherein the engineered IgG hinge region is heterologous to the Fc region and / or is mutated to provide a predetermined number of cysteine residues;

[0050] (c) introducing the at least one polynucleotide sequence into a cell;

[0051] (d) culturing the cell under conditions suitable for the expression of the at least one polynucleotide sequence;

[0052] (e) isolating the antibody expressed by the cell;

[0053] (ii) reducing the antibody with a reducing agent; and

[0054] (iii) conjugating the reduced antibody with the drug.

[0055] In some embodiments, the engineered IgG hinge region of the first polypeptide or the engineered IgG hinge region of each of the first polypeptide and the second polypeptide comprises at least two cysteine residues, preferably wherein the engineered IgG hinge region comprises three, four, five, six, seven, eight or nine cysteine residues.

[0056] In some embodiments, the engineered IgG hinge region of the first polypeptide comprises at least one additional cysteine residue compared to the native hinge region of the Fc region of the first polypeptide, preferably two, three, four, five, six, seven, eight or nine additional cysteine residues compared to the native hinge region of the Fc region of the first polypeptide.

[0057] In some embodiments, the engineered IgG hinge region of the first polypeptide comprises at least one fewer cysteine residue compared to the native hinge region of the Fc region of the first polypeptide, preferably two, three, four, five, six, seven, eight or nine fewer cysteine residues compared to the native hinge region of the Fc region of the first polypeptide.

[0058] In some embodiments, the engineered IgG hinge region of the second polypeptide comprises at least one additional cysteine residue compared to the native hinge region of the Fc region of the second polypeptide, preferably two, three, four, five, six, seven, eight or nine additional cysteine residues compared to the native hinge region of the Fc region of the second polypeptide. In some embodiments, the engineered IgG hinge region of the second polypeptide comprises at least one fewer cysteine residue compared to the native hinge region of the Fc region of the second polypeptide, preferably two, three, four, five, six, seven, eight or nine fewer cysteine residues compared to the native hinge region of the Fc region of the second polypeptide.

[0059] In some embodiments, the engineered IgG hinge region comprises a sequence having at least 70% identity to a sequence as set forth in any one of SEQ ID NOs: 1 , 2, 3, 4 or 5.

[0060] In some embodiments, the engineered IgG hinge region is an lgG1 hinge region.

[0061] In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 1 , or is a fragment thereof and / or a variant thereof comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 1.

[0062] In some embodiments, the engineered IgG hinge region is an lgG2 hinge region.

[0063] In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 2, or is a fragment thereof and / or a variant thereof comprising or an amino acid sequence having at least 70% identity to SEQ ID NO: 2.

[0064] In some embodiments, the engineered IgG hinge region is an lgG3 hinge region.

[0065] In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4, or is a fragment thereof and / or a variant thereof comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 3 or to SEQ ID NO: 4.

[0066] In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 6 or SEQ ID NO: 7, or is a fragment thereof and / or a variant thereof comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 6 or to SEQ ID NO: 7.

[0067] In some embodiments, the engineered IgG hinge region is an lgG4 hinge region.

[0068] In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 5, or is a fragment thereof and / or a variant thereof comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 5.

[0069] In some embodiments, the engineered IgG hinge region of the first polypeptide and / or second polypeptide comprises at least one amino acid modification to avoid O-glycosylation compared to the native hinge region of the Fc region of the first polypeptide and / or second polypeptide. In some embodiments, the at least one amino acid modification to avoid O-glycosylation is an amino acid substitution.

[0070] In some embodiments, the engineered IgG hinge region is an lgG3 hinge region and the at least one amino acid modification is selected from the group consisting of S241JJX, T241MMX and the combination thereof, wherein the numbering is according to the according to the Kabat numbering scheme.

[0071] In some embodiments, the engineered IgG hinge region is an lgG3 hinge region and the at least one amino acid modification is selected from the group consisting of S241FX, T241X and the combination thereof, wherein the numbering is according to the according to the Kabat numbering scheme.

[0072] In some embodiments, the engineered IgG hinge region is an lgG3 hinge region and the at least one amino acid modification is selected from the group consisting of S241UX, T241XX and the combination thereof, wherein the numbering is according to the according to the Kabat numbering scheme. In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in any one of SEQ ID NOs: 8-118, preferably an amino acid sequence as set forth in SEQ ID NO: 8, 9, 12, 13, 18, 51 , 67, 74, 84, 100, 112, 113 or 118, preferably an amino acid sequence as set forth in SEQ ID NO: 18, 51 , 67, 74, 84, 100, 112, 113 or 118.

[0073] In some embodiments, the engineered IgG hinge region is or is derived from a human IgG hinge region.

[0074] In some embodiments, the antibody is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, a monoclonal antibody, and a polyclonal antibody.

[0075] In some embodiments, the at least one variable domain of the first polypeptide, the second polypeptide or both the first polypeptide and the second polypeptide is specific for a cancer antigen.

[0076] In some embodiments, the antibody is conjugated to the drug by cysteine-based site-specific conjugation through all of the cysteine residues within the engineered IgG hinge region.

[0077] In some embodiments, the ADC has a drug-to-antibody-ratio (DAR) of from about 2 to about 12, preferably a DAR of from about 4 to about 8.

[0078] In some embodiments, the ADC has a drug-to-antibody-ratio (DAR) of about 2, 4, 6, 8, 10, or 12, preferably a DAR of about 8. In some embodiments, the drug is selected from the group consisting of a cytotoxic drug, an antimicrobial agent, or an immunomodulatory agent.

[0079] In some embodiments, the step of reducing the antibody in step (ii) is a partial reduction of the antibody to reduce the inter-chain disulphide bonds of the antibody.

[0080] In some embodiments, the reducing agent is dithiothreitol (DTT) or tris (2-carboxyethyl) phosphine (TCEP).

[0081] In some embodiments, the drug is in the form of a linker-drug conjugate comprising a thiolreactive group.

[0082] In some embodiments, the thiol-reactive group is selected from the group consisting of a maleimide, bromoacetamide, disulphide, a-haloacetamide, a-halocarbonyl, vinylsulfone, heteroaryl sulfone, thiosulfonate, electron deficient aryl halide, ethynylphosphonamidate, vinylphosphonite, palladiumoxidative-addition complex.

[0083] In some embodiments, step (iii) is performed using a thiol-reactive coupling strategy.

[0084] In some embodiments, the thiol-reactive coupling strategy is maleimide-based conjugation.

[0085] In some embodiments, the method of the invention provides homogenous ADCs.

[0086] In some embodiments, all of the cysteine residues within the engineered IgG hinge region form stable inter-chain disulphide bonds prior to step (ii).

[0087] In some embodiments, in step (iii) the antibody is conjugated to the drug by cysteine-based site-specific conjugation through all of the cysteine residues within the engineered IgG hinge region.

[0088] In a further aspect, the present invention provides an ADC obtained or obtainable by the methods of the invention.

[0089] In a further aspect, the present invention provides a pharmaceutical composition comprising the ADC according to the invention and a pharmaceutically acceptable carrier, excipient and / or diluent.

[0090] In a further aspect, the present invention provides an ADC of the invention ora pharmaceutical composition of the invention for use in therapy.

[0091] In a further aspect, the present invention provides an ADC of the invention, or a pharmaceutical composition of the invention for use in the treatment of a disease or condition selected from the group consisting of cancer, autoimmune disease, infection, infectious diseases, cardiovascular diseases and liver metabolic disorders.

[0092] BRIEF DESCRIPTION OF THE FIGURES

[0093] Figure 1 : Schematic of cysteine-mediated conjugation of partially reduced antibodies.

[0094] A. Cysteine-mediated conjugation of a partially reduced lgG1 antibody resulting in an ADC having a DAR of 8. B. Cysteine-mediated conjugation of a partially reduced VHH-Fc fusion resulting in an ADC having a DAR of 4. As VHH-Fcs do not exhibit light chains, two inter-chain disulphides are not present compared to conventional lgG1 antibodies, resulting in a maximal DAR of 4. C. Cysteine-mediated conjugation of a partially reduced, hinge-engineered VHH-Fc fusion resulting in an ADC having a DAR of 8. By engineering a total number of four interchain disulphides within the hinge region, this VHH-Fc design allows for an increased DAR compared to a conventional VHH-Fc fusion.

[0095] Figure 2: Internalization of VHH-Fc fusions. Internalization of VHH-Fcs into target positive tumour cells was investigated using a FACS-based assay.

[0096] Figure 3: Serum stability of VHH-Fc fusion proteins. dO = day 0 (Oh), d1 = day 1 (24h), d2 = day 2 (48h), d3 = day 3 (72h), d7 = day 7 (168h), d14 = day 14 (336h).

[0097] Figure 4: Serum stability of ADCs exhibiting lgG1- (A) or lgG3-(B)-derived hinge regions. Concentrations of the antibody portion or the full ADC are plotted against the incubation time in either human serum at 37°C or in PBS at 4°C.

[0098] Figure 5 - Cytotoxicity on antigen 1 positive and negative cells testing DAR4 lgG1- derived hinge containing ADCs in comparison to DAR 8 lgG3-derived hinge containing ADCs. Cytotoxicity evaluation of ADCs based on the A) A1 B3, B) A1 B4 and C) A1 B5 VHHs. In all cases the DAR4 and DAR8 ADCs were tested with different concentrations on targetpositive and -negative cells.

[0099] Figure 6 - Pharmacokinetic profile of VHH-Fc fusions exhibiting lgG1 or lgG3-derived hinge regions.

[0100] Figure 7 - In vivo stability of DAR 4 and DAR 8 ADCs.

[0101] Figure 8 - Cytotoxicity on antigen 3 positive cells testing lgG3-derived hinge containing ADCs with a DAR of 8. A-G) Reducing of viability of antigen 3 positive cells. H-l) Viability of antigen 1 positive cells when incubated with control antibodies or ADCs.

[0102] Figure 9 - Cytotoxicity on antigen 3 negative cells testing lgG3-derived hinge containing ADCs with a DAR of 8. A-G) Reducing of viability of antigen 3 negative cells. H- I) Viability of antigen 3 positive cells when incubated with control antibodies or ADCs. Figure 10 - Cytotoxic cell killing of antigen 1 positive cells using ADCs of a wide range of DARs. A-l) Reducing of viability of target positive cells in a concentration- and DAR- dependent manner. J-L) Viability of antigen 1 positive cells when incubated with control antibodies or ADCs.

[0103] Figure 11 - IC50 values in nanomolar and bottom values. The IC50 value states the concentration of ADC where the viability reached 50%. The Bottom value is the minimal viability in percent that is reached by the tested ADC.

[0104] Figure 12 - Cytotoxic cell killing of antigen 1 negative cells using ADCs of a wide range of DARs. A-l) Reducing of viability of target negative cells. J-L) Viability of antigen 1 negative cells when incubated with control antibodies or ADCs.

[0105] DETAILED DESCRIPTION OF THE INVENTION

[0106] Conventional ADC conjugation approaches rely on non-specific / stochastic coupling of druglinkers to lysines (about 40 residues per lgG1) or naturally occurring inter-chain disulphide forming cysteines within an antibody (about 8 residues per lgG1). These approaches often result in a heterogeneous profile of ADCs with a varying DAR between 2 to 4, leading to CMC challenges.

[0107] To overcome these disadvantages, next generation site-specific antibody-drug conjugation methods have been developed. To date, twelve ADCs have gained FDA approval for the treatment of various cancer types, of these, eight ADCs use cysteine-based site-specific conjugation strategies to form the product. ADCs generated using the next generation cysteine-based site-specific antibody-drug conjugation methods show high homogeneity, which is advantageous. However, existing cysteine-based site-specific antibody-drug conjugation methods (e.g. the Thiomab approach) require genetically engineering the antibody backbone to comprise free cysteine residues for conjugation, leading to CMC challenges (Kostova V et al., Pharmaceuticals, 2021 ; 14: 442) and might increase the immunogenicity of the product. For example, the cysteine mutations are formed as mixed disulphides with cysteine or glutathione, such that the resulting ADC is heterogeneous after production. Moreover, in the Thiomab approach, the reduction of the engineered antibody for conjugation results in partial reduction of the inter-chain disulphides as well as the cysteines genetically engineered into the antibody backbone. This necessitates the re-oxidization of the inter-chain disulphides after the reduction step and before the free cysteines which were genetically engineered into the antibody can be conjugated, i.e. complicates the CMC process.

[0108] The present inventors have developed a method for producing ADCs using cysteine-based site-specific conjugation without the need for incorporating artificial cysteine residues within the backbone of the antibody (potentially conserved regions of the antibody backbone). Hence, the immunogenicity profile of the product stays unaltered and no time intensive genetic engineering is needed. Moreover, the methods of producing an ADC provided herein are compatible with established cysteine-based site-specific conjugation strategies which have been used for FDA-approved ADCs.

[0109] In particular, the present inventors have engineered the IgG hinge region within the antibody to contain a predetermined number of cysteine residues. As described herein, varying the number of cysteine residues present within the hinge region of the antibodies permits the control of the DAR of the ADC. It is desirable to be able to control the DAR of an ADC, for example, to produce an ADC which is optimised for a specific therapeutic use by balancing efficacy and safety of the product. Engineering the IgG hinge region within the antibody to contain a predetermined number of cysteine residues enables the production of a homogenous ADC product with the desired DAR via cysteine-based site-specific conjugation strategies. Moreover, by utilising an engineered hinge region that is based upon a naturally occurring IgG hinge region, immunogenicity is reduced.

[0110] The present inventors have also demonstrated that modifications to avoid O-glycosylation are tolerated within the engineered IgG hinge regions. Furthermore, the present inventors have surprisingly found that, prior to conjugation, all of the cysteine residues within the engineered IgG hinge regions form stable inter-chain disulphide bonds, i.e. between the corresponding cysteine residues of the engineered IgG hinge regions of the first and second polypeptides of the antibody. This is advantageous, since both O-glycosylation and free cysteines will lead to CMC challenges of the later ADC, for example by increasing antibody heterogeneity.

[0111] The present inventors have surprisingly demonstrated that an ADC comprising an engineered IgG hinge region demonstrates preserved or elevated ADC properties, including stability, cell binding, internalization, antibody-dependent cellular cytotoxicity (ADCC) and complementdependent cytotoxicity (CDC).

[0112] Antibody Drug Conjugate

[0113] The present invention generally relates to antibody-drug conjugates (ADCs).

[0114] ADCs are a class of targeted therapeutics that improves both the selectivity and the activity (e.g. cytotoxic activity) of drugs, such as cancer drugs, by targeting the drugs to specific targets such as cancer cells. In general, ADCs comprise three main components: (i) an antibody (such as a monoclonal antibody) conjugated to (ii) a linker, which in turn is also conjugated to (iii) a cargo or payload (such as a cytotoxic or chemotherapeutic drug).

[0115] In a first aspect, the present invention provides an antibody-drug conjugate (ADC), wherein the antibody is conjugated to the drug by cysteine-based site-specific conjugation, wherein the antibody comprises a first polypeptide comprising an immunoglobulin fragment crystallisable (Fc) region, at least one variable domain N-terminal of the Fc region and an engineered IgG hinge region between the Fc region and the at least one variable domain, and wherein the engineered IgG hinge region is heterologous to the Fc region and / or is mutated to provide a predetermined number of cysteine residues.

[0116] In some embodiments, the antibody further comprises a second polypeptide comprising an Fc region and an engineered IgG hinge region N-terminal of the Fc region, wherein the engineered IgG hinge region is heterologous to the Fc region of the second polypeptide and / or is mutated to provide a predetermined number of cysteine residues, and wherein the engineered IgG hinge region of the first polypeptide and the engineered IgG hinge region of the second polypeptide are complementary engineered IgG hinge regions.

[0117] In one embodiment, the second polypeptide does not comprise a variable domain.

[0118] In some embodiments, the second polypeptide further comprises at least one variable domain N-terminal of the engineered IgG hinge region.

[0119] In some embodiments, the antibody is a VHH-Fc.

[0120] Accordingly, in a further aspect, the present invention provides an antibody-drug conjugate (ADC), wherein the antibody is conjugated to the drug by cysteine-based site-specific conjugation, wherein the antibody comprises a first polypeptide and a second polypeptide, wherein each of the first polypeptide and the second polypeptide comprises an immunoglobulin fragment crystallisable (Fc) region, at least one variable domain which is a VHH N-terminal of the Fc region and an engineered IgG hinge region between the Fc region and the at least one VHH, wherein the engineered IgG hinge region of the first polypeptide and the engineered IgG hinge region of the second polypeptide are complementary engineered IgG hinge regions, and wherein the engineered IgG hinge region is heterologous to the Fc region and / or is mutated to provide a predetermined number of cysteine residues.

[0121] Cysteine-based site-specific conjugation strategies are known in the art. For example, such strategies are reviewed by Kostova et al. and by Walsh et al. (Kostova V et al., Pharmaceuticals, 2021 ; 14: 442; and Walsh et al., Chem. Soc. Rev., 2021 ,50, 1305-1353). Suitably, the cysteine-based site-specific conjugation may be performed using any suitable method known in the art. Suitably, the cysteine-based site-specific conjugation may be performed as described herein (see Examples). Thus, the production of an ADC using cysteine-based site-specific conjugation in accordance with the invention is within the capabilities of a person of ordinary skill in the art.

[0122] In some embodiments, the antibody is conjugated to the drug by cysteine-based site-specific conjugation through cysteine residues within the engineered IgG hinge region.

[0123] In some embodiments, the antibody is conjugated to the drug by cysteine-based site-specific conjugation through all of the cysteine residues within the engineered IgG hinge region.

[0124] In some embodiments, it will generally be understood that each cysteine residue is conjugated to a different drug molecule, i.e. via 1 :1 cysteine residue to drug molecule binding.

[0125] In some embodiments, the antibody of the present invention is not conjugated to a drug or payload via any unpaired cysteine residues. In some embodiments, “unpaired cysteine residues” are generally understood as cysteine residues that are not present in the same or an equivalent position in both polypeptide chains of the antibody of the present invention. In some embodiments, the antibody of the present invention is exclusively conjugated to drugs or payloads via one or more pairs of cysteine residues. In some embodiments, the antibody of the present invention is conjugated to drugs or payloads via one or more cysteine residues comprised in the hinge regions that would otherwise be capable of forming an interchain disulphide bridge between the hinge regions.

[0126] In some embodiments, the ADC has a drug-to-antibody-ratio (DAR) of from about 2 to about 22, such as from about 2 to about 20, from about 2 to about 12, from about 4 to about 10, or from about 6 to about 8.

[0127] In some embodiments, the ADC has a drug-to-antibody-ratio (DAR) of from about 2 to about 12, preferably a DAR of from about 4 to about 8.

[0128] In some embodiments, the ADC has a drug-to-antibody-ratio (DAR) of about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 22. Suitably, the ADC has a DAR of about 4, 6, or 8. In some embodiments, the ADC has a drug-to-antibody-ratio (DAR) of about 2, 4, 6, 8, 10, or 12, preferably a DAR of about 8.

[0129] It will be understood that the term “drug-to-antibody ratio (DAR)” in the context of ADCs refers to the average number of drug molecules connected (i.e. conjugated) to a single antibody. Thus, the “DAR” can be considered as the average number of linked payload (e.g. cytotoxic drugs) molecules per antibody. The number of drug molecules per antibody molecule can be characterized by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays and HPLC.

[0130] In the context of the present invention, it will generally be understood that “DAR” referred to herein relates to the drug / payloads that are linked to the hinge regions, i.e. the heterologous / engineered hinge regions of the present invention. Thus, in the context of the present invention and disclosure, any reference to “DAR” herein does not account for possible contributions to DAR by other non-hinge region sequences / cysteines. For the avoidance of doubt, such contributions to overall DAR are well within the scope of the present invention, but may not be counted by any references to a “DAR” number herein. By way of example, an ADC of the present invention with a DAR of 4 comprises drugs / payloads linked to four cysteine residues in the engineered hinge regions thereof, and may comprise any number of (or zero) further drugs / payloads linked to non-hinge region sequences. Accordingly, in some embodiments, the DAR number in the context of the invention can be calculated by multiplying the number of cysteine residues that are present in one hinge region sequence by two.

[0131] Nevertheless, in certain other embodiments “DAR” is the overall DAR including all linked drug / payload molecules to the ADC.

[0132] In some embodiments, the ADC / antibody / polypeptide of the present invention does not form surface-exposed inter-heavy-chain disulphide bridges outside of the hinge region. “Inter- heavy-chain” refers to the disulphide bridges being between any two immunoglobulin heavy polypeptide chains present in an antibody. “Surface-exposed” refers to disulphide bridges capable of being reduced and contributing to DAR. In some embodiments, the ADC / antibody / polypeptide of the present invention does not comprise any disulphide bridges or cysteine residues capable of forming disulphide bridges outside of a) the hinge region(s), and optionally b) the residues involved in any KiH modifications. In some embodiments, this particularly applies to constructs / ADCs of the present invention that do not comprise a Fab.

[0133] The DAR is a key property used to measure the quality of an ADC because it can significantly affect ADC efficacy and CMC challenges for the ADC. It will also be understood that the DAR can affect the safety and therapeutic effectiveness of the ADC. In particular, the DAR value affects the efficacy of the drug, as low drug loading reduces the potency, while high drug loading can negatively affect pharmacokinetics and toxicity.

[0134] It is therefore advantageous to be able to control the DAR of an ADC product in order to provide an optimised ADC product having the desired properties including good therapeutic efficacy and safety. By “control the DAR” it is meant that the ADC product can be designed (e.g. engineered) to have a desired DAR. Thus, a tailor-made ADC product with a predetermined DAR can be produced. Suitably, the ADC product may also be homogenous.

[0135] As used herein, the terms “homogenous ADCs” and “homogenous ADC product” refer to ADCs having a substantially uniform DAR. Suitably, the ADCs of the invention and produced by the methods of the invention have a uniform DAR, i.e. have the same DAR, wherein the DAR is an integer value. Suitably, the ADCs of the invention and produced by the methods of the invention may be fully conjugated, i.e. all of the cysteine residues which form stable interchain disulphide bonds within the antibody (prior to reduction and conjugation) may be conjugated to the drug. Thus, the ADCs of the invention and produced by the methods of the invention may lack unconjugated or incompletely conjugated antibodies (i.e. lack antibodies having free cysteine residues). Suitably, the DAR value may correspond to the number of cysteine residues within the antibody which form stable inter-chain disulphide bonds within the antibody (prior to reduction and conjugation). Cysteine residues forming stable inter-chain disulphide bonds within the antibody (prior to reduction and conjugation) may be located within the hinge region(s) (e.g. the engineered IgG hinge region(s) of the invention) and within the LC, where present. Hence, the DAR value can be controlled for any given antibody format (e.g. antibody formats including a LC, such as an IgG antibody, and antibody formats which do not comprise a LC, such as a VHH-Fc antibody), by providing an engineered IgG hinge region comprising a predetermined number of cysteine residues.

[0136] As such, varying the number of cysteine residues present within the hinge region of the antibodies permits the control of the DAR of the ADC. Thus, the DAR directly correlates with the number of predetermined cysteine residues within the engineered IgG hinge region of the first polypeptide and, where present, the engineered IgG hinge region of the second polypeptide. Thus, the methods of the invention provide an ADC having a predetermined DAR through the use of the engineered IgG hinge region(s) as defined herein.

[0137] In one embodiment, the DAR value corresponds to the total number of cysteine residues within the antibody which form stable inter-chain disulphide bonds within the antibody prior to reduction and conjugation to the drug. In one embodiment, the DAR value corresponds to the total number of cysteine residues within the engineered IgG hinge region of the first polypeptide and, where present, the engineered IgG hinge region of the second polypeptide.

[0138] By way of example, Figure 1 provides a schematic overview of the production of ADCs via cysteine-based site-specific conjugation based upon a conventional lgG1 antibody (Figure 1A), a conventional VHH-Fc fusion (Figure 1 B) and an exemplary antibody in accordance with the invention comprising engineered IgG hinge regions as described herein (Figure 1C).

[0139] A conventional IgG 1 antibody displays four inter-chain disulphide bonds in total: two between the HCs and one between each HC with its respective LC. It is therefore possible to reduce the inter-chain disulphides to expose eight free cysteines that can be subsequently conjugated to the linker-payloads. Using such an approach, a homogenous product with a DAR of eight can be generated (Figure 1 A).

[0140] Antibody fragments such as a VHH, however, are limited in their number of disulphide bridges. A conventional VHH-Fc fusion (wherein each polypeptide chain comprises a VHH, a truncated lgG1 hinge and an lgG1 Fc), for example, exhibits two inter-chain disulphide bridges between the two HCs. Since no LCs are present in this antibody format, there are no inter-chain disulphide bridges between a HC and its respective LC and a truncated lgG1 hinge region (SEQ ID NO: 12) or a modified lgG1 hinge region (SEQ ID NO: 13) is used that comprises two cysteine residues per chain (as opposed to two cysteine residues per chain for the full-length lgG1 hinge region). It is therefore possible to reduce the inter-chain disulphides to expose four free cysteines that can be subsequently conjugated to the linker-payloads. Upon reduction and subsequent conjugation to a linker-payload conjugate via thiol-reactive coupling strategy, such as a maleimide-based conjugation, a homogenous product with a DAR of four may therefore be generated (Figure 1 B).

[0141] By contrast, by incorporating a total number of four inter-chain disulphides within an engineered IgG hinge region in accordance with the invention, an exemplary engineered VHH- Fc in accordance with the invention exhibits four inter-chain disulphide bridges between the two HCs. Since no LCs are present in this antibody format, there are no inter-chain disulphide bridges between a HC and its respective LC. It is therefore possible to reduce the inter-chain disulphides of this exemplary engineered VHH-Fc in accordance with the invention to expose eight free cysteines that can be subsequently conjugated to the linker-payloads. Upon reduction and subsequent conjugation to a linker-payload conjugate via thiol-reactive coupling strategy, such as a maleimide-based conjugation, a homogenous product with a DAR of eight may therefore be generated (Figure 1 C). Hence, the present invention enables the production of an ADC having a predetermined DAR. In the context of the exemplary ADC provided in Figure 1 C, the predetermined DAR is eight. The present invention is not limited to this exemplary ADC - the present invention enables the production of an ADC having any predetermined DAR, wherein the predetermined DAR is an integer which is 2 or a multiple thereof.

[0142] The antibody component and the drug component of the antibody-drug conjugate of the invention are linked (i.e. conjugated) to each other via the linker as defined herein.

[0143] Such linkers typically have chemically reactive groups at each end. These linkers can form a covalent attachment between two molecules, e.g. the antibody and the drug. Thus, the antibody and the drug may be covalently linked to a linker. Suitably, one region of the linker may bind to the antibody and another region of the linker may bind to the drug.

[0144] In some embodiments, the linker may be a cleavable linker. In some embodiments, the linker may be a maleimide tetrapeptide-based cleavable linker.

[0145] In some embodiments, the linker comprises a thiol-reactive group selected from the group consisting of a maleimide, bromoacetamide, disulphide, a-haloacetamide, a-halocarbonyl, vinylsulfone, heteroaryl sulfone, thiosulfonate, electron deficient aryl halide, ethynylphosphonamidate, vinylphosphonite, palladiumoxidative-addition complex, bissulfone, water-soluble allyl sulfone, thiol-yne bioconjugation with terminal alkyne or cyclooctyne, dibromo- (DBM) and dithio-maleimide (DTM), hybrid thiobromomaleimide (TBM), dibromopyridazinediones, divinylpyrimidine, and DiPODS (two oxadiazolyl methyl sulfone moieties connected by a phenyl group).

[0146] In some embodiments, the linker comprises a thiol-reactive group selected from the group consisting of a maleimide, bromoacetamide, disulphide, a-haloacetamide, a-halocarbonyl, vinylsulfone, heteroaryl sulfone, thiosulfonate, electron deficient aryl halide, ethynylphosphonamidate, vinylphosphonite, and palladiumoxidative-addition complex.

[0147] Linkers for use in a cysteine-based site-specific conjugation strategy in accordance with the invention are known in the art. The linker for use according to the present invention may be any suitable linker known in the art.

[0148] Without wishing to be bound by theory, the antibody-drug conjugate according to the invention may have (but is not limited to) one or more of the following features:

[0149] • High potency of payload;

[0150] High drug-to-antibody ratio; Stable linker-payload;

[0151] • Tumour-selective cleavable linker

[0152] • ADCC activity; and / or

[0153] • ADCC activity and bystander antitumour effect.

[0154] In some embodiments, the antibody or ADC of the present invention is stable in serum. In some embodiments, “stable” means not proteolytically cleaved. In some embodiments, the serum is human serum. In some embodiments, the stability is for at least two weeks at 37 degrees C. In some embodiments, the antibody or ADC of the present invention is stable in human serum for at least two weeks at 37 degrees C.

[0155] In some embodiments, the present invention provides a composition comprising the ADC of the invention with a high homogeneity of DAR, such as at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99% or about 100% homogeneity of DAR. In embodiments, it is understood that the homogeneity of DAR is the % of the ADC having the same DAR number, e.g. the same number of conjugated drug molecules.

[0156] In some embodiments, the ADC of the present invention binds specifically to a target antigen and has maintained affinity for its target antigen. By “maintained affinity”, it is understood that the ADC of the present invention has unchanged or substantially unchanged affinity for the target antigen when compared to the same antibody construct of the ADC which does not comprise conjugated drug molecules.

[0157] In some embodiments, the ADC of the present invention has maintained affinity for CD64. By “maintained affinity”, it is understood that the ADC of the present invention has unchanged or substantially unchanged affinity for CD64 when compared to the same antibody construct of the ADC which does not comprise conjugated drug molecules.

[0158] In some embodiments, the ADC of the present invention has maintained thermal stability. By “maintained thermal stability”, it is understood that the ADC of the present invention has unchanged or substantially unchanged thermal stability when compared to the same antibody construct of the ADC which does not comprise conjugated drug molecules.

[0159] Engineered IgG hinge region

[0160] As discussed above, the present invention permits the control of the DAR of the ADC by varying the number of cysteine residues present within the hinge region of the antibodies. Without wishing to be bound by theory, even though the inter-chain disulphides between the hinge regions of the antibody prior to conjugation stabilize the IgG molecule, they are not essential for the structural integrity of the antibody. Hence, it is possible to conjugate the antibody to the linker-drug via all of the cysteine residues within the engineered IgG hinge region without compromising the structural integrity of the antibody. The IgG hinge region may be any IgG hinge region defined in accordance with the present invention, e.g. an lgG1 , lgG2, lgG3, lgG4 or IgG pseudogene (e.g. IGHGP) hinge region, or any variant, derivative, fragment or truncation thereof in accordance with the present invention. The IgG hinge regions of the present invention may be of any species, orthologue or paralogue. Thus, hinge regions of all species are encompassed, including all animal, human, murine and other orthologues, but are preferably human IgG hinge regions or derived therefrom.

[0161] In one embodiment, the engineered IgG hinge region of the first polypeptide is heterologous to the Fc region of the first polypeptide. The use of the engineered IgG hinge region which is heterologous to the Fc region of the first polypeptide thereby provides the predetermined number of cysteine residues.

[0162] In one embodiment, the engineered IgG hinge region of the first polypeptide is homologous to the Fc region of the first polypeptide and is mutated to provide a predetermined number of cysteine residues. The use of the engineered IgG hinge region which is mutated as described herein thereby provides the predetermined number of cysteine residues.

[0163] In one embodiment, the engineered IgG hinge region of the first polypeptide is heterologous to the Fc region of the first polypeptide and is mutated to provide a predetermined number of cysteine residues. The use of the engineered IgG hinge region which is heterologous to the Fc region of the first polypeptide and mutated as described herein thereby provides the predetermined number of cysteine residues.

[0164] In one embodiment, the engineered IgG hinge region of the second polypeptide is heterologous to the Fc region of the second polypeptide. The use of the engineered IgG hinge region which is heterologous to the Fc region of the second polypeptide thereby provides the predetermined number of cysteine residues.

[0165] In one embodiment, the engineered IgG hinge region of the second polypeptide is homologous to the Fc region of the second polypeptide and is mutated to provide a predetermined number of cysteine residues. The use of the engineered IgG hinge region which is mutated as described herein thereby provides the predetermined number of cysteine residues.

[0166] In one embodiment, the engineered IgG hinge region of the second polypeptide is heterologous to the Fc region of the second polypeptide and is mutated to provide a predetermined number of cysteine residues. The use of the engineered IgG hinge region which is heterologous to the Fc region of the first polypeptide and mutated as described herein thereby provides the predetermined number of cysteine residues.

[0167] As used herein, the term “IgG hinge region” refers to the region of the native IgG chain which is denoted the hinge region. For example, for human IgG isotypes, the IgG hinge region may be denoted according to the Kabat numbering scheme.

[0168] As used herein, the term “engineered IgG hinge region” refers to an IgG hinge region that is heterologous to the Fc region of the first polypeptide or of the second polypeptide, that is mutated to provide a predetermined number of cysteine residues as described herein or that is both heterologous to the Fc region of the first polypeptide or of the second polypeptide and mutated to provide a predetermined number of cysteine residues as described herein.

[0169] As used herein, the term “heterologous to the Fc region” refers to an engineered IgG hinge region that is or is based upon the hinge region of a different immunoglobulin isotype to the Fc region of the polypeptide or to the Fc region variant (as described herein) of the polypeptide. For example, an lgG2 hinge, an lgG3 hinge and an lgG4 hinge are each heterologous to an I gG1 Fc region or a variant thereof; an IgG 1 hinge, an lgG3 hinge and an lgG4 hinge are each heterologous to an lgG2 Fc region or a variant thereof; and so on. The heterologous hinge region may additionally be mutated as described herein.

[0170] In embodiments, it will generally be understood that both hinge regions in the antibody / ADC of the present invention are of the same IgG isotype. In some embodiments, both hinge regions comprise the same number and relative position of cysteine residues. In some embodiments, both hinge regions comprise the same sequence.

[0171] In some embodiments, the polypeptide / antibody / ADC of the present invention is defined as comprising a pairing of hinge region and Fc region isotypes that does not occur in nature. The hinge region and / or Fc region may also be subject to additional modifications, substitutions etc. beyond this non-naturally occurring pairing of hinge region and Fc region isotypes.

[0172] In some embodiments, the Fc region is an lgG1 Fc region, derivative or fragment thereof, and the hinge regions are selected from lgG2, lgG3 or lgG4 hinge regions. In some embodiments, the Fc region is an lgG2 Fc region, derivative or fragment thereof, and the hinge regions are selected from lgG1 , lgG3 or lgG4 hinge regions. In some embodiments, the Fc region is an lgG3 Fc region, derivative or fragment thereof, and the hinge regions are selected from lgG1 , lgG2 or lgG4 hinge regions. In some embodiments, the Fc region is an lgG4 Fc region, derivative or fragment thereof, and the hinge regions are selected from lgG1 , lgG2 or lgG3 hinge regions. In some embodiments, the Fc region is an lgG1 Fc region, and the hinge regions are selected from lgG2, lgG3 or lgG4 hinge regions. In some embodiments, the Fc region is an lgG2 Fc region, and the hinge regions are selected from lgG1 , lgG3 or lgG4 hinge regions. In some embodiments, the Fc region is an lgG3 Fc region, and the hinge regions are selected from lgG1 , lgG2 or lgG4 hinge regions. In some embodiments, the Fc region is an lgG4 Fc region, and the hinge regions are selected from lgG1 , lgG2 or lgG3 hinge regions. In embodiments, it will generally be understood that both hinge regions are of the same IgG isotype.

[0173] In some embodiments, the hinge region is an lgG1 hinge region, derivative or fragment thereof, and the Fc region is not an IgG 1 Fc region, derivative or fragment thereof. In some embodiments, the hinge region is an lgG2 hinge region, derivative or fragment thereof, and the Fc region is not an lgG2 Fc region, derivative or fragment thereof. In some embodiments, the hinge region is an lgG3 hinge region, derivative or fragment thereof, and the Fc region is not an lgG3 Fc region, derivative or fragment thereof. In some embodiments, the hinge region is an lgG4 hinge region, derivative or fragment thereof, and the Fc region is not an lgG4 Fc region, derivative or fragment thereof.

[0174] As used herein, the terms “homologous to the Fc region” and “native hinge region of the Fc region” refers to a hinge region (e.g. an engineered IgG hinge region) that is the hinge region of the same immunoglobulin isotype to the Fc region of the polypeptide or to the Fc region variant (as described herein) of the polypeptide. For example, an IgG 1 hinge is homologous to an IgG 1 Fc region or a variant thereof; an lgG2 hinge is homologous to an lgG2 Fc region or a variant thereof; and so on. The homologous hinge region is mutated as described herein to form an engineered IgG hinge region in accordance with the invention.

[0175] In some embodiments, it will generally be understood herein that engineering the hinge regions of the polypeptide / antibody / ADC of the invention means that the amino acid sequence thereof is a sequence that does not occur in nature. Suitably, in some embodiments, the polypeptide / antibody / ADC of the invention comprises a non-naturally occurring sequence. Suitably, in some embodiments, the polypeptide / antibody / ADC of the invention comprises a sequence that does not occur in nature. Suitably, in some embodiments, the polypeptide / antibody / ADC of the invention comprises a synthetic sequence.

[0176] A native IgG hinge region may be mutated by addition, deletion and / or substitution of at least one residue present in the naturally-occurring hinge region to provide the engineered IgG hinge region having a preselected number of cysteine residues in accordance with the present invention. Suitably, the nucleotide sequence encoding a native IgG hinge region may be mutated by addition, deletion and / or substitution of at least one nucleotide present in the sequence encoding the naturally-occurring hinge region to encode the engineered IgG hinge region having a preselected number of cysteine residues in accordance with the present invention.

[0177] In some embodiments, the native IgG hinge region is mutated by addition to provide a predetermined number of cysteine residues.

[0178] In some embodiments, the native IgG hinge region is mutated by deletion to provide a predetermined number of cysteine residues. Thus, the engineered IgG hinge region may be a truncation of a native IgG hinge region. The native IgG hinge region may be truncated to provide the predetermined number of cysteine residues.

[0179] In some embodiments, the native IgG hinge region is mutated by substitution to provide a predetermined number of cysteine residues.

[0180] In some embodiments, the native IgG hinge region is mutated by addition and deletion to provide a predetermined number of cysteine residues.

[0181] In some embodiments, the native IgG hinge region is mutated by addition and substitution to provide a predetermined number of cysteine residues.

[0182] In some embodiments, the native IgG hinge region is mutated by deletion and substitution to provide a predetermined number of cysteine residues.

[0183] The mutation of a native IgG hinge region to provide the predetermined number of cysteine residues using conventional techniques in molecular biology is within the capabilities of a person of ordinary skill in the art. Generally speaking, suitable routine methods include directed mutagenesis, gene synthesis and recombinant DNA / RNA technology.

[0184] As used herein, the term “complementary engineered IgG hinge regions” means that the engineered IgG hinge regions of the first and second polypeptides have the same length and comprise cysteine residues at the same positions within the hinge region according to the Kabat numbering scheme. That the cysteine residues are located at the same positions within the hinge regions of the first and second polypeptides enables the cysteine residues within the engineered IgG hinge regions to form stable inter-chain disulphide bonds within the antibody prior to conjugation to the payload. Thus, the engineered IgG hinge regions of the first and second polypeptides each comprise the same number of cysteine residues, i.e. the predetermined number of cysteine residues for the engineered IgG hinge region of the first polypeptide and for the engineered IgG hinge region of the second polypeptide is identical. Suitably, the engineered IgG hinge regions of the first and second polypeptides may be identical. In a preferred embodiment, the engineered IgG hinge region of the first and / or second polypeptide comprises a predetermined number of cysteine residues.

[0185] In some embodiments, the engineered IgG hinge region of the first polypeptide comprises at least one cysteine residue. Suitably, the engineered IgG hinge region comprises two, three, four, five, six, seven, eight, nine, ten or eleven cysteine residues.

[0186] The engineered IgG hinge regions of the first polypeptide and the second polypeptide each comprise at least one cysteine residue. In some embodiments, the engineered IgG hinge regions each comprise at least two, three, four, five, six, seven, eight, nine, ten or eleven cysteine residues. In some embodiments, the engineered IgG hinge regions each comprise two, three, four, five, six, seven, eight, nine, ten or eleven cysteine residues. Thus, the predetermined number of cysteine residues may be one, two, three, four, five, six, seven, eight, nine, ten or eleven cysteine residues.

[0187] In one embodiment, the engineered IgG hinge regions each comprise at least two cysteine residues. In one embodiment, the engineered IgG hinge regions each comprise at least three cysteine residues. In one embodiment, the engineered IgG hinge regions each comprise at least four cysteine residues. In one embodiment, the engineered IgG hinge regions each comprise at least five cysteine residues. In one embodiment, the engineered IgG hinge regions each comprise at least six cysteine residues. In one embodiment, the engineered IgG hinge regions each comprise at least seven cysteine residues. In one embodiment, the engineered IgG hinge regions each comprise at least eight cysteine residues. In one embodiment, the engineered IgG hinge regions each comprise at least nine cysteine residues. In one embodiment, the engineered IgG hinge regions each comprise at least ten cysteine residues. In one embodiment, the engineered IgG hinge regions each comprise at least eleven cysteine residues.

[0188] The predetermined number of cysteine residues is determined by the desired DAR. For example, if the desired DAR (also termed the ‘predetermined DAR’) is eight, and the antibody does not comprise any LCs, then the predetermined number of cysteine residues for the engineered IgG hinge region of each of the first polypeptide and the second polypeptide is four. By way of further example, if the desired DAR (also termed the ‘predetermined DAR’) is ten, and the antibody does not comprise any LCs, then the predetermined number of cysteine residues for the engineered IgG hinge region of each of the first polypeptide and the second polypeptide is five.

[0189] In some embodiments, the engineered IgG hinge region of the first polypeptide comprises at least one additional cysteine residue compared to the native hinge region of the Fc region of the first polypeptide. Suitably, the engineered IgG hinge region of the first polypeptide comprises two, three, four, five, six, seven, eight or nine additional cysteine residues compared to the native hinge region of the Fc region of the first polypeptide.

[0190] In some embodiments, the engineered IgG hinge region of the first polypeptide comprises at least one fewer cysteine residue compared to the native hinge region of the Fc region of the first polypeptide. Suitably, the engineered IgG hinge region of the first polypeptide comprises two, three, four, five, six, seven, eight or nine fewer cysteine residues compared to the native hinge region of the Fc region of the first polypeptide.

[0191] In some embodiments, the engineered IgG hinge region of the second polypeptide comprises at least one additional cysteine residue compared to the native hinge region of the Fc region of the second polypeptide. Suitably, the engineered IgG hinge region of the second polypeptide comprises two, three, four, five, six, seven, eight or nine additional cysteine residues compared to the native hinge region of the Fc region of the second polypeptide.

[0192] In some embodiments, the engineered IgG hinge region of the second polypeptide comprises at least one fewer cysteine residue compared to the native hinge region of the Fc region of the second polypeptide. Suitably, the engineered IgG hinge region of the second polypeptide comprises two, three, four, five, six, seven, eight or nine fewer cysteine residues compared to the native hinge region of the Fc region of the second polypeptide.

[0193] In some embodiments, all of the cysteine residues within the engineered IgG hinge regions of the first and second polypeptides form stable inter-chain disulphide bonds prior to drugconjugation. In other words, the antibody does not comprise any free cysteine residues within the engineered IgG hinge regions of the first and second polypeptides. Thus, all of the cysteine residues within the engineered IgG hinge region of the first polypeptide may form stable interchain disulphide bonds with the corresponding cysteine residue (i.e. the cysteine residue at the same position according to the Kabat numbering system) within the engineered IgG hinge region of the second polypeptide. For an illustrative example of complete disulphide bond formation in an antibody format that does not comprise a LC, see the left hand panel of Figure 1 B or of Figure 10. Alternatively, all of the cysteine residues within the engineered IgG hinge region of the first polypeptide may form stable inter-chain disulphide bonds with the corresponding cysteine residue (i.e. cysteine residues at the same position according to the Kabat numbering system) within the engineered IgG hinge region of the second polypeptide and with at least one cysteine residue within the LC, where present. For an illustrative example of complete disulphide bond formation in an antibody format comprising a LC, see the left hand panel of Figure 1A. Free (unpaired) cysteines can negatively impact the chemistry, manufacturing and controls (CMC) development process of the antibody. These CMC challenges include, but are not limited to: antibody heterogeneity, low stability, aggregation, low solubility and reduced potency. Therefore, the ADCs of the invention have the advantage of avoiding these challenges associated with free cysteines.

[0194] Suitably, the engineered IgG hinge regions of the first and second polypeptides may be mutated as described herein such that they do not comprise any free cysteine residues prior to drug-conjugation. In this regard, the native human lgG1 hinge regions contain cysteine residues which form stable inter-chain disulphide bonds with the LC. For use with antibody formats that do not comprise a LC (e.g. a VHH-Fc), the native human lgG1 hinge regions may therefore be mutated as described herein to eliminate the cysteine residues (for example, by deletion or substitution) which do not form stable inter-chain disulphide bonds in the absence of a LC.

[0195] As described herein, the engineered IgG hinge region may be heterologous to the Fc region of the first polypeptide or of the second polypeptide. Thus, the engineered IgG hinge region may be a native lgG1 , lgG2, lgG3 or lgG4 hinge region, with the proviso that the engineered IgG hinge region is heterologous to the Fc region of the first polypeptide or of the second polypeptide. For example, the engineered IgG hinge region may be a native lgG1 hinge region if the Fc region if the first and second polypeptides is an lgG2 Fc region.

[0196] Accordingly, in some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in any one of SEQ ID NOs: 1 , 2, 3, 4, or 5.

[0197] The engineered IgG hinge region may be mutated to provide a predetermined number of cysteine residues by truncation of a native IgG hinge region. Thus, the engineered IgG hinge region may be a fragment of a native lgG1 , lgG2, lgG3 or lgG4 hinge region that provides a predetermined number of cysteine residues. The engineered IgG hinge region may be heterologous to the Fc region of the first polypeptide or the second polypeptide and be mutated to provide a predetermined number of cysteine residues by truncation of a native IgG hinge region that is heterologous to the Fc region of the first polypeptide or the second polypeptide.

[0198] Accordingly, in some embodiments, the engineered IgG hinge region is a fragment of an amino acid sequence as set forth in any one of SEQ ID NOs: 1 , 2, 3, 4, or 5. The fragment may have a length of between about 4 and about 60 (suitably, between about 5 and about 55, between about 10 and about 50, between about 15 and about 45, between about 20 and about 40, or between about 25 and about 35) amino acids. Suitably, the fragment may have a length of about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19 or about 20 amino acids. Suitably fragments are described herein (see, for example, Table 1). The engineered IgG hinge region may be mutated to provide a predetermined number of cysteine residues by substitution of one or more amino acids of a native IgG hinge region to provide a predetermined number of cysteine residues. For example, the native IgG hinge region may be mutated by substituting one or more cysteine residues with an amino acid residue other than cysteine in order to reduce the number of cysteine residues within the resulting engineered IgG hinge region. Alternatively or additionally, the native IgG hinge region may be mutated by substituting one or more non-cysteine amino acid residues with a cysteine residue in order to increase the number of cysteine residues within the resulting engineered IgG hinge region.

[0199] The engineered IgG hinge region may be heterologous to the Fc region of the first polypeptide or the second polypeptide and be mutated to provide a predetermined number of cysteine residues by substitution of one or more amino acids of a native IgG hinge region that is heterologous to the Fc region of the first polypeptide or the second polypeptide.

[0200] Accordingly, in some embodiments, the engineered IgG hinge region has an amino acid sequence comprising or consisting of an amino acid sequence having at least 70% (suitably, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity to any one of SEQ ID NOs: 1 , 2, 3, 4, or 5.

[0201] The engineered IgG hinge region may be mutated to provide a predetermined number of cysteine residues by truncation of and by substitution of one or more amino acids of a native IgG hinge region.

[0202] The engineered IgG hinge region may be heterologous to the Fc region of the first polypeptide or the second polypeptide and be mutated to provide a predetermined number of cysteine residues by truncation of and by substitution of one or more amino acids of a native IgG hinge region that is heterologous to the Fc region of the first polypeptide or the second polypeptide.

[0203] Accordingly, in some embodiments, the engineered IgG hinge region is a fragment and a variant of any one of SEQ I D NOs: 1 , 2, 3, 4 or 5 comprising or consisting of an amino acid sequence having at least 70% (suitably, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity to any one of SEQ ID NOs: 1 , 2, 3, 4, or 5.

[0204] In some embodiments, the engineered IgG hinge region is an lgG1 hinge region. Suitably, the engineered IgG hinge region is an lgG1 hinge region that has been mutated as described herein. An illustrative human lgG1 sequence is as follows:

[0205] EPKSCDKTHTCPPCP (SEQ ID NO: 1)

[0206] In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 1 , or is a fragment thereof and / or a variant thereof comprising or consisting of an amino acid sequence having at least 70% (suitably, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity to SEQ ID NO: 1 . Suitably, the variant comprises or consists of an amino acid sequence having at least 90% (suitably, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity to SEQ ID NO: 1 .

[0207] In some embodiments, the engineered IgG hinge region is an lgG2 hinge region. Suitably, the engineered IgG hinge region is an lgG2 hinge region that has been mutated as described herein.

[0208] An illustrative human lgG2 sequence is as follows:

[0209] ERKCCVECPPCP (SEQ ID NO: 2)

[0210] In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 2, or is a fragment thereof and / or a variant thereof comprising or consisting of an amino acid sequence having at least 70% (suitably, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity to SEQ ID NO: 2. Suitably, the variant comprises or consists of an amino acid sequence having at least 90% (suitably, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity to SEQ ID NO: 2.

[0211] In some embodiments, the engineered IgG hinge region is an lgG3 hinge region. Suitably, the engineered IgG hinge region is an lgG3 hinge region that has been mutated as described herein.

[0212] An illustrative human lgG3 hinge region sequence is as follows:

[0213] ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTP PPCPRCP (SEQ ID NO: 3)

[0214] A further illustrative human lgG3 hinge region sequence is as follows:

[0215] ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCP (SEQ ID NO: 4) In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4, or is a fragment thereof and / or a variant thereof comprising or consisting of an amino acid sequence having at least 70% (suitably, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity to SEQ ID NO: 3 or to SEQ ID NO: 4. Suitably, the variant comprises or consists of an amino acid sequence having at least 90% (suitably, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity to SEQ ID NO: 3 or SEQ ID NO: 4.

[0216] The human IGHG3*01 sequence (SEQ ID NO: 3) is arranged in four exons. The translated amino acid sequences of exons 2, 3 and 4 is identical. Any of the exon sequences may be used in the practice of the present invention. Thus, a fragment of SEQ ID NO: 3 may correspond to the sequence of exon 1 or of exon 2, 3 or 4.

[0217] An illustrative human lgG3 hinge region exon 1 sequence (translated to amino acids) is as follows:

[0218] ELKTPLGDTTHTCPRCP (SEQ ID NO: 6)

[0219] An illustrative human lgG3 hinge region exon 2, exon 3 or exon 4 sequence (translated to amino acids) is as follows:

[0220] EPKSCDTPPPCPRCP (SEQ ID NO: 7)

[0221] In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 6 or SEQ ID NO: 7, or is a fragment thereof and / or a variant thereof comprising or consisting of an amino acid sequence having at least 70% (suitably, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity to SEQ ID NO: 6 or to SEQ ID NO: 7. Suitably, the variant comprises or consists of an amino acid sequence having at least 90% (suitably, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity to SEQ ID NO: 6 or SEQ ID NO: 7.

[0222] In some embodiments, the engineered IgG hinge region is an lgG4 hinge region. Suitably, the engineered IgG hinge region is an lgG4 hinge region that has been mutated as described herein.

[0223] An illustrative human lgG4 hinge sequence is as follows: ESKYGPPCPSCP (SEQ ID NO: 5)

[0224] In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 5, or is a fragment thereof and / or a variant thereof comprising or consisting of an amino acid sequence having at least 70% (suitably, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity to SEQ ID NO: 5. Suitably, the variant comprises or consists of an amino acid sequence having at least 90% (suitably, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity to SEQ ID NO: 5.

[0225] It is known that the threonine and serine residues within the human lgG3 hinge are prone to O-glycosylation. The serine residues of the human lgG3 hinge as well as threonine and serine residues of other IgG isotype hinge regions may also be subject to O-glycosylation. Without wishing to be bound by theory, the O-glycosylation may lead to CMC challenges for the ADC, for example by increasing heterogeneity of the product. Therefore, it is desirable to avoid O- glycosylation of the antibody hinge region for ADC applications.

[0226] The modification of a native IgG hinge region to avoid O-glycosylation using conventional techniques in molecular biology is within the capabilities of a person of ordinary skill in the art following the teachings disclosed herein. Generally speaking, suitable routine methods include directed mutagenesis, gene synthesis and recombinant DNA / RNA technology.

[0227] In some embodiments, the engineered IgG hinge region of the first polypeptide and / or second polypeptide comprises at least one amino acid modification to avoid O-glycosylation compared to the native IgG hinge region of the Fc region of the first polypeptide and / or second polypeptide. Suitably, the at least one amino acid modification may be a deletion or a substitution of a serine and / or threonine residue. Suitably, the at least one amino acid modification may be a deletion or a substitution of at least one serine residue. Suitably, the at least one amino acid modification may be a deletion or a substitution of all of the serine residues within the native IgG hinge region. Suitably, the at least one amino acid modification may be a deletion or a substitution of at least one threonine residue. Suitably, the at least one amino acid modification may be a deletion or a substitution of all of the threonine residues within the native IgG hinge region. Suitably, the at least one amino acid modification may be a deletion or a substitution of at least one serine and at least one threonine residue. Suitably, the at least one amino acid modification may be a deletion or a substitution of all of the serine and threonine residues within the native IgG hinge region. In some embodiments, the at least one amino acid modification to avoid O-glycosylation is an amino acid substitution. In some embodiments, the amino acid substitution is to an amino acid residue that is not serine or threonine. In some embodiments, the amino acid substitution is to alanine. In some embodiments, the amino acid substitution is a serine to alanine substitution and / or a threonine to alanine substitution as applicable.

[0228] In some embodiments, the engineered IgG hinge region is an lgG3 hinge region and the at least one amino acid modification is selected from the group consisting of S241FX, T241IX and the combination thereof, wherein the numbering is according to the according to the Kabat numbering scheme, and wherein X is any amino acid except C, S or T.

[0229] In some embodiments, the engineered IgG hinge region is an lgG3 hinge region and the at least one amino acid modification is selected from the group consisting of S241UX, T241XX and the combination thereof, wherein the numbering is according to the according to the Kabat numbering scheme, and wherein X is any amino acid except C, S or T.

[0230] In some embodiments, the engineered IgG hinge region is an lgG3 hinge region and the at least one amino acid modification is selected from the group consisting of S241JJX, T241MMX and the combination thereof, wherein the numbering is according to the according to the Kabat numbering scheme, and wherein X is any amino acid except C, S or T.

[0231] In one embodiment, the engineered IgG hinge region is an lgG3 hinge region and the at least one amino acid modification is S241JJX, wherein the numbering is according to the according to the Kabat numbering scheme, and wherein X is any amino acid except C, S or T.

[0232] In some embodiments, the engineered IgG hinge region is an lgG3 hinge region and the at least one amino acid modification is T241MMX, wherein the numbering is according to the according to the Kabat numbering scheme, and wherein X is any amino acid except C or T.

[0233] In some embodiments, the engineered IgG hinge region is an lgG3 hinge region and the at least one amino acid modification is S241JJX and T241MMX, wherein the numbering is according to the according to the Kabat numbering scheme, and wherein X is any amino acid except C, S or T.

[0234] In some embodiments, the engineered IgG hinge region is an lgG3 hinge region and the at least one amino acid modification is selected from the group consisting of S241FX, T241X, S241UX, T241xX, S241JJX, T241MMX and the combination thereof, wherein the numbering is according to the according to the Kabat numbering scheme, and wherein X is any amino acid except C, S or T. Illustrative engineered IgG hinge region sequences which may be employed in the practice of the invention are detailed below in Table 1.

[0235] Table 1 - Illustrative engineered IgG hinge region sequences. The predetermined DAR of an ADC wherein the first and the second polypeptide each comprise the hinge sequence is also provided (referred to as “DAR” in the table).

[0236]

[0237]

[0238] In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in any one of SEQ ID NOs: 8-118, or is a fragment thereof and / or or a variant thereof comprising or consisting of an amino acid sequence having at least 70% (suitably, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity to any one of SEQ ID NOs: 8-118. Suitably, the variant comprises or consists of an amino acid sequence having at least 90% (suitably, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity to any one of SEQ ID NOs: 8-118. Suitable fragments of SEQ ID Nos: 8-118 for use according to the invention are described in Table 1.

[0239] In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in any one of SEQ ID NOs: 8-118, preferably an amino acid sequence as set forth in any one of SEQ ID NOs: 8, 9, 12, 13, 18, 51 , 67, 74, 84, 100, 113 or 118, more preferably an amino acid sequence as set forth in any one of SEQ I D NOs: 8, 9, 18, 51 , 67, 74, 84, 100, 113 or 118.

[0240] In some embodiments, the engineered IgG hinge region is a fragment of an amino acid sequence as set forth in any one of SEQ ID NOs: 8-118, preferably an amino acid sequence as set forth in any one of SEQ ID NOs: 8, 9, 12, 13, 18, 51 , 67, 74, 84, 100, 113 or 118, more preferably an amino acid sequence as set forth in any one of SEQ ID NOs: 8, 9, 18, 51 , 67, 74, 84, 100, 113 or 118.

[0241] In some embodiments, the engineered IgG hinge region comprises or consists of an amino acid sequence having at least 70% (suitably, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity to any one of SEQ ID NOs: 8- 118, preferably an amino acid sequence as set forth in any one of SEQ ID NOs: 8, 9, 12, 13, 18, 51 , 67, 74, 84, 100, 113 or 118, more preferably an amino acid sequence as set forth in any one of SEQ ID NOs: 8, 9, 18, 51 , 67, 74, 84, 100, 113 or 118.

[0242] In some embodiments, the engineered IgG hinge region is a fragment and a variant of an amino acid sequence as set forth in any one of SEQ ID NOs: 8-118, preferably an amino acid sequence as set forth in any one of SEQ ID NOs: 8, 9, 12, 13, 18, 51 , 67, 74, 84, 100, 113 or 118, more preferably an amino acid sequence as set forth in any one of SEQ ID NOs: 8, 9, 18, 51 , 67, 74, 84, 100, 113 or 118, wherein the engineered IgG hinge region comprises or consists of a sequence having at least at least 70% (suitably, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity thereto.

[0243] In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 8 or is a fragment thereof and / or or a variant thereof comprising or consisting of an amino acid sequence having at least 70% (suitably, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity thereto.

[0244] In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 9 or is a fragment thereof and / or or a variant thereof comprising or consisting of an amino acid sequence having at least 70% (suitably, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity thereto.

[0245] In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 12 or is a fragment thereof and / or or a variant thereof comprising or consisting of an amino acid sequence having at least 70% (suitably, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity thereto.

[0246] In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 13 or is a fragment thereof and / or or a variant thereof comprising or consisting of an amino acid sequence having at least 70% (suitably, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity thereto.

[0247] In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 18 or is a fragment thereof and / or or a variant thereof comprising or consisting of an amino acid sequence having at least 70% (suitably, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity thereto.

[0248] In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 51 or is a fragment thereof and / or or a variant thereof comprising or consisting of an amino acid sequence having at least 70% (suitably, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity thereto.

[0249] In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 67 or is a fragment thereof and / or or a variant thereof comprising or consisting of an amino acid sequence having at least 70% (suitably, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity thereto.

[0250] In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 74 or is a fragment thereof and / or or a variant thereof comprising or consisting of an amino acid sequence having at least 70% (suitably, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity thereto.

[0251] In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 84 or is a fragment thereof and / or or a variant thereof comprising or consisting of an amino acid sequence having at least 70% (suitably, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity thereto.

[0252] In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 100 or is a fragment thereof and / or or a variant thereof comprising or consisting of an amino acid sequence having at least 70% (suitably, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity thereto.

[0253] In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 113 or is a fragment thereof and / or or a variant thereof comprising or consisting of an amino acid sequence having at least 70% (suitably, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity thereto.

[0254] In some embodiments, the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 118 or is a fragment thereof and / or or a variant thereof comprising or consisting of an amino acid sequence having at least 70% (suitably, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) identity thereto. In some embodiments, the engineered IgG hinge region is or is derived from a human, murine, camelid, rabbit, sheep, goat or chicken IgG hinge region.

[0255] In some embodiments, the engineered IgG hinge region is or is derived from a human IgG hinge region. This provides the advantage that the engineered IgG hinge region is less immunogenic as compared to a non-human IgG hinge region sequence.

[0256] As used herein, the terms “derived from” or “based upon” a native (e.g. a human) IgG hinge region refer to a hinge region which has been mutated as described herein to provide a predetermined number of cysteine residues.

[0257] Antibody

[0258] As used herein, the term “antibody” may refer to a protein or polypeptide having an antigenbinding domain which comprises at least one complementarity determining region (CDR).

[0259] The term “complementarity determining region” or "CDR" generally refers to one of the e.g. 6 hypervariable regions within the variable domains of an antibody, e.g. 3 CDRs per variable domain / VHH, 6 CDRs in a standard VHH-Fc fusion construct and 12 CDRs in a standard antibody construct.

[0260] “Complementarity determining region” or “CDR” with regard to antigen-binding domain or antibody refers to a hypervariable region or a highly variable loop in the variable region of the heavy chain of the light chain of an antibody, which contribute primarily to antigen binding. CDRs can interact with the antigen conformation and largely determine binding to the antigen (although some framework regions are known to be involved in binding). The heavy chain variable region and the light chain variable region each contain 3 CDRs (heavy chain CDRs 1 , 2 and 3 and light chain CDRs 1 , 2 and 3, numbered from the amino to the carboxy terminus).

[0261] Techniques for preparing and using various antibody-based constructs and fragments are well known in the art.

[0262] As used herein, “antigen binding site” or “antigen-binding domain” means a protein or polypeptide which comprises at least one complementarity determining region (CDR). The antigen binding site may comprise 3 CDRs, i.e. be equivalent to that of a single domain antibody (sdAb) domain such as a VHH domain.

[0263] As used herein, the term “constant immunoglobulin domain” may refer to a constant domain of an immunoglobulin, e.g. a CH3 domain.

[0264] In conventional, full-length antibodies (e.g. IgG antibodies) that comprise four polypeptides - two light chains and two heavy chains - the Fc region forms a homodimer of the CH2-CH3 domains of each heavy chain polypeptide. As used herein, the term “fragment crystallisable (Fc) region” may refer to the Fc region of an immunoglobulin, which comprises the CH2-CH3 domains, or any fragments, truncations, derivatives or variants thereof. Truncations may include as little as one immunoglobulin constant domain or a fragment thereof. In some embodiments, the Fc region comprises or consists of a CH2 domain. In some embodiments, the Fc region comprises or consists of a CH3 domain. In some embodiments, the Fc region comprises or consists of a CH2 domain and CH3 domain. Preferably, the Fc regions of the invention are full-length or essentially full- length Fc regions. The Fc regions of the present invention include those in which known / standard / routine modifications have been made. The Fc regions of the present invention are not particularly limited by species, although human is preferred, Thus, Fc regions of all species are encompassed, including all animal, human, murine and other orthologues. Fc regions or Fc region-like sequences encoded by paralogues are also encompassed. Suitably, in embodiments of the invention the Fc region may be an lgG1 , lgG2, lgG3, lgG-4, IgA, IgE, IgM or IgD Fc region. Suitably, in embodiments of the invention the Fc region may be an lgG1 , lgG2, lgG3 or lgG4 Fc region. In a preferred embodiment, the Fc region is an lgG1 Fc region. In the context of the present invention, the first polypeptide and the second polypeptide each comprise an Fc region of the same immunoglobulin isotype. In some embodiments, the Fc region is an inactivated Fc region, e.g. a Fc region inactivated by mutation. In some embodiments, “inactivated” means the Fc region lacks some or all of one or more or all of the effector functions of a native Fc region. sdAbs are IgG molecules are found naturally in e.g. camelids (VHHs of IgG origin, such as IGG2 and lgG3 VHHs) and sharks (sbAb of the IgNAR type). Camelid sdAbs are devoid of the light chain and lack the first constant domain of the heavy chain (CH1) of conventional IgGs. Consequently, the antigen-binding fragment of sdAbs solely comprises a single variable domain, often referred to as a Variable Heavy domain of Heavy chain (VHH domain) (also referred to herein as a “single domain variable heavy immunoglobulin” domain). Thus, a VHH comprises 3 CDRs.

[0265] As used herein, the term “variable immunoglobulin domain” or “variable domain” may refer to a variable domain of an immunoglobulin, e.g. a VHH domain.

[0266] The immunoglobulin domains used in the invention are not limited to a specific sequence, and may comprise any suitable known immunoglobulin domains.

[0267] In some embodiments, the at least one variable domain of the first polypeptide and / or the second polypeptide is selected from the group consisting of a single-chain variable fragment (scFv); an Fab; an Fab’; an F(ab)’2; an Fv; a single domain antibody (sdAb); a VHH; a single chain variable domain; a designed ankyrin repeat protein (DARPin); and an aptamer. In some embodiments, wherein an Fab is present in the first and / or second polypeptide, an additional upper hinge region is present in said polypeptide, e.g. N-terminal of the engineered hinge region thereon. It will generally be understood that the additional upper hinge region allows disulphide linkage with the light chain of the Fab. For the avoidance of doubt, this disulphide linkage / cysteines involved therein do not count towards the DAR or cysteine residues of the engineered hinge region for the purposes of the present invention.

[0268] In some embodiments, the at least one variable domain of the first polypeptide and / or the second polypeptide is selected from the group consisting of a single-chain variable fragment (scFv); an Fab; an Fv; a single domain antibody (sdAb); a VHH; a single chain variable domain; and a designed ankyrin repeat protein (DARPin).

[0269] In one embodiment, the at least one variable domain of the first polypeptide is a variable domain of the same type as the at least one variable domain of the second polypeptide. For example, the variable domain N-terminal of the Fc region of the first polypeptide may be a VHH and the variable domain N-terminal of the Fc region of the second polypeptide may be a VHH.

[0270] In one embodiment, the at least one variable domain of the first polypeptide is of a different type to the at least one variable domain of the second polypeptide. For example, the variable domain N-terminal of the Fc region of the first polypeptide may be an scFv and the variable domain N-terminal of the Fc region of the second polypeptide may be a VHH. Suitably, the antibody may be produced using CrossMAb technology (Schaefer et al., PNAS, 2011 , 108: 11187-92; Takahashi et al., Cell, 1982, 29: 671-679; Grunert et al., ACS Omega 2022, 7, 4, 3671-3679; and Klein et al., mAbs, 2016, 8: 1010-1020).

[0271] In some embodiments, the antibody is selected from the group consisting of a full-length immunoglobulin, a scFv-Fc, a Fab-Fc, a Fab’-Fc, a F(ab)’2-Fc, an Fv-Fc, a sdAb-Fc, or a VHH- Fc.

[0272] In some embodiments, the antibody is selected from the group consisting of a full-length immunoglobulin, a scFv-Fc, a Fab-Fc, an Fv-Fc, a sdAb-Fc, or a VHH-Fc.

[0273] In some preferred embodiments, the antibody is a VHH-Fc. In some preferred embodiments, the antibody is a Fab-Fc. In some preferred embodiments, the antibody is a bispecific antibody. In some preferred embodiments, the bispecific antibody comprises a Fab and VHH. In some preferred embodiments, the bispecific antibody comprises a first VHH-Fc polypeptide chain and a second Fab-Fc polypeptide chain. In some preferred embodiments, the bispecific antibody comprises a first VHH-Fc polypeptide chain and a second VHH-Fc polypeptide chain. In some preferred embodiments, the bispecific antibody comprises a first Fab-Fc polypeptide chain and a second Fab-Fc polypeptide chain.

[0274] In some embodiments, the antibody is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, a monoclonal antibody, and a polyclonal antibody.

[0275] The term "chimeric antibody" generally refers to an antibody obtained by fusing a variable region of a non-human antibody and a constant region of a human antibody, which can reduce an immune response induced by the non-human antibody. The non-human antibody may be, for example, a murine, camelid, rabbit, sheep, goat or chicken antibody. By way of example, for establishment of a chimeric antibody, a hybridoma secreting a specific monoclonal antibody can be established, and a variable region gene is cloned from the mouse hybridoma cells; then a constant region gene of human antibody can be cloned as required, and the mouse variable region gene and the human constant region gene are connected to form a chimeric gene; then the chimeric gene is inserted into an expression vector, wherein chimeric antibody molecules can be expressed in a eukaryotic system or a prokaryotic system.

[0276] The term "humanized antibody", also referred to as CDR-grafted antibody, generally refers to an antibody produced by grafting mouse CDR sequences into a human antibody variable region framework, i.e. , an antibody produced in a different type of human germline antibody framework sequence. Therefore, the humanised mouse antibodies are more able to elicit an immune response in a human. Such framework sequences can be obtained from public DNA databases or disclosed references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be obtained from the "VBase" human germline sequence database. Further, if needed, antibody hinge and constant domain sequences can be derived from the IMGT database.

[0277] The term "fully humanized antibody", "fully human antibody" or "completely human antibody", which may also be known as "fully humanized monoclonal antibody", may have both humanized variable region and constant region so as to eliminate immunogenicity and toxic side effects. The development of monoclonal antibodies has four stages, namely murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully humanized monoclonal antibodies. The antibodies or ligands described herein can be fully humanized monoclonal antibodies. Relevant technologies for the preparation of fully human antibodies may be: human hybridoma technology, EBV-transformed B-lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology, single B-cell antibody preparation technology, and the like. A “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the antibodies composing the population are identical except for possible naturally occurring mutations that may be present in minor amounts. A monoclonal antibody is highly specific and targets a single antigen epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies targeting (or specific for) different epitopes. The modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies, and is not to be construed as producing the antibody by any particular method.

[0278] In some embodiments, the sequence of the antibody may be defined using Kabat numbering (Kabat E.A. et al., (1991)).

[0279] Antibodies may be obtained by techniques comprising immunizing an animal with a target antigen and isolating the antibody from serum. Monoclonal antibodies may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al. , J. Mol. Biol. 222:581-597 (1991), for example.

[0280] In an embodiment, according to the number of antigen-binding domains, e.g. VHH domains, that are present, the antibody for use according to the present invention may be monovalent, bivalent, trivalent, tetravalent or pentavalent. Similarly, in an embodiment, according to the number of different antigen-binding domains, e.g. VHH domains, that are present, the antibody for use according to the invention may be monospecific or multispecific, e.g., bispecific, trispecific, tetraspecific etc.

[0281] Any suitable multispecific or multivalent antibody format which is known in the art may be used in the practice of the present invention. Suitably, the bispecific antibody may be an IgG-scFv, IgG-sdAb, IgG-VHH, scFv-Fc-scFv, KiH-IgG, KA-body, KiH-Fc-Fab / scFv.

[0282] The variable domains (e.g. VHH domains) used in the invention are not particularly limiting, and may comprise any antigen binding site that is specific for a selected target protein. Methods for determining binding specificity of an antibody to a particular antigen are known in the art include, but are not limited to, by biolayer interferometry (BLI), surface plasmon resonance (SPR) analysis (e.g. using a BIAcore instrument), ELISA, western blot, in situ hybridisation, immunohistochemistry, flow cytometry, Forster resonance energy transfer (FRET), phage display libraries, yeast two-hybrid screens, co-immunoprecipitation, bimolecular fluorescence complementation and tandem affinity purification. Binding affinity can also be determined using methods such as BLI, SPR analysis (e.g. using a BIAcore instrument), flow cytometry, fluorescence quenching, isothermal titration calorimetry.

[0283] Methods for providing variable domains, such as sdAbs (which comprise VHH domains), against a specific target are known in the art (see Caussinus et al.’, Nat Struct Mol Biol; 2011 ; 19(1); 117-121 & Fulcher et al. ', Open Biol; 2016; 6(10); pii 160255). Further, methods to isolate antigen-specific VHHs from immune or semisynthetic libraries using phage, yeast, or ribosome display are established in the art (see Muyldermans J Biotechnol. 2001 Jun; 74(4):277-302. & Dufner et al. Trends Biotechnol. 2006 Nov; 24(11):523-9).

[0284] By way of example, a VHH can be obtained by immunisation of e.g. dromedaries, camels, llamas or alpacas with the desired antigen and subsequent isolation of the mRNA coding for VHHs. Single domain shark variable domain of new antigen receptor (VNAR) antibodies are also known and suitable for use according to the present invention as an alternative sdAb to a VHH domain. Hence, by way of further example, a VNAR can be obtained by immunisation of sharks with the desired antigen and subsequent isolation of the mRNA coding for VNARs. Reverse transcription and PCR can then be used to generate a library of VHHs or VNARs. Standard screening techniques such as phage display and ribosome display may be used to identify the suitable clones binding the antigen of interest.

[0285] Once the most potent clones have been identified, their DNA sequence may be optimized, for example to improve their stability towards enzymes. Humanisation may also be performed.

[0286] VHHs and VNARs may be expressed in a cell using conventional vectors, such as those described herein.

[0287] The ability of the antibody to specifically bind its target may be determined using methods which are known in the art. For example, determination of binding may be performed e.g. by biolayer interferometry (BLI), surface plasmon resonance (SPR) analysis (using a BIAcore instrument), western blot, flow cytometry, in situ hybridisation and / or microscopy. Suitably, determination of binding affinity may be performed by e.g. by biolayer interferometry (BLI), surface plasmon resonance (SPR) analysis (using a BIAcore instrument) and / or flow cytometry. Suitably, determination of binding affinity may be performed as described herein (see Example 3).

[0288] In one embodiment, variable domains of the first and the second polypeptide have the same binding specificity.

[0289] In one embodiment, the variable domains of the first and the second polypeptide have different binding specificities.

[0290] In an embodiment, the variable domain(s) (e.g. VHH domain(s)) of the first polypeptide are specific for a first target and the variable domain(s) (e.g. VHH domain(s)) of the second polypeptide are specific for a second target. In a preferred embodiment, the first and second targets are different. In an embodiment, accordingly, the antibody comprising the first and second polypeptides is a heterodimer. In an alternative embodiment, the variable domain(s) (e.g. VHH domain(s)) of the first polypeptide are specific for the same target as the variable domain(s) (e.g. VHH domain(s)) of the second polypeptide. In an embodiment, accordingly, the antibody comprising the first and second polypeptides is a homodimer.

[0291] In an embodiment, the first polypeptide further comprises at least one binding moiety or a cognate of a binding moiety C-terminal of the Fc region. In an embodiment, the at least one binding moiety or cognate is immediately C-terminal of the Fc region. Suitably, the first polypeptide further comprises a binding moiety C-terminal of the Fc region. Suitably, the first polypeptide further comprises a cognate of a binding moiety C-terminal of the Fc region.

[0292] In an embodiment, the second polypeptide further comprises at least one binding moiety or a cognate of a binding moiety C-terminal of the Fc region. In an embodiment, the at least one binding moiety or cognate is immediately C-terminal of the Fc region. Suitably, the second polypeptide further comprises a binding moiety C-terminal of the Fc region. Suitably, the second polypeptide further comprises a cognate of a binding moiety C-terminal of the Fc region.

[0293] In an embodiment, the first polypeptide further comprises a binding moiety or a cognate of a binding moiety C-terminal of the Fc region of the first polypeptide and the second polypeptide further comprises a binding moiety or a cognate of a binding moiety C-terminal of the Fc region of the second polypeptide. In an embodiment, each of the binding moieties or cognates is immediately C-terminal of the Fc region of the respective polypeptide sequence. Suitably, the each of the first polypeptide and the second polypeptide further comprise a binding moiety C- terminal of the Fc region of the respective polypeptide. Suitably, each of the first polypeptide and the second polypeptide further comprise a cognate of a binding moiety C-terminal of the Fc region of the respective polypeptide. Suitably, the first polypeptide further comprises a binding moiety C-terminal of the Fc region of the first polypeptide and the second polypeptide further comprises a cognate of a binding moiety C-terminal of the Fc region of the second polypeptide. Suitably, the first polypeptide further comprises a cognate of a binding moiety C- terminal of the Fc region of the first polypeptide and the second polypeptide further comprises a binding moiety C-terminal of the Fc region of the second polypeptide.

[0294] By “N-terminal of the Fc region” it is meant that the variable domain or engineered IgG hinge region is located N-terminal of the Fc region by any means known in the art. In an embodiment, the engineered IgG hinge region is fused directly to the N-terminus of the Fc region, i.e. the engineered IgG hinge region may be immediately adjacent to the Fc region. However, in an embodiment, the engineered IgG hinge region is connected to the N-terminus of the Fc region by a linking sequence as described herein. The variable domain is connected to the N- terminus of the Fc region by an engineered IgG hinge sequence as described herein.

[0295] By “N-terminal of the engineered IgG hinge region” it is meant that the variable domain is located N-terminal of the engineered IgG hinge region by any means known in the art. In an embodiment, the variable domain is fused directly to the N-terminus of the engineered IgG hinge region, i.e. the variable domain may be immediately adjacent to the engineered IgG hinge region. However, in an embodiment, the variable domain is connected to the N-terminus of the engineered IgG hinge region by a linking sequence as described herein.

[0296] By “C-terminal of the Fc region” it is meant that the binding moiety or cognate of a binding moiety is located C-terminal of the Fc region by any means known in the art. In an embodiment, the binding moiety or cognate of a binding moiety is fused directly to the C- terminus of the Fc region, i.e. the binding moiety or cognate of a binding moiety may be immediately adjacent to the Fc region. However, in an embodiment, the binding moiety or cognate of a binding moiety is connected to the C-terminus of the Fc region by a linking sequence as described herein.

[0297] The linking sequence of the first and / or second polypeptide may comprise one or more linkers (e.g. glycine-serine (GS) linkers), such as those linkers that are widely known in the art. In an embodiment, the linking sequence comprises a GS linker. In an embodiment, the linking sequence consists of a GS linker. In an embodiment, the linking sequence is GS. In a preferred embodiment, the linking sequence is devoid of cysteine residues. Thus, the linking sequences of the first and second polypeptides may not form any inter-chain disulphide bonds. Preferably, the linking sequences of the first and second polypeptides do not form inter-domain disulphide bonds.

[0298] The binding moiety of the first and / or second polypeptide may be any suitable binding entity which is capable of specifically binding to a target, such as a target polypeptide sequence. Numerous binding moieties are known in the art, including those based on the antigen binding site of an antibody, antibody mimetics, and T-cell receptors. For example, the binding moiety may comprise: a variable immunoglobulin domain; an antigen binding site of an antibody; a single-chain variable fragment (scFv); a Fab; an Fv; a single domain antibody (sdAb); a VHH; a single chain variable domain (which may be a VH or VL chain, having 3 CDRs); or an artificial single binder such as a Darpin (designed ankyrin repeat protein). In a preferred embodiment, the binding moiety is an antigen-binding fragment of an antibody. In a preferred embodiment, the antigen-binding fragment of an antibody is a VHH. The cognate of a binding moiety may be any motif that is specifically recognised by a binding moiety. Numerous cognates of binding moieties are provided in the art. In an embodiment, the cognate is an antigen, an epitope or a polypeptide tag that is specifically recognised by an antibody or an antigen-binding fragment of an antibody. In a preferred embodiment, the cognate is a polypeptide tag. In a preferred embodiment, the cognate is a polypeptide tag that is specifically recognised by an antigen-binding fragment of an antibody.

[0299] In an embodiment, the binding moiety that is C-terminal of the Fc region of the first polypeptide and / or the second polypeptide is preferably a VHH domain and is specific for a third target. In an embodiment, the third target is different to the first and second targets. In an embodiment, the binding moieties that are C-terminal of the Fc region of each of the first polypeptide and the second polypeptide are preferably VHH domains and are specific for a third target and a fourth target. In an embodiment, each of the first, second, third and fourth targets are different.

[0300] In an embodiment, the cognate of a binding moiety that is C-terminal of the Fc region of the first polypeptide and / or the second polypeptide is not specifically recognised by any of the variable domains (e.g. VHH domains) and binding moieties, where present, of the first and second polypeptides.

[0301] In an embodiment, a binding moiety or a variable domain (e.g. a VHH) “specific for” a target or “specifically recognising” a target refers to binding to the target with an affinity equivalent to that of a functional antibody fragment. In an embodiment, the targets are target antigens.

[0302] In an embodiment in which the first and / or second polypeptides contain a plurality of variable domains and / or binding moieties (e.g. VHH domains), each variable domain and / or binding moiety (e.g. VHH domain) in the same polypeptide may be specific for the same target. In an embodiment in which the first and / or second polypeptides contain a plurality of variable domains and / or binding moieties (e.g. VHH domains), each variable domain and / or binding moiety (e.g. VHH domain) in the same polypeptide may be specific for a different target.

[0303] Any suitable Fc region which is known in the art may be employed in the practice of the present invention. In some embodiments, one or more amino acid modifications may be introduced into an Fc region of an antibody provided herein, thus producing an Fc region variant. The Fc region variant may comprise a human Fc region sequence (such as the human lgG1 , lgG2, I gG3, or lgG4 Fc region) comprising an amino acid modification (such as substitution) at one or more amino acid positions. For example, Fc engineering for modulated effector functions is known in the art. Fc modifications include, but are not limited to, silencing effector functions, elevating effector functions, and elongating or shortening half-life. A review of Fc modifications is provided by Liu et al. (Liu et al., Antibodies, 2020, 9: 64). In an embodiment, the first and second polypeptides comprise modifications that enhance the formation of a dimer, preferably a heterodimer, comprising one monomer of the first polypeptide and one monomer of the second polypeptide. In an embodiment, the modifications can be those modifications which are widely known in the art of antibody technology for the purpose of (hetero)dimerisation between antibody chains (e.g. “knobs-into-holes” (KiH) modifications). In a typical embodiment, the modifications are present in the CH3 domains of the first and second polypeptides.

[0304] In one embodiment, one or more of the CH3 domains of the first polypeptide and / or one or more of the CH3 domains of the second polypeptide comprise a modification that enhances the formation of the dimer comprising the first and second polypeptide. In an embodiment wherein the first and second polypeptides each comprise more than one CH3 domain, all of the CH3 domains may comprise such modifications.

[0305] As used herein, the term “enhances the formation of the dimer comprising the first and second polypeptide” includes promoting dimerisation of the first and second polypeptides. Thus, in an embodiment, the modifications promote dimerisation of the first and second polypeptides. In an embodiment, the modifications promote heterodimerisation of the first and second polypeptides. By heterodimerisation it is meant that the first polypeptide is different to the second polypeptide, and the first polypeptide will form a dimer with the second polypeptide, but the first polypeptide will not form a dimer with the first polypeptide and the second polypeptide will not form a dimer with the second polypeptide. In an embodiment, the modifications promote homodimerisation of the first and second polypeptides. By homodimerisation it is meant that the first polypeptide is the same as the second polypeptide, and the first polypeptide will form a dimer with the second polypeptide.

[0306] Various means of promoting dimerisation of two domains (e.g. homo- or hetero-dimerisation) are known in the art. In an embodiment, the two domains may comprise a modification that enhances formation of dimer, e.g. a modification that increases the affinity of the two domains, promotes and / or enables the formation of one or more disulphide bonds, reduces steric hindrance to the dimerisation, promotes electrostatic and / or hydrophilic / hydrophobic interactions between the two domains, or any combination thereof.

[0307] Any suitable technology for promoting the dimerisation of Fc regions (e.g. CH3 domains), i.e. of at least one of the CH3 domains of the first polypeptide and at least one of the CH3 domains of the second polypeptide, may be employed in the practice of the invention.

[0308] Preferably, the modification that enhances the formation of a (hetero-)dimer between the Fc regions (e.g. CH3 domains) is KiH technology (Ridgeway et al., Protein Engineering, Design and Selection, 1996, 9: 617-621 and Merchant et al., Nat Biotechnol, 1998, 16: 677-681). The KiH technology is based on an engineered pair of CH3 domains that heterodimerises (CH3 heterodimer) in which asymmetric hydrophobic mutations are introduced between the homodimeric CH3 domain. KiH involves introducing mutations that create a protuberance (“knob”) in the interface of the first CH3 domain and a corresponding cavity (“hole”) in the interface of the second CH3 domain, such that the protuberance can be positioned in the cavity to promote heterodimer assembly and hinder homodimer formation. KiH variants therefore thermodynamically favour the formation of heterodimers rather than homodimers.

[0309] By way of further example, the present invention may employ: the strand-exchange engineered domain (SEED) CH3 dimers (Davis et al., Protein Eng Des Sei., 2010, 23:195- 202); electrostatic steering employing DD-KK variants with asymmetric electrostatic interactions (Gunasekaran et al., J Biol Chem., 2010 , 285: 19637-46): Azymetric technologies by Zymeworks (https: / / www.zvmeworks.com / technologies / azymetric / ): Bispecific

[0310] Engagement by Antibodies based on the T-cell receptor (BEAT) (Skegro et al., J Biol Chem., 2017, 292: 9745-9759): Fast-lg and ART-lg (https: / / www.chugai- pharm.co.jp / english / profile / rd / technologies.html): DEKK dimerisation technology

[0311] (https: / / merus.nl / technology / multiclonics-platform / and Nardis et al, J Biol Chem., 2017, 292: 14706-14717): HA-TF variants with asymmetric hydrophobic interactions (Moore et al., MAbs, 2011 , 3: 546-57): computationally designed CH3 interfaces, such as the 7.8.60 design (Leaver-Fay et al., Structure, 2016, 24: 641-651): EW-RVT variants, which were designed to replace the conserved electrostatic interactions with asymmetric hydrophobic interactions and to add asymmetric long-range electrostatic interactions at the rim of the heterodimeric CH3 interface (Choi et al., Mol Cancer Then, 2013, 12: 2748-59): and the K370ECH3A-E357NCH3B mutations employed in the “A107” variant which replaced the homodimer-favouring electrostatic interactions with heterodimer-stabilizing hydrogen bonds.

[0312] Any suitable KiH modifications known in the art may be employed in the practice of the present invention. For example, the T366W (“knob”) modification and the T366S, L368A and Y407V (“hole”) modifications may be used (according to Ell residue numbering (Edelman et al., Proc Natl Acad Sci U S A., 1969, 63: 78-85)).

[0313] The variable domains (e.g. VHH domains) used in the invention are not particularly limiting, and may comprise any antigen binding site that is specific for a selected target protein. The selected target protein may be a cancer antigen, an immune cell marker, or a non-human cell antigen. In some embodiments, the at least one variable domain of the first polypeptide, the second polypeptide or both the first polypeptide and the second polypeptide is specific for a cancer antigen.

[0314] Various tumour associated antigens (TAA) are known in the art. In an embodiment, the variable domains (e.g. VHH domains) of the present invention are capable of specifically binding to a TAA.

[0315] In some embodiments, the at least one variable domain of the first polypeptide, the second polypeptide or both the first polypeptide and the second polypeptide is specific for an immune cell marker. Various immune cell markers are known in the art. For example, anti-CD163 ADCs that target macrophages are known in the art (Liu et al., Expert Opinion on Biological Therapy, 2016, 16: 591-593). By way of further example, ADCs targeting CXCR4 are known in the art. CXCR4 is highly expressed on T cells, B-cells, and monocytes, as well as hematopoietic stem cells, with minimal to no expression on non-hematopoietic cells (Liu et al., Expert Opinion on Biological Therapy, 2016, 16: 591-593).

[0316] Moreover, ADCs targeting non-human cells are known in the art. For example, an antibodyantibiotic conjugate (AAC) targeting intracellular Streptococcus aureus has been reported (Liu et al., Expert Opinion on Biological Therapy, 2016, 16: 591-593).

[0317] Drug

[0318] The drug to which the antibody is conjugated may also be referred to as a “cargo” or “payload”. The payload used in the invention is not particularly limiting, and may comprise any therapeutic or diagnostic agent that can be conjugated to an antibody. In addition to using synthetic cytotoxins, coupling of other payloads with site-specific antibody conjugation has also been reported. These other payloads include non-cytotoxic compounds, such as proteins / peptides, glycans, lipids, and nucleic acids.

[0319] Suitably, the payload may be a small molecule, an oligonucleotide (e.g. RNA or DNA), a peptide, a dye (e.g. IRDye 700DX), a cytotoxic drug, a chemotherapeutic drug, an antimicrobial agent (such as an antibiotic), a protein degrader, an enzyme inhibitor, a protein ligand, or an immunomodulatory agent. Suitable payloads are known in the art (see, for example, Liu et al. , Expert Opinion on Biological Therapy, 2016, 16: 591-593; and Zhijia Wang et al., Acta Pharmaceutica Sinica B, 2023,13: 4025-4059). Suitably, in some embodiments, the payload may be a topoisomerase inhibitor, i.e. a chemical compound that inhibits at least one topoisomerase. Suitably, in some embodiments, the payload may be a topoisomerase I inhibitor, such as irinotecan, topotecan, belotecan, deruxtecan, exatecan or camptothecin. Suitably, in some embodiments, the payload may be a topoisomerase II inhibitor, such as etoposide, doxorubicin, or epirubicin.

[0320] In some embodiments, the drug is selected from the group consisting of a cytotoxic drug, a chemotherapeutic drug, an antimicrobial agent (such as an antibiotic), or an immunomodulatory agent.

[0321] The term cytotoxic or chemotherapeutic drug refers to a drug that reduces or eliminates the viability of a cell. Suitable cytotoxic or chemotherapeutic drugs will be known in the art.

[0322] The term “cytotoxic drug” generally refers to a toxic drug. Suitably, the cytotoxic drug may be a chemical molecule that is potent enough to disrupt the normal growth of a tumour cell exposed thereto. Cytotoxic drugs can kill tumour cells at a sufficiently high concentration. The “cytotoxic drug” may include toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes, toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes.

[0323] For example, the cytotoxic drug may be selected from the group consisting of Auristatins, Maytansinoids, Tubulysins, Taxoids, Calicheamicins, Amatoxins, Pyrrolobenzodiazepines, Camptothecins and derivatives thereof. Suitably, the cytotoxic drug may be MMAE, MMAF, DM1 , DM4, duocarmycin, Dxd, SN38, exatecan, doxorubicin, SG3199, or Taxol.

[0324] Method of Producing an Antibody Drug Conjugate

[0325] In a further aspect, the present invention provides a method of producing an antibody-drug conjugate (ADC) having a predetermined drug-to-antibody ratio (DAR), comprising the steps of:

[0326] (i) providing an antibody as defined herein;

[0327] (ii) reducing the antibody with a reducing agent; and

[0328] (iii) conjugating the reduced antibody with the drug.

[0329] In a further aspect, the present invention provides a method of producing an antibody-drug conjugate (ADC) having a predetermined drug-to-antibody ratio (DAR), comprising the steps of:

[0330] (i) (a) providing at least one polynucleotide sequence encoding an antibody comprising a first polypeptide comprising an immunoglobulin fragment crystallisable (Fc) region and at least one variable domain N-terminal of the Fc region; (b) modifying the at least one polynucleotide sequence to encode an engineered IgG hinge region between the Fc region and the at least one variable domain, wherein the engineered IgG hinge region is heterologous to the Fc region and / or is mutated to provide a predetermined number of cysteine residues;

[0331] (c) introducing the at least one polynucleotide sequence into a cell;

[0332] (d) culturing the cell under conditions suitable for the expression of the at least one polynucleotide sequence;

[0333] (e) isolating the antibody expressed by the cell;

[0334] (ii) reducing the antibody with a reducing agent; and

[0335] (iii) conjugating the reduced antibody with the drug.

[0336] In some embodiments, the antibody further comprises a second polypeptide comprising an Fc region, and step (i)(b) further comprises modifying the at least one polynucleotide sequence to encode an engineered IgG hinge region N-terminal of the Fc region of the second polypeptide, wherein the engineered IgG hinge region is heterologous to the Fc region of the second polypeptide and / or is mutated to provide a predetermined number of cysteine residues, and wherein the engineered IgG hinge region of the first polypeptide and the engineered IgG hinge region of the second polypeptide are complementary engineered IgG hinge regions.

[0337] In some embodiments, the second polypeptide further comprises at least one variable domain N-terminal of the engineered IgG hinge region.

[0338] In a further aspect, the present invention provides a method of producing an antibody-drug conjugate (ADC) having a predetermined drug-to-antibody ratio (DAR), comprising the steps of:

[0339] (i) (a) providing at least one polynucleotide sequence encoding an antibody wherein the antibody comprises a first polypeptide and a second polypeptide, wherein each of the first polypeptide and the second polypeptide comprises an immunoglobulin fragment crystallisable (Fc) region and at least one variable domain which is a VHH N-terminal of the Fc region;

[0340] (b) modifying the at least one polynucleotide sequence to encode an engineered IgG hinge region between the Fc region and the at least one variable domain of each of the first polypeptide and the second polypeptide, wherein the engineered IgG hinge region of the first polypeptide and the engineered IgG hinge region of the second polypeptide are complementary engineered IgG hinge regions, and wherein the engineered IgG hinge region is heterologous to the Fc region and / or is mutated to provide a predetermined number of cysteine residues; (c) introducing the at least one polynucleotide sequence into a cell;

[0341] (d) culturing the cell under conditions suitable for the expression of the at least one polynucleotide sequence;

[0342] (e) isolating the antibody expressed by the cell;

[0343] (ii) reducing the antibody with a reducing agent; and

[0344] (iii) conjugating the reduced antibody with the drug.

[0345] The at least one polynucleotide sequence may be modified to encode an engineered IgG hinge region by introducing a nucleic acid sequence encoding an engineered IgG hinge region into the at least one polynucleotide sequence. Alternatively, the at least one polynucleotide sequence may be modified to mutate a native IgG hinge region to provide an engineered IgG hinge region in accordance with the invention as described herein.

[0346] The modification of the at least one polynucleotide sequence to encode an engineered IgG hinge region according to the invention using conventional techniques in molecular biology is within the capabilities of a person of ordinary skill in the art. Generally speaking, suitable routine methods include directed mutagenesis, gene synthesis and recombinant DNA / RNA technology.

[0347] The introduction of at least one polynucleotide sequence encoding a component of the present invention into a cell using conventional molecular and cell biology techniques is within the capabilities of a person of ordinary skill in the art. For example, a vector or an expression cassette could be used.

[0348] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. In accordance with the present invention, and by way of example, some vectors used in recombinant nucleic acid techniques allow entities, such as a segment of nucleic acid (e.g. a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into and expressed by a target cell. The vector may facilitate the integration of the nucleotide sequence(s) encoding the antibody of the invention to maintain the nucleotide sequence encoding the antibody of the invention and its expression within the target cell. Expression cassettes as described herein comprise regions of nucleic acid containing sequences capable of being transcribed.

[0349] The cysteine-based site-specific conjugation may be performed using any suitable method known in the art and as described herein.

[0350] In some embodiments, the step of reducing the antibody in step (ii) is a partial reduction of the antibody. Suitably, cysteine-based site-specific conjugation strategies involve partial reduction of the antibody to reduce the inter-chain disulphide bonds of the antibody. This directed reduction of the antibody enables the control of the DAR of the product, i.e. by selectively reducing the inter-chain disulphide bonds of the antibody conjugation of the antibody to the linker-drug will occur via the free cysteine residues generated in the partial reduction step. This assists in the production of a homogenous ADC product with a uniform DAR.

[0351] In some embodiments, the step of reducing the antibody in step (ii) is a partial reduction of the antibody to reduce the inter-chain disulphide bonds of the antibody.

[0352] In some embodiments, the reducing agent is dithiothreitol (DTT) or tris (2-carboxyethyl) phosphine (TCEP).

[0353] In some embodiments, the drug is in the form of a linker-drug conjugate comprising a thiolreactive group. The linker may be as described herein.

[0354] In some embodiments, the linker comprises a thiol-reactive group selected from the group consisting of a maleimide, bromoacetamide, disulphide, a-haloacetamide, a-halocarbonyl, vinylsulfone, heteroaryl sulfone, thiosulfonate, electron deficient aryl halide, ethynylphosphonamidate, vinylphosphonite, palladiumoxidative-addition complex, bissulfone, water-soluble allyl sulfone, thiol-yne bioconjugation with terminal alkyne or cyclooctyne, dibromo- (DBM) and dithio-maleimide (DTM), hybrid thiobromomaleimide (TBM), dibromopyridazinediones, divinylpyrimidine, and DiPODS (two oxadiazolyl methyl sulfone moieties connected by a phenyl group).

[0355] In some embodiments, the thiol-reactive group is selected from the group consisting of a maleimide, bromoacetamide, disulphide, a-haloacetamide, a-halocarbonyl, vinylsulfone, heteroaryl sulfone, thiosulfonate, electron deficient aryl halide, ethynylphosphonamidate, vinylphosphonite, palladiumoxidative-addition complex.

[0356] In some embodiments, step (iii) is performed using a thiol-reactive coupling strategy.

[0357] In some embodiments, the thiol-reactive coupling strategy is maleimide-based conjugation.

[0358] In some embodiments, the method of the invention provides homogenous ADCs.

[0359] In some embodiments, all of the cysteine residues within the engineered IgG hinge region form stable inter-chain disulphide bonds prior to step (ii).

[0360] In some embodiments, in step (iii) the antibody is conjugated to the drug by cysteine-based site-specific conjugation through all of the cysteine residues within the engineered IgG hinge region. In a further aspect, the present invention provides an ADC obtained or obtainable by the methods of the invention.

[0361] Pharmaceutical composition

[0362] In a further aspect, the present invention provides a pharmaceutical composition comprising the ADC according to the invention.

[0363] The pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and / or compounds. Such a formulation may, for example, be in a form suitable for intravenous infusion.

[0364] Accordingly, in a further aspect, the present invention provides a pharmaceutical composition comprising the ADC according to the invention and a pharmaceutically acceptable carrier, excipient and / or diluent.

[0365] Therapeutic use

[0366] In a further aspect, the present invention provides an ADC of the invention ora pharmaceutical composition of the invention for use in therapy.

[0367] In a further aspect, the invention provides an ADC of the invention or a pharmaceutical composition of the invention for use in a method of therapy or a diagnostic method.

[0368] In a further aspect, the invention provides the use of an ADC of the invention or a pharmaceutical composition of the invention for the manufacture of a medicament or diagnostic agent.

[0369] In a further aspect, the invention provides a method of therapy or a diagnostic method comprising administering the ADC of the invention or pharmaceutical composition of the invention to a subject.

[0370] A method for therapy includes a method for treating a disease as well as a method for preventing a disease.

[0371] A method for treating a disease relates to the therapeutic use of an ADC or pharmaceutical composition of the invention. In this respect, the ADC or pharmaceutical composition according to the invention may be administered to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease and / or to slow down, reduce or block the progression of the disease. The method for preventing a disease relates to the prophylactic use of an ADC or pharmaceutical composition of the invention. In this respect, the ADC or pharmaceutical composition according to the invention may be administered to a subject who has not yet contracted the disease and / or who is not showing any symptoms of the disease to prevent or impair the cause of the disease or to reduce or prevent development of at least one symptom associated with the disease. The subject may have a predisposition for, or be thought to be at risk of developing, the disease.

[0372] The disease to be treated and / or prevented may be cancer.

[0373] Suitably, the cancer may be a solid tumour or a liquid tumour.

[0374] The cancer may be a cancer such as neuroblastoma, prostate cancer, bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell), leukaemia, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, and thyroid cancer.

[0375] In a further aspect, the present invention provides an ADC of the invention, or a pharmaceutical composition of the invention for use in the treatment of a disease or condition selected from the group consisting of cancer, autoimmune disease, infection, infectious diseases, cardiovascular diseases and liver metabolic disorders.

[0376] The therapeutic or diagnostic use an ADCs is well-known in the art (see, for example, Liu et al., Expert Opinion on Biological Therapy, 2016, 16: 591-593; and Zhijia Wang et al., Acta Pharmaceutica Sinica B, 2023,13: 4025-4059).

[0377] In some embodiments, the subject of the medical uses and methods of treatment according to the present invention may be a mammal.

[0378] In some embodiments, the subject may be a human.

[0379] In some embodiments, the subject may alternatively be a non-human mammal, including for example, a primate, a monkey, a dog, a cat, a horse, a cow, a sheep, a pig, a rabbit, a rat, or a mouse.

[0380] In some embodiments, the subject may be a patient, such as a human patient.

[0381] In some embodiments, the subject may suffer from and / or have been diagnosed with one or more cancer(s).

[0382] In some embodiments, the subject may suffer from and / or have been diagnosed with an autoimmune disease, infection, infectious diseases, cardiovascular diseases or liver metabolic disorders. Nucleic acid sequences and particles

[0383] In a further aspect, the invention provides one or more nucleic acid sequences capable of expressing the antibody for use according to the invention.

[0384] In a further aspect, the invention provides one or more nucleic acid sequences capable of expressing the first polypeptide sequence as defined herein.

[0385] In a further aspect, the invention provides one or more nucleic acid sequences capable of expressing the second polypeptide sequence as defined herein.

[0386] In a further aspect, the invention provides one or more nucleic acid sequences capable of expressing the first polypeptide sequence and the second polypeptide sequence as defined herein.

[0387] According to the present disclosure, terms such as “capable of expressing”, “nucleic acid expressing” and “nucleic acid encoding” or similar terms are used interchangeably herein and with respect to a particular peptide or polypeptide mean that the nucleic acid, if present in the appropriate environment, e.g. within a cell, can be expressed to produce said peptide or polypeptide.

[0388] Suitably, the nucleic acid sequences capable of expressing the antibody for use according to the invention may comprise a plurality of nucleic acid sequences which encode components of the construct such as the first polypeptide, second polypeptide, Fc region, variable domain, VHH domain, engineered IgG hinge region and / or linking sequence as described herein.

[0389] It will be understood by the skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed. Suitably, the polynucleotides of the present invention are codon optimised to enable expression in a mammalian cell, in particular a cell as described herein.

[0390] Nucleic acids according to the invention may comprise DNA and / or RNA. The nucleic acids may be single-stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3’ and / or 5’ ends of the molecule. Herein, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.

[0391] In a further aspect, the present invention provides a vector comprising the one or more nucleic acid sequences of the present invention.

[0392] In an embodiment, the vector comprises a plurality of nucleic acid sequences which encode different components as provided by the present invention. For example, in an embodiment the vector comprises a first nucleic acid sequence which encodes the first polypeptide and a second nucleic acid sequence which encodes the second polypeptide of the invention.

[0393] In embodiments, the vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon based vector or synthetic mRNA.

[0394] In an embodiment, the vector is capable of transfecting or transducing a cell.

[0395] The one or more nucleic acid sequences of the present invention may be present in particles comprising (i) the one or more nucleic acid sequences and (ii) at least one cationic or cationically ionizable compound such as a polymer or lipid complexing the one or more nucleic acid sequences. Electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged nucleic acid are involved in particle formation. This results in complexation and spontaneous formation of nucleic acid particles comprising the one or more nucleic acid sequences.

[0396] Accordingly, in a further aspect, the invention provides a nucleic acid particle comprising the nucleic acid sequence(s) according to the invention.

[0397] Different types of nucleic acid containing particles have been described previously to be suitable for delivery of nucleic acid (e.g. RNA) in particulate form (cf. , e.g., Kaczmarek, J. C. et al., 2017, Genome Medicine 9, 60). For non-viral delivery vehicles, nanoparticle encapsulation of nucleic acids physically protects nucleic acids from degradation and, depending on the specific chemistry, can aid in cellular uptake and endosomal escape.

[0398] In the context of the present disclosure, the term "particle" relates to a structured entity formed by molecules or molecule complexes, in particular particle forming compounds. In some embodiments, the particle contains an envelope (e.g., one or more layers or lamellas) made of one or more types of amphiphilic substances (e.g., amphiphilic lipids). In this context, the expression "amphiphilic substance" means that the substance possesses both hydrophilic and lipophilic properties. The envelope may also comprise additional substances (e.g., additional lipids) which do not have to be amphiphilic. In some embodiments, the term "particle" relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure. According to the present disclosure, the term "particle" includes nanoparticles. Nucleic acid particles (such as RNA particles and / or DNA particles) include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations.

[0399] In general, a lipoplex (LPX) is obtainable from mixing two aqueous phases, namely a phase comprising nucleic acid (such as RNA and / or DNA) and a phase comprising a dispersion of lipids. In some embodiments, the lipid phase comprises liposomes.

[0400] In general, a lipid nanoparticle (LNP) is obtainable from direct mixing of nucleic acid (such as RNA and / or DNA) in an aqueous phase with lipids in a phase comprising an organic solvent, such as ethanol. In that case, lipids or lipid mixtures can be used for particle formation, which do not form lamellar (bilayer) phases in water.

[0401] In some embodiments, LNPs comprise or consist of a cationic / ionisable lipid and helper lipids such as phospholipids, cholesterol, and / or polyethylene glycol (PEG) lipids. In some embodiments, in the nucleic acid LNPs (such as DNA LNPs) described herein the nucleic acid (such as DNA) is bound by ionisable lipid that occupies the central core of the LNP. In some embodiments, PEG lipid forms the surface of the LNP, along with phospholipids. In some embodiments, the surface comprises a bilayer. In some embodiments, cholesterol and ionisable lipid in charged and uncharged forms can be distributed throughout the LNP.

[0402] In some embodiments, nucleic acid (such as RNA and / or DNA, e.g., mRNA) may be noncovalently associated with a particle as described herein. In embodiments, the nucleic acid (such as RNA and / or DNA, especially mRNA) may be adhered to the outer surface of the particle (surface nucleic acid) and / or may be contained in the particle (encapsulated nucleic acid (such as encapsulated DNA).

[0403] In a further aspect, the invention provides a cell comprising the nucleic acid sequence(s) or nucleic acid particle according to the invention.

[0404] Kits

[0405] In a further aspect, the invention provides a kit comprising the polypeptide, antibody, nucleic acid sequence(s), nucleic acid particle or cell of the present invention.

[0406] In some embodiments, the kit further comprises instructions for conjugating one or more drug or payload molecules to the polypeptide or antibody of the present invention.

[0407] Where present, in some embodiments, the kit further comprises instructions for expressing the nucleic acid (sequences) or nucleic acid particle of the present invention.

[0408] In some embodiments, the kit further comprises instructions for making an ADC. General definitions

[0409] This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5’ to 3’ orientation; amino acid sequences are written left to right in amino (N) to carboxy (C) orientation, respectively.

[0410] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

[0411] The term “polypeptide” is used in the conventional sense to mean a series of amino acids, typically L-amino acids, connected one to the other, typically by peptide bonds between the a- amino and carboxyl groups of adjacent amino acids. The term “polypeptide” is used interchangeably with the terms “amino acid sequence”, “peptide” and / or “protein”. The term “residues” is used to refer to amino acids in an amino acid sequence.

[0412] The term “variant” in relation to a polypeptide refers to a polypeptide that has an equivalent function to the amino acid sequences described herein, but which includes one or more amino acid substitutions, insertions or deletions.

[0413] As used herein, the terms “polynucleotide”, “nucleotide”, “nucleic acid sequence” and “nucleic acid” are intended to be synonymous with each other.

[0414] The terms “variant”, “homologue” or “derivative” in relation to a nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence.

[0415] “Sequence identity” between two nucleic acid or protein sequences indicates the percentage of nucleotides that are identical between the sequences. The terms “% identical” and “% identity” or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or “window of comparison”, in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981 , Ads App. Math. 2, 482, with the aid of the global homology algorithm by Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website (e.g., at blast. ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK _LOC=align2seq). In some embodiments, the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match / Mismatch Scores set to 1 , -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. In some embodiments, the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1 ; and (vi) conditional compositional score matrix adjustment.

[0416] Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.

[0417] In some embodiments, the degree of similarity or identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments continuous nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

[0418] In some embodiments, “isolated” means removed (e.g., purified) from the natural state or from an artificial composition, such as a composition from a production process. For example, a nucleic acid, peptide or polypeptide naturally present in a living animal is not “isolated”, but the same nucleic acid, peptide or polypeptide partially or completely separated from the coexisting materials of its natural state is “isolated”. An isolated nucleic acid, peptide or polypeptide can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0419] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence.

[0420] In the context of the present disclosure, the term “transcription” relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA (especially mRNA). Subsequently, the RNA may be translated into peptide or polypeptide.

[0421] With respect to RNA, the term “expression” or “translation” relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or polypeptide.

[0422] The term “nucleic acid” comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. A nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule. A nucleic acid can be isolated. The term “isolated nucleic acid” means, according to the present disclosure, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis.

[0423] The term “nucleoside” (abbreviated herein as “N”) relates to compounds which can be thought of as nucleotides without a phosphate group. While a nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[0424] The five standard nucleosides which usually make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine and guanosine. The five nucleosides are commonly abbreviated to their one letter codes II, A, T, C and G, respectively. However, thymidine is more commonly written as “dT” (“d” represents “deoxy”) as it contains a 2’-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) and not ribonucleic acid (RNA). Conversely, uridine is found in RNA and not DNA. The remaining three nucleosides may be found in both RNA and DNA. In RNA, they would be represented as A, C and G, whereas in DNA they would be represented as dA, dC and dG.

[0425] A modified purine (A or G) or pyrimidine (C, T, or II) base moiety is preferably modified by one or more alkyl groups, more preferably one or more C1-4 alkyl groups, even more preferably one or more methyl groups. Particular examples of modified purine or pyrimidine base moieties include N7-alkyl-guanine, N6-alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil, and N(1)- alkyl-uracil, such as N7-C1-4 alkyl-guanine, N6-C1-4 alkyl-adenine, 5-C1-4 alkyl-cytosine, 5- C1-4 alkyl-uracil, and N(1)-C1-4 alkyl-uracil, preferably N7-methyl-guanine, N6-methyl- adenine, 5-methyl-cytosine, 5-methyl-uracil, N1-methyl-pseudouridine, and N(1)-methyl- uracil.

[0426] Herein, the term “DNA” relates to a nucleic acid molecule which includes deoxyribonucleotide residues. In preferred embodiments, the DNA contains all or a majority of deoxyribonucleotide residues. As used herein, “deoxyribonucleotide” refers to a nucleotide which lacks a hydroxyl group at the 2’-position of a p-D-ribofuranosyl group. DNA encompasses without limitation, double stranded DNA, single stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal DNA nucleotides or to the end(s) of DNA. It is also contemplated herein that nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, these altered DNAs are considered analogs of naturally-occurring DNA. A molecule contains “a majority of deoxyribonucleotide residues” if the content of deoxyribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0427] DNA may be recombinant DNA and may be obtained by cloning of a nucleic acid, in particular cDNA. The cDNA may be obtained by reverse transcription of RNA.

[0428] The term “RNA” relates to a nucleic acid molecule which includes ribonucleotide residues. In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues. As used herein, “ribonucleotide” refers to a nucleotide with a hydroxyl group at the 2’-position of a p-D- ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered / modified nucleotides can be referred to as analogs of naturally occurring nucleotides, and the corresponding RNAs containing such altered / modified nucleotides (i.e., altered / modified RNAs) can be referred to as analogs of naturally occurring RNAs. A molecule contains “a majority of ribonucleotide residues” if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0429] “RNA” includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), selfamplifying RNA (saRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA) and immunostimulatory RNA (isRNA). In some embodiments, “RNA” refers to mRNA.

[0430] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0431] The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms “comprising”, “comprises” and “comprised of’ also include the term “consisting of’.

[0432] Embodiments of the invention

[0433] Various preferred features and embodiments of the present invention will now be described with reference to the following numbered paragraphs:

[0434] 1. A polypeptide comprising an immunoglobulin fragment crystallisable (Fc) region, at least one variable domain N-terminal of the Fc region and an engineered IgG hinge region between the Fc region and the at least one variable domain, and wherein the engineered IgG hinge region is heterologous to the Fc region and / or is mutated to provide a predetermined number of cysteine residues.

[0435] 2. An antibody comprising a first polypeptide comprising an Fc region, at least one variable domain N-terminal of the Fc region and an engineered IgG hinge region between the Fc region and the at least one variable domain, and wherein the engineered IgG hinge region is heterologous to the Fc region and / or is mutated to provide a predetermined number of cysteine residues.

[0436] 3. An antibody-drug conjugate (ADC), wherein the antibody is conjugated to the drug by cysteine-based site-specific conjugation, wherein the antibody comprises a first polypeptide comprising an immunoglobulin fragment crystallisable (Fc) region, at least one variable domain N-terminal of the Fc region and an engineered IgG hinge region between the Fc region and the at least one variable domain, and wherein the engineered IgG hinge region is heterologous to the Fc region and / or is mutated to provide a predetermined number of cysteine residues.

[0437] 4. The polypeptide, antibody or ADC according to any one of paragraphs 1 to 3, wherein the at least one variable domain is selected from the group consisting of a single-chain variable fragment (scFv); a Fab; a Fab’; a F(ab)’2; an Fv; a single domain antibody (sdAb); a VHH; a single chain variable domain; a designed ankyrin repeat protein (DARPin); and an aptamer.

[0438] 5. The antibody or ADC according to any one of paragraphs 2 to 4, wherein the antibody is selected from the group consisting of a full-length immunoglobulin, a scFv-Fc, a Fab-Fc, an Fv-Fc, a sdAb-Fc, or a VHH-Fc.

[0439] 6. The antibody or ADC according to any one paragraphs 2 to 5, wherein the antibody is a VHH-Fc.

[0440] 7. The antibody or ADC according to any one of paragraphs 2 to 6, wherein the antibody further comprises a second polypeptide comprising an Fc region and an engineered IgG hinge region N-terminal of the Fc region, wherein the engineered IgG hinge region is heterologous to the Fc region of the second polypeptide and / or is mutated to provide a predetermined number of cysteine residues, and wherein the engineered IgG hinge region of the first polypeptide and the engineered IgG hinge region of the second polypeptide are complementary engineered IgG hinge regions. 8. The antibody or ADC according to paragraph 7, wherein the second polypeptide further comprises at last one variable domain N-terminal of the engineered IgG hinge region, preferably wherein the at least one variable domain is selected from the group consisting of a single-chain variable fragment (scFv); an Fab; an Fab’; an F(ab)’2; an Fv; a single domain antibody (sdAb); a VHH; a single chain variable domain; a designed ankyrin repeat protein (DARPin); and an aptamer; more preferably wherein the at least one variable domain is a VHH.

[0441] 9. The antibody or ADC according to paragraph 8, wherein the ADC comprises one of the following: a) a first polypeptide comprising a VHH and a second polypeptide comprising a VHH; b) a first polypeptide comprising a VHH and a second polypeptide comprising a Fab; c) first polypeptide comprising a Fab and a second polypeptide comprising a VHH; or d) a first polypeptide comprising a Fab and a second polypeptide comprising a Fab.

[0442] 10. An antibody-drug conjugate (ADC), wherein the antibody is conjugated to the drug by cysteine-based site-specific conjugation, wherein the antibody comprises a first polypeptide and a second polypeptide, wherein each of the first polypeptide and the second polypeptide comprises an immunoglobulin fragment crystallisable (Fc) region, at least one variable domain which is a VHH N-terminal of the Fc region and an engineered IgG hinge region between the Fc region and the at least one VHH, wherein the engineered IgG hinge region of the first polypeptide and the engineered IgG hinge region of the second polypeptide are complementary engineered IgG hinge regions, and wherein the engineered IgG hinge region is heterologous to the Fc region and / or is mutated to provide a predetermined number of cysteine residues.

[0443] 11 . The polypeptide, antibody or ADC according to any one of the preceding paragraphs, wherein the engineered IgG hinge region of each polypeptide is heterologous to the Fc region of said polypeptide

[0444] 12. The polypeptide, antibody or ADC according to any one of the preceding paragraphs, wherein the engineered IgG hinge region of the first polypeptide or the engineered IgG hinge region of each of the first polypeptide and the second polypeptide comprises at least one cysteine residue, preferably wherein the engineered IgG hinge region comprises two, three, four, five, six, seven, eight or nine cysteine residues.

[0445] 13. The polypeptide, antibody or ADC according to any one of the preceding paragraphs, wherein the engineered IgG hinge region of the first polypeptide comprises at least one additional cysteine residue compared to the native hinge region of the Fc region of the first polypeptide, preferably two, three, four, five, six, seven, eight or nine additional cysteine residues compared to the native hinge region of the Fc region of the first polypeptide.

[0446] 14. The polypeptide, antibody or ADC according to any one of the preceding paragraphs, wherein the engineered IgG hinge region of the first polypeptide comprises at least one fewer cysteine residue compared to the native hinge region of the Fc region of the first polypeptide, preferably two, three, four, five, six, seven, eight or nine fewer cysteine residues compared to the native hinge region of the Fc region of the first polypeptide.

[0447] 15. The polypeptide, antibody or ADC according to any one of paragraphs 7-14, wherein the engineered IgG hinge region of the second polypeptide comprises at least one additional cysteine residue compared to the native hinge region of the Fc region of the second polypeptide, preferably two, three, four, five, six, seven, eight or nine additional cysteine residues compared to the native hinge region of the Fc region of the second polypeptide.

[0448] 16. The polypeptide, antibody or ADC according to any one of paragraphs 7-14, wherein the engineered IgG hinge region of the second polypeptide comprises at least one fewer cysteine residue compared to the native hinge region of the Fc region of the second polypeptide, preferably two, three, four, five, six, seven, eight or nine fewer cysteine residues compared to the native hinge region of the Fc region of the second polypeptide.

[0449] 17. The polypeptide, antibody or ADC according to any one of the preceding paragraphs, wherein the engineered IgG hinge region comprises a sequence having at least 70% identity to a sequence as set forth in any one of SEQ I D NOs: 1 , 2, 3, 4 or 5.

[0450] 18. The polypeptide, antibody or ADC according to any one of the preceding paragraphs, wherein the engineered IgG hinge region is an lgG1 hinge region.

[0451] 19. The polypeptide, antibody or ADC according to any one of the preceding paragraphs, wherein the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 1 , or is a fragment thereof and / or a variant thereof comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 1. 20. The polypeptide, antibody or ADC according any one of paragraphs 1-17, wherein the engineered IgG hinge region is an lgG2 hinge region.

[0452] 21. The polypeptide, antibody or ADC according to any one of paragraphs 1-17 or 20, wherein the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 2, or is a fragment thereof and / or a variant thereof comprising or an amino acid sequence having at least 70% identity to SEQ ID NO: 2.

[0453] 22. The polypeptide, antibody or ADC according to any one of paragraphs 1-17, wherein the engineered IgG hinge region is an lgG3 hinge region.

[0454] 23. The polypeptide, antibody or ADC according to any one of paragraphs 1-17 or 22, wherein the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4, or is a fragment thereof and / or a variant thereof comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 3 or to SEQ ID NO: 4.

[0455] 24. The polypeptide, antibody or ADC according to any one paragraphs 1-17, 22 or 23, wherein the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 6 or SEQ ID NO: 7, or is a fragment thereof and / or a variant thereof comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 6 or to SEQ ID NO: 7.

[0456] 25. The polypeptide, antibody or ADC according to any one of paragraphs 1-17, wherein the engineered IgG hinge region is an lgG4 hinge region.

[0457] 26. The polypeptide, antibody or ADC according to any one of paragraphs 1-17 or 25, wherein the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 5, or is a fragment thereof and / or a variant thereof comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 5.

[0458] 27. The polypeptide, antibody or ADC according to any one of the preceding paragraphs, wherein the engineered IgG hinge region has an amino acid sequence as set forth in any one of SEQ ID NOs: 8-118, preferably an amino acid sequence as set forth in SEQ ID NO: 8, 9, 12, 13, 18, 51 , 67, 74, 84, 100, 113 or 118.

[0459] 28. The polypeptide, antibody or ADC according to any one of the preceding paragraphs, wherein the engineered IgG hinge region of the first polypeptide and / or second polypeptide comprises at least one amino acid modification to avoid O-glycosylation compared to the native IgG hinge region of the Fc region of the first polypeptide and / or second polypeptide.

[0460] 29. The polypeptide, antibody or ADC according to paragraph 28, wherein the at least one amino acid modification to avoid O-glycosylation is an amino acid substitution. 30. The polypeptide, antibody or ADC according to any one of the preceding paragraphs, wherein the engineered IgG hinge region has an amino acid sequence as set forth in any one of SEQ ID NOs: 34-44, 51-61 , 64-67, 70-75, 78-87, 90-111 or 113.

[0461] 31 . The polypeptide, antibody or ADC according to any one of paragraphs 28-30, wherein the engineered IgG hinge region is an lgG3 hinge region and the at least one amino acid modification is selected from the group consisting of S241JJX, T241MMX and the combination thereof, wherein the numbering is according to the according to the Kabat numbering scheme.

[0462] 32. The polypeptide, antibody or ADC according to any one of the preceding paragraphs, wherein the engineered IgG hinge region is or is derived from a human IgG hinge region.

[0463] 33. The polypeptide, antibody or ADC according to any one of the preceding paragraphs, wherein the antibody is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, a monoclonal antibody, and a polyclonal antibody.

[0464] 34. The polypeptide, antibody or ADC according to any one of the preceding paragraphs, wherein the at least one variable domain of the first polypeptide, the second polypeptide or both the first polypeptide and the second polypeptide is specific for a cancer antigen.

[0465] 35. The polypeptide, antibody or ADC according to any one of the preceding paragraphs, wherein the antibody is conjugated to the drug by cysteine-based site-specific conjugation through all of the cysteine residues within the engineered IgG hinge region.

[0466] 36. The polypeptide, antibody or ADC according to any one of the preceding paragraphs, wherein the ADC has a drug-to-antibody-ratio (DAR) of from about 2 to about 12, preferably a DAR of from about 4 to about 8.

[0467] 37. The polypeptide, antibody or ADC according to any one of the preceding paragraphs, wherein the ADC has a drug-to-antibody-ratio (DAR) of about 2, 4, 6, 8, 10, or 12, preferably a DAR of about 8.

[0468] 38. The polypeptide, antibody or ADC according to any one of the preceding paragraphs, wherein the drug is selected from the group consisting of a cytotoxic drug, an antimicrobial agent, or an immunomodulatory agent.

[0469] 39. A method of producing an antibody-drug conjugate (ADC) having a predetermined drug-to-antibody ratio (DAR), comprising the steps of:

[0470] (i) providing an antibody as defined in any one of paragraphs 2-38;

[0471] (ii) reducing the antibody with a reducing agent; and (iii) conjugating the reduced antibody with the drug.

[0472] 40. A method of producing an antibody-drug conjugate (ADC) having a predetermined drug-to-antibody ratio (DAR), comprising the steps of:

[0473] (i) (a) providing at least one polynucleotide sequence encoding an antibody comprising a first polypeptide comprising an immunoglobulin fragment crystallisable (Fc) region and at least one variable domain N-terminal of the Fc region;

[0474] (b) modifying the at least one polynucleotide sequence to encode an engineered IgG hinge region between the Fc region and the at least one variable domain of the first polypeptide, wherein the engineered IgG hinge region is heterologous to the Fc region and / or is mutated to provide a predetermined number of cysteine residues;

[0475] (c) introducing the at least one polynucleotide sequence into a cell;

[0476] (d) culturing the cell under conditions suitable for the expression of the at least one polynucleotide sequence;

[0477] (e) isolating the antibody expressed by the cell;

[0478] (ii) reducing the antibody with a reducing agent; and

[0479] (iii) conjugating the reduced antibody with the drug.

[0480] 41. A method of producing an antibody-drug conjugate (ADC) having a predetermined drug-to-antibody ratio (DAR), comprising the steps of:

[0481] (i) (a) providing at least one polynucleotide sequence encoding an antibody wherein the antibody comprises a first polypeptide and a second polypeptide, wherein each of the first polypeptide and the second polypeptide comprises an immunoglobulin fragment crystallisable (Fc) region and at least one variable domain which is a VHH N-terminal of the Fc region;

[0482] (b) modifying the at least one polynucleotide sequence to encode an engineered IgG hinge region between the Fc region and the at least one variable domain of each of the first polypeptide and the second polypeptide, wherein the engineered IgG hinge region of the first polypeptide and the engineered IgG hinge region of the second polypeptide are complementary engineered IgG hinge regions, and wherein the engineered IgG hinge region is heterologous to the Fc region and / or is mutated to provide a predetermined number of cysteine residues;

[0483] (c) introducing the at least one polynucleotide sequence into a cell;

[0484] (d) culturing the cell under conditions suitable for the expression of the at least one polynucleotide sequence; (e) isolating the antibody expressed by the cell;

[0485] (ii) reducing the antibody with a reducing agent; and

[0486] (iii) conjugating the reduced antibody with the drug.

[0487] 42. The method according to any one of paragraphs 39-41 , wherein the step of reducing the antibody in step (ii) is a partial reduction of the antibody to reduce the inter-chain disulphide bonds of the antibody.

[0488] 43. The method according to any one of paragraphs 39-42, wherein the reducing agent is dithiothreitol (DTT) or tris (2-carboxyethyl) phosphine (TCEP).

[0489] 44. The method according to any one of paragraphs 39-43, wherein the drug is in the form of a linker-drug conjugate comprising a thiol-reactive group.

[0490] 45. The method according to paragraph 44, wherein the thiol-reactive group is selected from the group consisting of a maleimide, bromoacetamide, disulphide, a-haloacetamide, a- halocarbonyl, vinylsulfone, heteroaryl sulfone, thiosulfonate, electron deficient aryl halide, ethynylphosphonamidate, vinylphosphonite, palladiumoxidative-addition complex.

[0491] 46. The method according to any one of paragraphs 39-45, wherein step (iii) is performed using a thiol-reactive coupling strategy.

[0492] 47. The method according to paragraph 46, wherein the thiol-reactive coupling strategy is maleimide-based conjugation.

[0493] 48. The method according to any one of paragraphs 39-47, wherein the method provides homogenous ADCs.

[0494] 49. The method according to any one of paragraphs 40 or 42-48, wherein the antibody further comprises a second polypeptide comprising an Fc region, and wherein step (i) (b) further comprises modifying the at least one polynucleotide sequence to encode an engineered IgG hinge region N-terminal of the Fc region of the second polypeptide, wherein the engineered IgG hinge region is heterologous to the Fc region of the second polypeptide and / or is mutated to provide a predetermined number of cysteine residues, and wherein the engineered IgG hinge region of the first polypeptide and the engineered IgG hinge region of the second polypeptide are complementary engineered IgG hinge regions.

[0495] 50. The method according to paragraph 49, wherein the second polypeptide further comprises at last one variable domain N-terminal of the engineered IgG hinge region, preferably wherein the at least one variable domain is selected from the group consisting of a single-chain variable fragment (scFv); an Fab; an Fab’; an F(ab)’2; an Fv; a single domain antibody (sdAb); a VHH; a single chain variable domain; a designed ankyrin repeat protein (DARPin); and an aptamer; more preferably wherein the at least one variable domain is a VHH.

[0496] 51. The method according to any one paragraphs 40-50, wherein the engineered IgG hinge region of the first polypeptide or the engineered IgG hinge region of the first polypeptide and the second polypeptide comprises at least one cysteine residue, preferably wherein the engineered IgG hinge region of the first polypeptide or the engineered IgG hinge region of the first polypeptide and the second polypeptide comprises two, three, four, five, six, seven, eight or nine cysteine residues.

[0497] 52. The method according to any one of paragraphs 40-51 , wherein the engineered IgG hinge region of the first polypeptide comprises at least one additional cysteine residue compared to the native hinge region of the Fc region of the first polypeptide, preferably two, three, four, five, six, seven, eight or nine additional cysteine residues compared to the native hinge region of the Fc region of the first polypeptide.

[0498] 53. The method according to any one of paragraphs 40-51 , wherein the engineered IgG hinge region of the first polypeptide comprises at least one fewer cysteine residue compared to the native hinge region of the Fc region of the first polypeptide, preferably two, three, four, five, six, seven, eight or nine fewer cysteine residues compared to the native hinge region of the Fc region of the first polypeptide.

[0499] 54. The method according to any one of paragraphs 42 or 49-53, wherein the engineered IgG hinge region of the second polypeptide comprises at least one additional cysteine residue compared to the native hinge region of the Fc region of the second polypeptide, preferably two, three, four, five, six, seven, eight or nine additional cysteine residues compared to the native hinge region of the Fc region of the second polypeptide.

[0500] 55. The method according to any one of paragraphs 42 or 49-53, wherein the engineered IgG hinge region of the second polypeptide comprises at least one fewer cysteine residue compared to the native hinge region of the Fc region of the second polypeptide, preferably two, three, four, five, six, seven, eight or nine fewer cysteine residues compared to the native hinge region of the Fc region of the second polypeptide.

[0501] 56. The method according to any one of paragraphs 40-55, wherein the engineered IgG hinge region comprises a sequence having at least 70% identity to a sequence as set forth in any one of SEQ ID NOs: 1 , 2, 3, 4 or 5. 57. The method according to any one of paragraphs 40-56, wherein the engineered IgG hinge region is an lgG1 hinge region.

[0502] 58. The method according to any one of paragraphs 40-57, wherein the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 1 , or is a fragment thereof and / or a variant thereof comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 1.

[0503] 59. The method according any one of paragraphs 40-56, wherein the engineered IgG hinge region is an lgG2 hinge region.

[0504] 60. The method according to any one of paragraphs 40-56 or 59, wherein the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 2, or is a fragment thereof and / or a variant thereof comprising or an amino acid sequence having at least 70% identity to SEQ ID NO: 2.

[0505] 61. The method according to any one of paragraphs 40-56, wherein the engineered IgG hinge region is an lgG3 hinge region.

[0506] 62. The method according to any one paragraphs 40-56 or 61 , wherein the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 39 or SEQ ID NO: 40, or is a fragment thereof and / or a variant thereof comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 3, to SEQ ID NO: 4, to SEQ ID NO: 39 or to SEQ ID NO: 40.

[0507] 63. The method according to any one of paragraphs 40-56, wherein the engineered IgG hinge region is an lgG4 hinge region.

[0508] 64. The method according to any one of paragraphs 40-56 or 63, wherein the engineered IgG hinge region has an amino acid sequence as set forth in SEQ ID NO: 5, or is a fragment thereof and / or a variant thereof comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 5.

[0509] 65. The method according to any one of paragraphs 40-64, wherein the engineered IgG hinge region has an amino acid sequence as set forth in any one of SEQ ID NOs: 8-118, preferably an amino acid sequence as set forth in SEQ ID NO: 8, 9, 12, 13, 18, 51 , 67, 74, 84, 100, 113 or 118.

[0510] 66. The method according to any one of paragraphs 40-65, wherein the engineered IgG hinge region of the first polypeptide and / or second polypeptide comprises at least one amino acid modification to avoid O-glycosylation compared to the native hinge region of the Fc region of the first polypeptide and / or second polypeptide.

[0511] 67. The method according to paragraph 66, wherein the at least one amino acid modification to avoid O-glycosylation is an amino acid substitution.

[0512] 68. The method according to any one of paragraphs 40-67, wherein the engineered IgG hinge region has an amino acid sequence as set forth in any one of SEQ ID NOs: 34-44, 51- 61 , 64-67, 70-75, 78-87, 90-111 or 113.

[0513] 69. The method according to any one of paragraphs 66-68, wherein the engineered IgG hinge region is an lgG3 hinge region and the at least one amino acid modification is selected from the group consisting of S241JJX, T241MMX and the combination thereof, wherein the numbering is according to the according to the Kabat numbering scheme.

[0514] 70. The method according to any one of paragraphs 40-69, wherein all of the cysteine residues within the engineered IgG hinge region form stable inter-chain disulphide bonds prior to step (ii).

[0515] 71. The method according to any one of paragraphs 40-70, wherein the engineered IgG hinge region is or is derived from a human IgG hinge region.

[0516] 72. The method according to any one of paragraphs 40 or 42-71 , wherein the antibody is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, a monoclonal antibody, and a polyclonal antibody.

[0517] 73. The method according to any one of paragraphs 40 or 42-72, wherein the antibody is selected from the group consisting of a full-length immunoglobulin, a scFv-Fc, a Fab-Fc, an Fv-Fc, a sdAb-Fc, or a VHH-Fc.

[0518] 74. The method according to paragraph 73, wherein the antibody is a VHH-Fc.

[0519] 75. The method according to any one of paragraphs 40 or 42-74, wherein the at least one variable domain of the first polypeptide and / or second polypeptide is selected from the group consisting of a single-chain variable fragment (scFv); an Fab; an Fab’; an F(ab)’2; an Fv; a single domain antibody (sdAb); a VHH; a single chain variable domain; a designed ankyrin repeat protein (DARPin); and an aptamer.

[0520] 76. The method according to any one of paragraphs 40-75, wherein the at least one variable domain of the first polypeptide, the second polypeptide or both the first polypeptide and the second polypeptide is specific for a cancer antigen. 77. The method according to any one of paragraphs 40-76, wherein in step (iii) the antibody is conjugated to the drug by cysteine-based site-specific conjugation through all of the cysteine residues within the engineered IgG hinge region.

[0521] 78. The method according to any one of paragraphs 40-77, wherein the ADC has a drug- to-antibody-ratio (DAR) of from about 2 to about 12, preferably a DAR of from about 4 to about 8.

[0522] 79. The method according to any one of paragraphs 40-78, wherein the ADC has a drug- to-antibody-ratio (DAR) of about 2, 4, 6, 8, 10, or 12, preferably a DAR of about 8.

[0523] 80. The method according to any one of paragraphs 40-79, wherein the drug is selected from the group consisting of a cytotoxic drug, an antimicrobial agent, or an immunomodulatory agent.

[0524] 81 . An ADC obtained or obtainable by the method according to any one of paragraphs 39- 80.

[0525] 82. A pharmaceutical composition comprising the ADC according to any one of paragraphs 3-38 or 81 and a pharmaceutically acceptable carrier, excipient and / or diluent.

[0526] 83. The ADC according to any one of paragraphs 3-38 or 81 , or the pharmaceutical composition according to paragraph 82, for use in therapy or for use in a diagnostic method.

[0527] 84. The ADC according to any one of paragraphs 3-38 or 81 , or the pharmaceutical composition according to paragraph 82, for use in the treatment of a disease or condition selected from the group consisting of cancer, autoimmune disease, infection, infectious diseases, cardiovascular diseases and liver metabolic disorders.

[0528] 85. One or more nucleic acid sequence(s) capable of expressing the polypeptide or antibody of any one of paragraphs 1 to 38 or 81.

[0529] 86. A nucleic acid particle comprising the nucleic acid sequence(s) according to paragraph 85.

[0530] 87. A cell comprising the construct, nucleic acid sequences or nucleic acid particle according to any one of paragraphs 1 to 38, 81 , 85 or 86.

[0531] 88. A method of making an ADC, comprising: i) expressing the nucleic acid sequence(s) or nucleic acid particle of paragraph 85 or 86 to produce an antibody; ii) reducing the antibody with a reducing agent; and iii) conjugating the reduced antibody with the drug.

[0532] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0533] The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

[0534] Further embodiments of the invention relating to the lgG3-derived hinge

[0535] Certain preferred embodiments of the present invention relate to the provision of an ADC comprising an lgG3 or lgG3-derived hinge region and optionally an engineered or heterologous (i.e. non-lgG3 or lgG3-derived) Fc region. These embodiments can be understood in conjunction with the other embodiments / details of the present invention herein, and also in conjunction with the following numbered paragraphs:

[0536] A1. A polypeptide comprising an immunoglobulin fragment crystallisable (Fc) region, at least one variable domain N-terminal of the Fc region and an lgG3 hinge region, fragment or derivative thereof between the Fc region and the at least one variable domain.

[0537] A2. An antibody comprising a first polypeptide comprising an Fc region, at least one variable domain N-terminal of the Fc region and an lgG3 hinge region, fragment or derivative thereof between the Fc region and the at least one variable domain.

[0538] A3. An antibody-drug conjugate (ADC), wherein the antibody is conjugated to the drug by cysteine-based site-specific conjugation, wherein the antibody comprises a first polypeptide comprising an Fc region, at least one variable domain N-terminal of the Fc region and an lgG3 hinge region, fragment or derivative thereof between the Fc region and the at least one variable domain.

[0539] A4. The polypeptide, antibody or ADC of any one of paragraphs A1 to A3, wherein the IgG hinge region is an engineered IgG hinge region heterologous to the Fc region and / or is mutated to provide a predetermined number of cysteine residues. A5. The polypeptide, antibody or ADC of any one of paragraphs A1 to A4, wherein the Fc region is not an lgG3 Fc region, variant, fragment or derivative thereof, optionally wherein the Fc region is an I gG 1 Fc region, an lgG2 Fc region or an lgG4 Fc region.

[0540] A6. The antibody or ADC according to any one of paragraphs A2 to A5, wherein the antibody further comprises a second polypeptide comprising an Fc region and an lgG3 hinge region, fragment or derivative thereof N-terminal of the Fc region, optionally wherein the hinge region is an engineered IgG hinge region heterologous to the Fc region of the second polypeptide and / or is mutated to provide a predetermined number of cysteine residues, and wherein the IgG hinge region of the first polypeptide and the IgG hinge region of the second polypeptide are complementary IgG hinge regions.

[0541] A7. The antibody or ADC according to paragraph A6, wherein the second polypeptide further comprises at least one variable domain N-terminal of the engineered IgG hinge region.

[0542] A8. An antibody-drug conjugate (ADC), wherein the antibody is conjugated to the drug by cysteine-based site-specific conjugation, wherein the antibody comprises a first polypeptide and a second polypeptide, wherein each of the first polypeptide and the second polypeptide comprises an immunoglobulin fragment crystallisable (Fc) region, at least one variable domain which is a VHH N-terminal of the Fc region and an lgG3 hinge region, derivative or fragment thereof between the Fc region and the at least one VHH, wherein the IgG hinge region of the first polypeptide and the IgG hinge region of the second polypeptide are complementary IgG hinge regions, and optionally wherein the IgG hinge region is an engineered IgG hinge region heterologous to the Fc region and / or is mutated to provide a predetermined number of cysteine residues.

[0543] A9. The polypeptide, antibody or ADC according to any one of the preceding A# paragraphs, wherein the engineered IgG hinge region of the first polypeptide comprises at least one additional cysteine residue compared to the native lgG3 hinge region, optionally wherein the engineered IgG hinge region of the second polypeptide comprises at least one additional cysteine residue compared to the native lgG3 hinge region.

[0544] A10. The polypeptide, antibody or ADC according to any one of the preceding A# paragraphs, wherein the engineered IgG hinge region of the first polypeptide comprises at least one fewer cysteine residue compared to the native lgG3 hinge region, optionally wherein the engineered IgG hinge region of the second polypeptide comprises at least one fewer cysteine residue compared to the native lgG3 hinge region.

[0545] A11. The polypeptide, antibody or ADC according to any one of the preceding A# paragraphs, wherein the IgG hinge region has:

[0546] (i) an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4, or is a fragment thereof and / or a variant thereof comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 3 or to SEQ ID NO: 4; or

[0547] (ii) an amino acid sequence as set forth in SEQ ID NO: 6 or SEQ ID NO: 7, or is a fragment thereof and / or a variant thereof comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 6 or to SEQ ID NO: 7; or

[0548] A12. The polypeptide, antibody or ADC according to any one of the preceding A# paragraphs, wherein the IgG hinge region has an amino acid sequence as set forth in any one of SEQ ID NOs: 28 to 113.

[0549] A13. The polypeptide, antibody or ADC according to any one of the preceding A# paragraphs, wherein the IgG hinge region of the first polypeptide and / or second polypeptide comprises at least one amino acid modification to avoid O-glycosylation compared to the native lgG3 hinge region.

[0550] A14. The polypeptide, antibody or ADC according to any one of the preceding A# paragraphs, wherein the IgG hinge region is or is derived from a human lgG3 hinge region.

[0551] A15. The ADC according to any one of paragraphs A3 to A14, wherein the antibody is conjugated to the drug by cysteine-based site-specific conjugation through all of the cysteine residues within the IgG hinge region.

[0552] A16. The ADC according to any one of paragraphs A3 to A15, wherein the ADC has a drug- to-antibody-ratio (DAR) of from about 2 to about 12, preferably a DAR of from about 4 to about 8.

[0553] A17. The ADC according to any one of paragraphs A3 to A16, wherein the ADC has a drug- to-antibody-ratio (DAR) of about 2, 4, 6, 8, 10, or 12, preferably a DAR of about 8.

[0554] A18. The polypeptide, antibody or ADC of any one of paragraphs A1 to A17, wherein the variable domain N-terminal of the Fc region in the first and / or first and second polypeptides is a VHH. A19. The polypeptide, antibody or ADC according to any one of paragraphs A1 to A18, wherein the antibody or ADC is stable in serum.

[0555] A20. The polypeptide, antibody or ADC according to any one of paragraphs A1 to A19, for use in a method of therapy or a diagnostic method.

[0556] Other aspects of the present invention, e.g. relating to methods, compositions and nucleic acids are equally applicable to the lgG3 hinge embodiments of the present invention as set forth in this section.

[0557] EXAMPLES

[0558] Materials and Methods

[0559] Expression of recombinant antibodies and VHH-Fc fusion proteins

[0560] Human codon optimized antibody and VHH-Fc sequences comprising engineered and / or heterologous hinge sequences were generated by gene synthesis and cloned into pTWIST expression vector (Twist Bioscience). For recombinant expression of the antibodies and VHH- Fc fusions, Expi293 cells were transfected with ExpiFectamine (ThermoFisher Scientific) using the manufacturer’s protocol. Culture supernatants were harvested from Expi293 producer cell lines after 4-5 days, centrifuged and recombinant proteins were used for further characterization and purification.

[0561] Conjugation of toxin payload to antibodies and VHH-Fc fusion proteins

[0562] To a solution of the antibody / VHH-Fc fusion protein of interest (1-20 mg / ml, 1.0 equiv) in 20 mM phosphate buffer is added a 100 mM EDTA buffer solution to a final concentration of 1 mM. The pH of the solution is adjusted to 7.0-8.0 with 0.5 M disodium hydrogen phosphate. Then a freshly prepared 25 mM solution of TCEP hydrochloride (3.0-6.0 equiv, adjusted for desired DAR value) in 20 mM phosphate buffer, pH 7.0-8.0 is added to the antibody solution and gently mixed. After 60-90 min, a 30 mM solution of the linker-payload (5.0-10.0 equiv, adjusted for desired DAR value) in DMSO is added to the antibody solution. The resulting solution is gently mixed and incubated at 22-37 °C. After 2 h, the reaction is purified and the buffer exchanged using ultrafiltration or gel filtration with 20 mM histidine buffer, pH 6.0 or another suitable buffer to afford the antibody drug conjugate. The drug-to-antibody-ratio (DAR) was analyzed using hydrophobic interaction chromatography or mass spectrometry.

[0563] Western Blot for aggregate detection Western Blot analysis was performed using the cell culture supernatant of the previously cultivated Expi293 cells as sample. Western Blots were performed using the Jess device from BioTechne.

[0564] The supernatant was 1 :10 diluted and applied to the machine according to the manufacturer’s protocol. The separation was performed under non-reducing conditions and the subsequent detection was performed using an anti-human HRP antibody.

[0565] Affinity to the target antigen

[0566] For affinity measurements an Octet HTX device from Sartorius was used.

[0567] Commercially available AHC biosensors were soaked for at least 10 min in kinetic buffer (KB) purchased from the instrument manufacturer. Following a 60 sec baseline in KB, the antibody / VHH-Fc proteins of interest (or corresponding ADCs) were loaded onto the sensors for 300 sec or until a threshold of 0.7-1 nm in response was reached. Following a 120 sec baseline in KB, association to antigen 1 was measured over 600 sec using seven different concentrations of the antigen starting at 50 nM in a 1 :1 serial dilution series down to 0.78125 nM. As a reference, KB without the antigen was measured. The dissociation was acquired over 600 sec in KB. The signal of the reference well was subtracted from the signal of all other biosensors and the signals of these processed data were aligned to the average of the baseline before the association step. For inter-step correction, the data were aligned to the dissociation step and Savitzky-Golay filtering was applied to all curves. Association and dissociations were globally fitted using a 1 :1 Langmuir binding model.

[0568] Avidity to the target antigen

[0569] For avidity measurements an Octet HTX device from Sartorius was used.

[0570] Commercially available Ni-NTA or SAX2.0 biosensors were soaked for at least 10 min in kinetic buffer (KB) purchased from the instrument manufacturer. Following a 60 sec baseline in KB, the His-tagged or biotinylated antigen was loaded for 300 sec or until a threshold of 1 nm in response was reached. Following a 120 sec baseline in KB, association to the Fc- containing protein of interest or the ADC was measured over 600 sec using seven different concentrations starting at 50 nM in a 1 :1 serial dilution series down to 0.78125 nM. As a reference, KB without the test item was measured. The dissociation was acquired over 600 sec in KB. The signal of the reference well was subtracted from the signal of all other biosensors and the signals of these processed data were aligned to the average of the baseline before the association step. For inter-step correction, the data were aligned to the dissociation step and Savitzky-Golay filtering was applied to all curves. Association and dissociations were globally fitted using a 1 :1 Langmuir binding model.

[0571] Affinity to CD64

[0572] For affinity measurements an Octet HTX device from Sartorius was used.

[0573] Commercially available Ni-NTA biosensors were soaked for at least 10 min in kinetic buffer (KB) purchased from the instrument manufacturer. Following a 60 sec baseline in KB, the His- tagged CD64 was loaded for 300 sec or until a threshold of 1 nm in response was reached. Following a 120 sec baseline in KB, association to the antibody / VHH-Fc / ADC of interest was measured over 600 sec using seven different concentrations of the test item starting at 50 nM in a 1 :1 serial dilution series down to 0.78125 nM. As a reference, KB without the test item was measured. The dissociation was acquired over 600 sec in KB. The signal of the reference well was subtracted from the signal of all other biosensors and the signals of these processed data were aligned to the average of the baseline before the association step. For inter-step correction, the data were aligned to the dissociation step and Savitzky-Golay filtering was applied to all curves. Association and dissociations were globally fitted using a 1 :1 Langmuir binding model.

[0574] Affinity to FcRn

[0575] To determine binding to FcRn, SAX2.0 biosensors were soaked for 10 min in KB, followed by a 60 sec baseline in KB. Loading of biotinylated human FcRn was done over 300 sec or until a threshold of 1.5 nm in response was reached. After subsequent quenching in a 100 pg / mL biocytin solution, a baseline for 120 sec in 100 mM sodium phosphate, 150 mM NaCI, 0.05% Tween-20, pH6.0 buffer (KB-pH6) was measured. The Fc-containing test item was diluted in KB-pH6 in a 1 :1 serial dilution from 1600 nM down to 25 nM. Association was measured over 60 sec followed by dissociation in KB-pH6 over 60 sec. The signal of the reference well was subtracted from the signal of all other biosensors and the signals of these processed data were aligned to the average of the second baseline. For inter-step correction, the data were aligned to the dissociation step and Savitzky-Golay filtering was applied to all curves. Association and dissociations were globally fitted using a 1 :1 Langmuir binding model. As FcRn interactions show a heterogeneous binding behaviour to Fc-parts of antibodies, only the first 5 or 10 sec of the dissociation were fitted.

[0576] Dynamic Light Scattering (DLS) and Nano Differential Scanning Fluorimetry (NanoDSF)

[0577] Thermal stabilities (via NanoDSF) and size distribution (via DLS) were investigated using the Prometheus PANTA from NanoTemper. 10 pL of antibodyA / HH-Fc protein or ADC sample was loaded into capillaries. After loading, the capillaries were mounted into the instrument and the DLS analysis was performed. To assess thermal stability, a heat ramp of 1 °C / min from 25°C to 95°C was subsequently applied to all samples. During this process the intrinsic fluorescence of the proteins was measured at 350 nm and 330 nm. The ratio was plotted against the temperature and the first derivative was calculated. Minima and maxima corresponded to the TM values.

[0578] Size Exclusion Chromatography (SEC)

[0579] Aggregation analysis was performed via SEC utilizing an Agilent Infinity II HPLC and a Biozen 1.8 pm dSEC-2, 200 A LC column (300 x 4.6 mm). Flowrates were adjusted to 0.25 mL / min, resulting in approximately 255 bar pressure. As mobile phase 0.2 M potassium phosphate, 250 mM KCI, pH 6.2, 5% acetonitrile was used. Each run took 20 min excluding a 2-3 min wash step between each analysis. 10 pL of the antibody / VHH-Fc fusion protein or ADC test items were applied and detected by absorption at 280 nm.

[0580] EC50 determination

[0581] Antigen-positive or -negative cells were seeded in a 96-well microtiter plate (round button) to a final density of 2x105and incubated in the presence of different concentrations of the antibody / VHH-Fc fusion or ADC for 30 min at 4°C. A concentration range of 90 nM, down to 123 pM was covered. Afterwards, cells were washed twice with FACS-buffer (1x phosphate- buffer saline + 10 mL 0.5 M ethylenediaminetetraacetic acid + 10 mL Fetal Bovine Serum (FBS) and once with 1x phosphate-buffer saline and subsequently incubated with an antihuman APC detection antibody or an anti-toxin AF64 detection antibody for 30 min at 4°C. After another washing step with FACS-buffer, cells were fixated with BD Fixative and analysed with the FACS celesta (BD Biosciences). An excitation laser at 633 nm was used to detect the APC signals. Mean fluorescence intensities (MFI) values were plotted against the concentration of the antibody / VHH-Fc or ADC and the resulting data points were fitted using a non-linear fit, resulting in the EC50 values of the respective antibody / VHH-Fc or ADC.

[0582] FACS internalization

[0583] Antigen-positive cells were seeded in FACS tubes to a final density of 2x105and incubated in the presence of 100 nM or 500 nM VHH-Fc fusion for 30 min at 4°C.

[0584] Afterwards, cells were washed twice with FACS-buffer and once with 1x phosphate-buffer saline and were subsequently re-suspended in 1 mL growth medium. Cells were incubated for either 30 min, 1 h, 2h or 4h at either 37°C or on ice. Cells were washed twice with FACS buffer and once with PBS before an anti-human APC detection antibody was applied for 30 min. After another washing step with FACS-buffer and PBS, cells were fixated with BD Fixative and analysed with the FACS celesta (BD Biosciences). Excitation laser at 633 nm was used to detect the APC signals. The MFI values of the 4°C samples were compared with their respective 37°C counterpart to visualize the internalization-mediated difference in fluorescence intensity.

[0585] Mass spectrometry of unconjugated antibodies

[0586] For intact mass determination, 10 pg of each VHH-Fc fusion / antibody was optionally deglycosylated in the original buffer by overnight incubation with PNGase F at 37°C. A volume corresponding to 1 pg (de-)glycosylated protein was separated on a Waters Acquity l-Class LIPLC by reversed-phase chromatography on a Waters BEH C4 column maintained at 80°C. Proteins were eluted with a linear gradient of 15-30% acetonitrile over 12 minutes, followed by an increase to 95% acetonitrile in 2 min. 0.1 % (v / v) formic acid was used as modifier in all solvents. Mass spectra in the m / z-range of 500-4000 were acquired on a Waters Xevo G2-XS QTOF. Data analysis was performed in ProteinMetrics Inc. Byos software. Raw mass spectra were integrated over the entire elution range and deconvoluted with settings appropriate for the m / z range and instrument resolution. For the annotation of peaks in the deconvoluted mass spectra, homodimers with all possible disulphide-bridges formed and asparagine to aspartic acid converted by de-glycosylation was assumed.

[0587] Serum stability

[0588] To verify serum stability, a 0.5 mg / mL solution of the test item in either PBS (control) or human serum was incubated at either 4°C (control) or 37°C. At certain time points samples were taken and frozen until the analysis of the test item took place.

[0589] Mass spectrometry for DAR determination

[0590] Sample Preparation: Antibody-Drug conjugates were reduced in their original buffer by the addition of Tris(2-carboxyethyl)phosphine hydrochloride to a final concentration of 10 mM and incubation at 40°C for 45 minutes. Reduced samples were directly subjected to LC-MS measurement.

[0591] Liquid Chromatography - High Resolution Mass Spectrometry: A total mass of 1 pg reduced ADC was separated on an Acquity l-Class LIPLC system equipped with a 2.1 x 50 mm Acquity LIPLC Protein BEH C4 column (Waters Inc.) at 80°C. A linear gradient from 10 - 50% acetonitrile in water was run over 7 min with a flow rate of 0.4 ml / min. 0.1% (v / v) formic acid was used as a modifier in all solvents. Proteins eluting from the column were analyzed on a Waters Xevo G2-XS QTOF mass spectrometer operated in positive ion, sensitivity mode. Full scan mass spectra were acquired from 400 to 4500 m / z with Glu-Fibrinopeptide peptide B as a lockmass.

[0592] LC-MS Data Analysis: Raw mass spectra were integrated over the chromatographic peak of the eluting protein and deconvoluted using Byos (Protein Metrics Inc.). Deconvoluted “neutral” mass peaks were annotated using the average molecular weight calculated from the amino acid sequence under the following assumptions: No disulfide bridges formed; Glutamine on protein N-terminus converted to pyroglutamate; C-terminal Lysine clipped. The following variable modifications were considered for the annotation: max. 1 Glycan (GOF, G1 F or G2F); max. 6 payload. A tolerance of 15 Da was allowed for annotation of the deconvoluted masses. The distribution of Drug-Antibody Ratios was determined based on the relative intensity of the deconvoluted mass peaks.

[0593] Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays

[0594] For CDC assays, antigen positive CHO-K1 cells were used as target cells, while antigen negative CHO-K1 cells served as control cells. Target and control cells were incubated with human serum (final concentration 25%) and anti-antigen antibodies at various concentrations ranging from 50,000 ng / mL to 28.9 ng / mL for four hours. An antibody specifically binding to a different, “control target” was used as a positive control for Antigen-negative CHO-K1 cells. CDC activity was determined using the CellTiter-Glo® 2.0 assay, which measures luminescence resulting from ATP-dependent luciferin oxidation, in accordance with the manufacturer's instructions.

[0595] For ADCC assays, luciferase-positive cell line served as the antigen-positive target cell line, while another luciferase-positive cell line served as the antigen-negative control. Human PBMCs from a healthy donor were thawed and pre-cultivated for 48 h prior to the assay and were used as effector cells at E:T ratio of 40:1. Target, control and effector cells were incubated for 24 hours with anti-antigen standard antibody as well as anti-antigen antibodies at various concentrations ranging from 50,000 ng / mL to 0.01 ng / mL. An antibody specifically binding to a different, “control target” was used as a positive control for the antigen negative cells. Specific lysis was quantified by measuring the luciferase activity in the remaining viable tumor cells following the addition of Promega Luciferin Reagent. A decrease in luminescence indicates lysis of target cells, reflecting the efficacy of the antibody in inducing ADCC.

[0596] Cytotoxicity assays

[0597] Target positive and target negative HEK cells were seeded at a density of 5000 and 4000 cells per well in a white 96 well plate. After incubation at 37° for 24 hours, cells were treated with 5x serial dilutions of the ADCs. Cell viability was measured as ATP concentration 72 hours after treatment by adding the CellTiterGlo-Reagent (Promega) and subsequent luminescence measurement using a Clario Star Plus plate reader. Cell viability was analyzed as % of untreated cells. Dose-response curves and curve fitting were performed using GraphPad Prism (9.5.1).

[0598] In vivo stability analysis of the unlabeled antibody and ADC in mice

[0599] The PK of the ADC in serum was evaluated by administering the unlabeled antibody at doses of 10 mg / kg to female Balb / c mice (7-8 weeks old) via the tail vein. The drug solution consisted of unlabeled antibody or ADC in phosphate-buffered saline was administered at a volume of 100 pL. Five minutes, 1 h, 4 h, 8 h, 1 day, 3 days, and 5 days after injection, blood was collected from the vena facialis.

[0600] Measurement of the unlabeled antibody and ADC in serum samples by GyrosELISA

[0601] The ADC and total antibody concentration in serum were determined using the Gyros xPand ELISA method. Data was generated using Gyrolab Generic TK or PK disks. Serum samples were centrifuged for four minutes at 4500 rpm at 4°C and diluted 1 :10 in Reagent E in 96-well plates. A standard curve and controls were prepared by diluting the test items in Reagent E with 10% serum from untreated mice. The standard, controls, and biotin-labeled capture reagent were transferred to a 96-well plate. The same was done with the fluorophore-labeled detection antibody. The master plate was prepared according to Gyrolab Manager specs. The Gyrolab 20HC (TK) or 1000HC (PK) disks were placed in the Gyros xPand ELISA. Design and evaluation were done in Gyrolab Manager and Evaluator.

[0602] In these experiments the antigen was biotinylated as a capture agent and diluted to 0.1 mg / mL in PBS-T. An Alexa647-labeled anti-human IgG antibody (Reagent B of Gyrolab Generic PK kit) was used to detect total antibody in the solution, but it could not distinguish between ADC and unconjugated antibody. Therefore, an anti-toxin antibody was also used to quantify the ADC. This labeled anti-toxin antibody was diluted to 25 nM in Rexxip F. By comparing the two measurements (setup with anti-human vs. anti-toxin), it is however possible to differentiate between the total amount of antibodies and the ADC, and thus to draw conclusions about the stability of the ADC.

[0603] Antibody and ADC nomenclature

[0604] Herein, multiple antibodies, including VHH-Fcfusions, Fab:VHH-Fc bispecifics and VHH:VHH- Fc bispecifics are described, as well as toxin-conjugated ADC versions thereof. For monospecific antibodies, the nomenclature comprises an “AxBy” prefix. This “Ax” determines the antigen, “By” determines the binder, wherein “x” and “y” are numbers. The following infix describes the design of the hinge region, e.g. where “D1” refers to the design 1 and D3 to the design 3 (Table 2). As a suffix, the isotype of the utilized Fc is stated, e.g. IgGI Fc for a Fc portion which is classified as of the lgG1 isotype. For ADCs, but not antibodies, an additional suffix is given, stating the drug-to-antibody ration for the molecule in question. Consequently, the ADC “A1 B3- D3- IgGIFc- DA R8” refers to an ADC specific for antigen 1 , comprising the binder no. 3, exhibits a hinge region according to the design 3, an lgG1-Fc portion and is conjugated with 8 toxins per antibody (DAR of 8). Bispecifcs (bs), due to their binding capabilities to two antigens, exhibit a prefix which is consecutively numbered starting at bs1.

[0605] Table 2 - Infixes refereeing to the hinge region and translation to the corresponding Seq ID NO. Example 1 - Western blot analysis of cell culture supernatant of different VHH-Fc fusions

[0606] The present inventors elucidated for the first time the utilisation of ADCs comprising an engineered IgG hinge region having a predetermined number of cysteine residues to control the DAR. In particular, to create an ADC comprising an engineered IgG hinge region, the present inventors engineered a VHH-Fc fusion comprising an lgG1 Fc region and a heterologous hinge region. The inventors utilised a human lgG2-derived hinge region or a human lgG3-derived hinge region as the heterologous hinge region. Human IgG hinge region sequences were used to minimize immunogenicity. Furthermore, to provide a homogenous ADC product, the inventors modified the lgG3-derived hinge region to replace the serine and threonine residues which are subject to O-glycosylation with alanine residues. The inventors also compared the VHH-Fcs comprising an engineered IgG hinge with conventional VHH-Fcs comprising an lgG1 Fc region and a truncated lgG1 hinge. Hence, a conventional VHH-Fc antibody comprising a truncated IgG 1 hinge as well as antibodies comprising engineered IgG hinge regions were produced.

[0607] The present inventors generated antibodies comprising 5 different VHH domains (A1 B1-5), each specific for the same target antigen, to investigate the effects of the engineered IgG hinge regions regardless of the VHH used or the specificity of the VHHs.

[0608] All 5 VHHs binding to the target antigen were produced transiently in ExpiHEK cells as VHH- Fc fusions comprising either a truncated lgG1 hinge (D1), or an lgG2- (D2) or an lgG3- (D3) derived hinge region, resulting in 15 constructs in total. Thus, the inventors first generated 5 conventional VHH-Fcs comprising an lgG1 Fc region and a truncated lgG1 hinge region for comparison. The present inventors then generated 10 VHH-Fcs comprising an lgG1 Fc region and an engineered IgG hinge region: 5 VHH-Fcs comprising an lgG2-derived hinge region and 5 VHH-Fcs comprising an lgG3-derived hinge region.

[0609] The IgG hinge regions are as follows:

[0610] Truncated lgG1 hinge: DKTHTCPPCP (SEQ ID NO: 12) lgG2-derived hinge: ERKCCVECPPCP (SEQ ID NO: 2) lgG3-derived hinge: PRCPEPKACDAPPPCPRCP (SEQ ID NO: 112)

[0611] The conventional VHH-Fcs (comprising the truncated lgG1 hinge SEQ ID NO: 12) would provide an ADC having a DAR of 4 following cysteine-based site-specific conjugation. The VHH-Fcs comprising an engineered lgG2- or lgG3-derived hinge region would provide an ADC having a DAR of 8 following cysteine-based site-specific conjugation. Thus, the VHH-Fcs comprising an engineered lgG2- or lgG3-derived hinge region provide antibodies which have been optimised for a higher DAR.

[0612] The present inventors then performed analysis of aggregation by Western blot using nonreduced samples. The cell culture supernatants, each containing one of the 15 VHH-Fc fusions, were analysed. Detection was carried out through use of an anti-human antibody.

[0613] By utilizing the lgG1 hinge and the lgG3-derived hinge, all VHH-Fcs were successfully produced and did not show any signs of aggregates, dimers or fragmentation products.

[0614] The lgG2-derived hinge led to aggregates or dimer formation in some constructs. One exception was the VHH-Fc2 variant (A1 B2-D2-lgG1 Fc), which mainly yielded the desired monomer band.

[0615] As such, shown herein are constructs with hinge regions derived from each of lgG2 and lgG3 that are capable of forming monomers. Thus, the VHH-Fc antibodies comprising an engineered IgG hinge region performed comparably to the conventional VHH-Fcs, regardless of the specificity of the VHHs. Moreover, the inventors have shown that modifications to avoid O-glycosylation are tolerated in the hinge regions. As shown below, all of the tested constructs are functional regardless of aggregate or dimer formation.

[0616] Example 2 - Biophysical characterization of VHH-Fc fusions

[0617] To further validate the VHH-Fc fusions comprising an engineered IgG hinge region, the present inventors performed biophysical characterization of the 15 VHH-Fc fusions described above (the 5 conventional VHH-Fcs as well as the 10 VHH-Fcs comprising an engineered IgG hinge regions derived from an lgG2 hinge or an lgG3 hinge).

[0618] Thermal denaturation values (determined by NanoDSF), and SEC purity are depicted for the VHH-Fc fusions below, in Table 3. DLS results were also determined (data not shown).

[0619] Stability analysis of each of the VHH-Fc fusions was carried out using Nano Differential Scanning Fluorimetry (NanoDSF). In general, the stabilities appear mainly mediated by the VHH candidate and are less dependent on the hinge region architecture. High stabilities with TM values of up to 76 °C were observed.

[0620] SEC analysis further confirms that monodispersity is very high for the lgG1 hinge and the lgG3-derived hinge candidates, and shown in some lgG2-derived variants.

[0621] Thus, the biophysical characterizations confirm the results presented above in Example 1. Table 3 - Biophysical characterization of VHH-Fc fusions comparing lgG1-, lgG2- and lgG3-derived hinge regions. SEC purity and thermal denaturation values determined by NanoDSF are depicted for the VHH-Fc fusions.

[0622] Example 3 - Affinity of VHH-Fc fusions To further validate the VHH-Fcs, the present inventors performed affinity determination for each of the 15 VHH-Fcs described above (the 5 conventional VHH-Fcs as well as the 10 VHH- Fcs comprising an engineered IgG hinge regions derived from an lgG2 hinge or an lgG3 hinge).

[0623] An Octet HTX device was used to determine the affinity of the 15 different IgG-derived hinge VHH-Fc fusions to: (i) the target antigen of the VHH-Fcs and, (ii) huCD64. Associations and dissociations were globally fitted using a 1 :1 Langmuir binding model, the results of which are shown in Table 4 below.

[0624] As indicated below, all three hinge regions mediate identical binding to the antigen, underlining that specificity and affinity is not altered upon switching to another hinge sequence. All targetantigen candidates bound the target antigen and CD64 with high affinity. This suggests that, as FcyR binding is not compromised by the engineered hinge sequences and that ADCC and CDC activities were preserved for antibodies comprising the engineered and heterologous IgG hinge regions.

[0625] There appears to be no negative influence upon adaptation of a heterologous hinge for allowing the achievement of higher DARs. Moreover, there appears to be no negative influence upon modification of the hinge to avoid O-glycosylation. Further, comparable results are obtained with 5 different VHHs for each design, i.e. regardless of the specificity of the VHHs. CD64 binding is also shown to be independent from the utilized hinge region. This allows the hinge design to be chosen solely on DAR and not on other properties.

[0626] Table 4 - Affinity of VHH-Fc fusions comprising lgG1-, lgG2 and lgG3-hinges towards antigen 1 and CD64. Affinity to the primary antigen of the VHH-Fcs as well as to CD64 was determined using BLI.

[0627] Example 4 - Cell binding of VHH-Fc fusions

[0628] To further validate the VHH-Fcs, the present inventors analysed cell binding for VHH-Fcs described above comprising the lgG1 hinge and lgG3-derived hinge regions.

[0629] Target positive tumor cells were stained with different concentrations of VHH-Fcs comprising lgG1 hinges and lgG3-derived hinges. Mean fluorescence intensities (MFI) values were plotted against the concentration of the VHH-Fc and the resulting data points were fitted using a non-linear fit, resulting in the EC50 values of the respective VHH-Fc. These values are shown in Table 5 below.

[0630] These experiments revealed a majority of sub-nanomolar to single digit nanomolar EC50 values on target cells. The different hinge regions mediate identical binding to the antigenpositive cells. As such, EC50 is not impacted by optimization of the hinge region for higher DARs (i.e. utilizing a heterologous hinge region), modification to avoid O-glycosylation or the specificity of the VHHs. This further emphasizes that one may choose the hinge design solely on DAR and not on other properties.

[0631] Table 5 - Cell binding of VHH-Fc fusions. Target positive tumor cells were stained with different concentrations of VHH-Fcs and EC50 values were determined.

[0632] Example 5 - Internalization of VHH-Fc fusions

[0633] To further validate the VHH-Fcs, the present inventors assessed internalization for the VHH- Fcs described above comprising lgG1 hinges and lgG3-derived hinges.

[0634] Internalization of VHH-Fcs comprising lgG1 hinges and lgG3-derived hinges into target positive tumor cells was investigated using a FACS-based assay which confirmed fast internalization of all candidates independent from the engineered Hinge region.

[0635] Internalization was studied at four different time points (0.5, 1 , 2, and 4 hours). As a control, candidates were incubated on ice to reduce internalization.

[0636] Internalization was examined by incubation at 37°C followed by FACS analysis. All candidates clearly show strong and rapid internalization, detectable after just 30 min (Figure 2).

[0637] It is clear from Figure 2 that all tested hinge regions mediate identical internalization to the antigen-positive cells. Internalization is not detrimentally impacted by optimization of the hinge region for higher DARs or modification to avoid O-glycosylation, since the different hinge regions tested showed the same internalization for a given VHH. This again emphasizes that one may choose the hinge design solely on DAR and not on other properties.

[0638] Example 6 - Intact mass spectrometry data of VHH-Fc fusion proteins

[0639] The intact masses of deglycosylated VHH-Fcs comprising lgG1 hinges and lgG3-derived hinge regions were determined using mass spectrometry.

[0640] The calculated mass and deconvoluted (experimental) mass are displayed below in Table 6. The intact masses are extremely close in value to the calculated masses. This confirms that the disulphide bridges are closed, as expected, and there is no appearance of mispaired interchain disulphides. A high resolution was achieved, making the presence of unpaired hinge cysteines extremely unlikely. Table 6 - Intact mass data of VHH-Fc fusion proteins. Intact masses of deglycosylated

[0641] VHH-Fc fusion proteins containing IgG 1 hinges and lgG3-derived hinge regions.

[0642] Example 7 - Serum stability of VHH-Fc fusion proteins

[0643] Hinge regions, especially of the lgG3 type, are considered prone for proteolytic cleavage (Baici et al., 1980 DOI: 10.1111 / j.1365-3083.1980.tb00039.x, Truner et al., 1970 DOI: 10.1038 / 225853b0 , Virella and Parkhouse et al., 1971 DOI: 10.1016 / 0019-2791 (71)90478- 2). Cleavage in the hinge region will result in the separation of the VHH, the antigen-specific binding moiety from the Fc portion, which is responsible for a prolonged half-life as well as effector function. Further, as the hinge region represents the location of the toxin conjugation as demonstrated in later examples, cleavage in the hinge region might impact the DAR in an unfavourable manner. Consequently, any cleavage occurring in the antibody, especially within the hinge region, may have a detrimental effect on the efficacy of the ADC. To investigate the stability of the antibodies containing lgG1- and lgG3-derived hinges, antibodies exhibiting the D1 or D3 hinge designs (A1 B3-D1-lgG1 Fc and A1 B3-D3-lgG1 Fc) were incubated in human serum at 37°C for up to two weeks. The human serum contains all proteins, including proteases, found in the serum of live humans and consequently is a suitable source of the proteases that the antibody will encounter in vivo. As a control, the same antibodies were incubated in PBS at 4°C for the same time frame.

[0644] To verify whether the antibody is intact, antigen 1 expressing cells were stained using the serum-incubated antibodies and binding was verified using an anti-human antibody. Only when the hinge region remains uncleaved and intact, the binding of the VHH to the antigen can be verified by detecting the human Fc portion. On target positive CHO cells, no loss of binding was observed over the course of incubation in serum at 37°C, very similar to the binding profile observed in PBS. While the data using antigen 1 expressing tumor cells displayed more background noise, the day one and day 14 samples showed comparable MFI values, underlining the stability of the IgG 1 - and lgG3-hinge antibodies in human serum for a prolonged period of time (Fig. 3).

[0645] Surprisingly, and despite literature sources indicating proteolytic liability of the lgG3-derived hinge, no cleavage was observed over the course of the experiment. These unexpected findings allowed for the further investigation of the heterologous hinge technology for ADC development.

[0646] Example 8 - ADC generation using antibodies targeting antigen 1

[0647] As the VHH-Fc fusion proteins tested in the prior examples exhibited very favourable properties, those were subsequently tested in conjugation experiments in order to generate ADCs. Herein, a Topoisomerase I (Topol) inhibitor payload in conjunction to a thiol reactive moiety was tested as a linker-payload. After partial reduction of the antibody, resulting in opening the inter-chain disulfide bridges, the free cysteines within the hinge sequence were used as conjugation sites. Consequently, the inventors hypothesized that the number of cysteines within the hinge, predetermined by the design of the antibody and the hinge, predetermines the DAR the ADC would achieve. The resulting ADCs were analysed by mass spectrometry to experimentally determine the drug to antibody ratio (Table 7).

[0648] Table 7 - Depiction of the anticipated and achieved DARs of the tested VHH-Fc-based ADCs targeting antigen 1 as determined by mass spectrometry.

[0649] In all cases the anticipated DAR was achieved with very high homogeneity. Here the present inventors demonstrate for the first time that the DAR of a VHH-Fc-based ADC can be stirred to higher values by adopting a heterologous hinge region while simultaneously achieving a highly homogeneous product.

[0650] Example 9 - Characterization of ADCs targeting antigen 1

[0651] The ADCs exhibiting the D3 design generated in the prior experiment represent for the first time ADCs in which the DAR was controlled by utilizing a heterogeneous hinge region. As hinge regions are surface exposed, there is a possibility that conjugations to payloads could influence aggregation behaviour. Furthermore, the special orientation of the binding moieties relatively to one another was altered upon opening the closely packed disulfide bridges and introducing linker-payload molecules might mediate special steric effects. Those alterations could impact the binding properties of the ADC. To verify full functionality of the generated ADCs of the present invention, the biophysical characterization which was performed on the unconjugated parental antibodies were consequently repeated for the ADCs (Table 8).

[0652] Table 8 - Biophysical characterization of VHH-Fc ADCs targeting antigen 1. SEC purity and thermal denaturation values determined by NanoDSF are depicted for the VHH-Fc fusions.

[0653] The conjugation, and the associated reduction of the stabilizing inter-chain disulfide bonds, reduced as expected the overall stability of the ADCs to a minor degree. However, surprisingly, no hinge- or DAR-dependent loss of stability was observed. This indicates that the constructs of the present invention can be further developed for the utilization as ADCs, and that the heterologous lgG3-derived hinge presented by the inventors was not inferior compared to the state-of-the-art hinge of the lgG1 type. Further, analytical SEC revealed high purity for all test items, even for the higher DAR 8 ADCs. Next, the binding properties of the antibodies were evaluated analogous to prior experiments (Table 9).

[0654] Table 9 - Affinity of VHH-Fc-based ADCs targeting antigenl . Affinity to the primary antigen of the VHH-Fcs as well as to CD64 was determined using BLI.

[0655] Again, high affinity binding was verified for all ADCs, with no notable DAR-dependent differences. In some instances, the off-rate was so slow at it exceeded instrument limits. Further, cell binding assays were performed to verify whether the retained binding to the recombinant target protein translated to retained binding to target-positive cells (Table 10). In this experiment, either the antibody-portion of the ADC (using the anti-human detection antibody) or the full ADC (using the anti-toxin detection antibody) was utilized to determine two EC50 values per test item.

[0656] Table 10 - Cell binding of VHH-Fc ADCs on antigen 1 positive cells. Target positive tumor cells were stained with different concentrations of VHH-Fcs and EC50 values were determined.

[0657] In general, a slightly weaker binding of the ADCs in comparison to the unconjugated antibodies was observed. However, again, neither a DAR- nor Hinge-dependent difference in the EC50 values were observed.

[0658] In summary, well behaving ADCs were generated comprising the predetermined DAR of 8 in a highly homogeneous manner by choosing the adequate hinge sequence. This was achieved without compromising on stability, binding affinity or monomer content of the ADC product.

[0659] Example 10 - ADC serum stability

[0660] In Example 7, the inventors verified the stability of the antibodies in human serum and observed that these molecules are surprisingly not prone to proteolytic cleavage. However, in these antibodies the hinge inter-chain disulfides were closed. This might have stabilized the hinge and consequently a hinge which is not structurally constrained by disulfides, as found in the ADCs, might be more prone to proteolytic cleavage. Thus, the inventors strived to verify the serum stability of the conjugated ADC. For this experiment, an incubation time of three weeks was chosen to better reflect the expected half-life these ADCs should have in an in vivo setting. ill After incubation in PBS (4°C) or human serum (37°C) for the given times, the samples were analysed by Gyros ELISA. Either the full protein was detected by capturing with biotinylated antigen 1 followed by detection using an anti-human AF647 antibody, or the full ADC was detected by performing the same capture reaction but detect with an anti-toxin AF647 antibody (Fig. 4).

[0661] The inventors observed no drop in concentration for either the antibody or the toxin detecting measurements. This underlines that the ADC remains fully functional and intact during the incubation in serum: Neither was the hinge sequence cleaved nor was the toxin released from the ADC. These findings were true for the state-of-the-art lgG1 -derived hinge (DAR 4; Fig. 4A) as well as for the heterologous lgG3-derived hinge exhibiting a DAR of 8 (Fig. 4B). Therefore, no DAR or Hinge-dependent effects were observed underlining the suitability of the D3 hinge design for therapeutic development.

[0662] Example 11 - Cytotoxicity assessment of ADCs

[0663] One of the most important properties of ADCs is a target-specific and potent killing of targetpositive cells. Consequently, the inventors investigated cytotoxicity by using antigen 1 positive HEK cells. For the ADC to show its effect, it needs to bind to the antigen, be internalized and allow for toxin release which ultimately kills the target cells. By investigating cytotoxicity, all these parameters are verified simultaneously. Furthermore, by also performing this experiment on target negative cells, the specificity of the ADC was investigated, as only unspecific binding should result in a drop of cell viability in the target negative cells. Further, free Exatecan, a Topoisomerase I inhibitor, was tested along with the ADCs, as well as another DAR 8 ADC which is not specific for antigen 1.

[0664] All ADCs mediated potent, target-specific, dose- and DAR-dependent killing. While Exatecan mediated cell killing in target-positive and -negative cells, only the test items showed specificity for target-positive cells. Target negative cells were only killed at very high concentration, a profile also observed with the non-binding isotype control (DAR 8). The VHH-Fc based ADCs showed a clear dose-dependency where in all cases the DAR 8 ADCs kill with lower IC50 values compared to their DAR 4 counter parts (T able 11 , Figure 5).

[0665] Table 11 - IC50 values of test items on target positive cells.

[0666]

[0667] In summary, the inventors demonstrated that the lgG3-derived heterologous hinge can be utilized to achieve ADCs with elevated DARs compared to the state-of-the-art lgG1 -derived hinge and thereby mediating a more potent cytotoxicity on target positive cells.

[0668] Example 12 - ADCC and CDC activity

[0669] Besides cell killing mediated by the cytotoxic payload conjugated to the antibody, ADCs can further mediate killing in toxin-independent manners. Among these is the antibody-dependent cell-mediated cytotoxicity (ADCC) and the complement-dependent cytotoxicity (CDC). To investigate these properties, ADCC and CDC assays were performed with the unconjugated antibodies as well as with the ADCs exhibiting D1- and D3-design hinges. In either case, the ADCC and CDC activities were confirmed, underlining that conjugation to the payload does not abolish these antibody-dependent effector functions for either the lgG1- or the lgG3- derived hinges.

[0670] Example 13 - Antibody PK and ADC stability in vivo

[0671] The inventors strived to investigate whether the modification of the hinge resulted in altered pharmacokinetic behavior. Consequently A1 B3-D1-lgG1 Fc and A1 B3-D3-lgG1 Fc were tested in PK studies and serum samples collected at various time points. To ensure the accuracy of the results, the LLOD (lower limit of detection) and LLOQ (lower limit of quantification) of the GyrosELISA setup, which was utilized to quantify the levels of both antibodies, are also included (Figure 6). The concentration of both antibodies, A1 B3-D1-lgG1 Fc and A1 B3-D3- IgGI Fc, in serum over time exhibits a nearly identical profile. This indicates that the modification of the hinge region does not affect the PK profile of the antibodies in any way. As the detection setup in this experiment is based on antigen 1 binding of the VHHs and detection using the Fc, it further proven that no cleavage of the hinge region occurs in vivo. In the next study, the inventors utilized the ADC versions of both antibodies, exhibiting a DAR of either 4 or 8 for in vivo experiments (Figure 7).

[0672] The objective of this study was to analyze the stability of the antibody-drug conjugates (ADCs) A1 B3-D1-lgG1 Fc-DAR4 and A1 B3-D3-lgG1 Fc-DAR8. To this end, the concentration of total antibodies (using an anti-human antibody for detection) and the concentration of the ADC (using an anti-toxin antibody for detection) were measured. The results of the present study demonstrate a clear correlation between the proportion of ADCs and the total antibody proportion in the serum. The latter is present in greater concentration between time points of 0.1 and 8 hours. However, at later time points, the concentration of ADC in the serum declines slightly in both constructs. This highlights that the D3-hinge containing antibody was not deconjugating in vivo and forms a stable ADC comparable with the state-of-the-art D1 construct.

[0673] Example 14 - Characterization on VHH-Fc fusion proteins against a second antigen

[0674] The promising results of the antigen 1 binding antibodies and ADCs encouraged the inventors to verify their protein engineering approach on a further set of VHHs binding to a second, different antigen (antigen 2). Analogously, standard lgG1-derived hinge sequences (D1) were used as controls as well as lgG3-derived hinges (D3) allowing for higher DARs in combination with 5 different VHHs. The proteins were successfully produced in high yields and were analysed biophysically after purification (Table 12).

[0675] Table 12 - Biophysical characterization of VHH-Fc fusion proteins targeting antigen 2. SEC purity and thermal denaturation values determined by NanoDSF are depicted for the VHH-Fc fusions.

[0676] Once again, the differences in SEC purity and thermal stability are exclusively mediated by the binding moieties with no notable differences between the hinge designs. The same was true for antigen affinity to the recombinant protein (Table 13) and to antigen 2 positive cells (Table 14)

[0677] Table 13 - Affinity of VHH-Fc fusions targeting antigen 2. Affinity to the primary antigen of the VHH-Fcs determined using BLI.

[0678] Table 14 - Cell binding of VHH-Fc ADCs targeting antigen 2. Target positive tumor cells were stained with different concentrations of VHH-Fcs and EC50 values were determined.

[0679] In summary, the inventors observed with a second set of binders - targeting a different antigen than had previously been assessed - that the hinge designs, capable of different DARs when conjugation is performed, behave identically on the protein level, and that the variable sequences are determining the properties of the antibody rather than the chosen hinge region. This further supports a concept of the present invention of basing the hinge design on the anticipated DAR and not on other parameters, thus allowing for a high flexibility during drug development.

[0680] Example 15 - Characterization on VHH-Fc fusion proteins against a third antigen

[0681] The promising results of the antigen 1 and 2 binding antibodies and ADCs encouraged the inventors to verify their observations by using yet a third set of VHHs targeting a third different antigen (antigen 3). Herein, nine different VHHs were tested in the VHH-Fc fusion format either exhibiting a conventional lgG1-derived hinge (D1), or the engineered lgG3 derived hinge (D3) as tested in prior experiments. After successful production of these antibodies, biophysical characterizations were performed (Table 15).

[0682] Table 15 - Biophysical characterization of VHH-Fc fusions targeting antigen 3. SEC purity and thermal denaturation values determined by NanoDSF are depicted for the VHH-Fc fusions.

[0683]

[0684] In the presented set of binders, the TM values between both hinge designs again does not differ in a significant manner. While some exhibited lower purity, there was no clear trend between both hinge designs observable. While the lgG1 -derived hinge yielded higher purity using the A3B1 VHH compared to the lgG3-derived one, the opposite effect was observed for the A3B7 binder. Consequently, the biochemical properties of the VHHs themselves were responsible for the behaviour of the antibody in any given format, rather than the employed hinge sequence.

[0685] For antigen 3, affinity measurements were performed either in the affinity setup, where the bivalent antibody is immobilized and the monomeric antigen is used as analyte, or in the avidity setup, where the antigen is immobilized, and the VHH-Fc is used as analyte (Table 16). As the lgG3-derived hinge is longer in sequence, an altered affinity could be expected, especially in the avidity setup. These changes in binding based on the hinge sequence are known in the art (Chiu et al., 2019 DOI: 10.3390 / antib8040055, Roux et al., 1998 PMID: 9780179).

[0686] Table 16 - Affinity and avidity measurements of VHH-Fc fusion binding to antigen 3.

[0687] Affinity and avidity to the primary antigen of the VHH-Fcs determined using BLI.

[0688] While the avidity experiments in general resulted in high affinity values, as expected, no differences based on the hinge design was observed. Especially in the avidity setup, the off- rates observed surpassed instrument limits. These findings further underlined the modularity of the heterologous hinge approach. This was further underlined by determination of the EC50 values on antigen 3 positive tumor cells (Table 17).

[0689] Table 17 - Cell binding of VHH-Fc fusions on antigen 3 positive cells. Target positive tumor cells were stained with different concentrations of VHH-Fcs and EC50 values were determined.

[0690]

[0691] Also in this set of binders the inventors strived to verify the successful closure of the interchain disulfide by mass spectrometry. (Table 18). As for antigen 1 before, the intact mass was confirmed and no unpaired cysteines in the hinge region were detected. This further highlighted the suitability of these designs for therapeutic antibody and ADC development.

[0692] Table 18 - Intact mass data of VHH-Fc fusion proteins targeting antigen 3. Intact masses of de-glycosylated VHH-Fc fusion proteins containing lgG1-derived hinges and lgG3-derived hinge regions.

[0693]

[0694] Example 16 - ADC generation targeting antigen 3

[0695] As for antigen 1 , a selected set of antibodies exhibiting the lgG3-derived hinge (D3) targeting antigen 3 were tested in conjugation experiments to Topoisomerase I inhibitors via thiolreactive coupling. Mass spectrometry was utilized to determine the DAR of the resulting ADCs. In all cases, the anticipated DAR of 8 was achieved with very high homogeneity (Table 19).

[0696] Table 19 - Depiction of the anticipated and achieved DARs of the tested VHH-Fc fusions targeting antigen 3 as determined by mass spectrometry.

[0697] In all cases the DAR of 8 was achieved with high homogeneity similar as observed for the antigen 1 targeting ADCs before. As state-of-the-art lgG1-derived VHH-Fc fusions are only capable of a DAR of 4, the inventors have now shown with a wide range of different binders that the anticipated DAR of 8 can reliability be achieved with a high homogeneity of up to 100% in the final ADCs when the heterologous hinge approach is utilized.

[0698] Example 17 - ADC characterization targeting antigen 3

[0699] The generated ADCs against antigen 3 were characterized for their biophysical properties. The fluorescence of the toxin interfered with the determination of the exact melting temperatures as it overlapped with the intrinsic fluorescence of the protein that was measured during unfolding, and consequently approximate values observed by eye based on the raw data were used for certain datapoints (marked as * in Table 20).

[0700] Table 20 - Biophysical characterization of VHH-Fc ADCs targeting antigen 3. SEC purity and thermal denaturation values determined by NanoDSF are depicted for the VHH-Fc fusions. While the ADCs exhibit notable thermal stabilities, also favourable aggregation profiles were observed. Furthermore, binding affinities were determined as with the unconjugated antibodies before in an affinity-based and an avidity-based setup (Table 21).

[0701] Table 21 - Affinity of VHH-Fc ADCs targeting antigen 3. Affinity and avidity to the primary antigen of the VHH-Fc ADCs determined using BLI.

[0702] For verification of binding to antigen 3 positive tumour cells, FACS experiments were conducted. The detection of the bound ADC was performed either via an anti-human antibody, detecting the antibody part of the ADC, or via an anti-toxin antibody, detecting the full ADC when bound to the antigen of interest. For both detection methods EC50 values were determined (Table 22). Comparable EC50 values between both detection methods were observed.

[0703] Table 22 - Cell binding of VHH-Fc ADCs targeting antigen 3. Target positive tumor cells were stained with different concentrations of VHH-Fcs and EC50 values were determined.

[0704] In summary, the inventors demonstrated with a different set of binders, that ADCs with a DAR of 8, generated by utilizing a heterologous lgG3-hinge, behave favourably in conjugation, stability, purity and binding assays. These results further underlined the modularity of the approach and emphasizes that the hinge regions of ADCs of the present invention can be solely chosen on the basis of the anticipated DAR, without compromising in other aspects.

[0705] Example 18 - Cytotoxicity assessment of ADCs targeting antigen 3

[0706] To verify the full functionality of the ADCs targeting antigen 3, cytotoxicity experiments utilizing antigen 3 positive cells were conducted by the inventors. As controls, a DAR 0 (unconjugated antibody) control was included as well as a non-binding DAR 8 ADC control. The experiment was independently performed two times and the mean values as well as the standard deviation of both independent experiments are depicted for target positive cells in figure 8 and table 23.

[0707] Table 23 - IC50 values of antigen 3 targeting ADCs. The IC50 value states the concentration of ADC where the viability reached 50%.

[0708] Sub-nanomolar IC50 values were achieved for all ADC, underlining the potency of these candidates. Additionally, the specificity was investigated by testing the cytotoxic effects using antigen 3 negative cells (figure 9). No unspecific killing events at relevant concentrations were observed. In summary, efficient and target-specific kill was observed for all ADCs. While target negative cells showed only an expected drop in viability at extremely high ADC concentrations, target positive cells were killed in a dose-dependent manner with sub-nanomolar IC50 values. This underlines that the inventive approach to use heterologous hinge regions to generate ADCs which mediate efficient killing is not limited to a particular target antigen (e.g. antigen 1), but represents a platform solution that is transferable to other antigens.

[0709] Example 19 - Bispecific Fab:VHH-Fc fusion comprising heterologous hinge regions

[0710] Above, the present inventors demonstrated the suitability of heterologous hinge-Fc pairings and lgG3-derived hinges to generate VHH-Fc fusion ADCs with DARs higher than the conventional design comprising an lgG1-derived hinge region.

[0711] To further evaluate the platform and to broaden the potential application of this approach, the inventors aimed to generate bispecific antibodies exhibiting modified hinge regions according to the present invention. Thus, bispecifics were constructed where one binding moiety was a Fab fragment, derived from an lgG1 antibody, and the second binding moiety was a VHH. Both binding moieties were fused to the N-terminal end of a hinge region, which itself, as in prior constructs, was fused to the Fc of an lgG1 antibody. The heterodimerization of the Fc part was enforced by utilizing the ‘knobs into holes’ technology. As Fabs exhibit an inter-heavy- light-chain disulfide bond, in these constructs the potential DAR is not only mediated by the inter-heavy-chain disulfide in the hinge region. An overview of the potential DARs mediated by the Hinge region and the overall possible DAR, which is the DAR of the Hinge +2 due to the Fab fragment, is depicted in Table 24.

[0712] Table 24 - Overview of the designs utilized in the test items and the expected DARs can be achieved with these bispecific antibodies. The bispecific antibodies were successfully produced in mammalian cells and after purification biophysically analysed (Table 25).

[0713] Table 25 - Biophysical characterization of Fab:VHH-Fc bispecific antibodies. Thermal denaturation values determined by NanoDSF are depicted for the bispecific antibodies.

[0714] As expected, no hinge-dependent differences in these bispecific antibodies were revealed and very good thermal stabilities were observed. Further, the binding properties of the bispecific antibodies to their respective antigens were elucidated (Table 26).

[0715] Table 26 - Affinity of Fab:VHH-Fc bispecific antibodies. Affinity to both antigens were determined using BLI. As observed before, the binding affinities to the antigens are independent to the hinge regions. Further, the binding to Fc receptors were elucidated, showing that neither CD64 nor FcRn binding were compromised by utilizing an lgG3-derived hinge (Table 27)

[0716] Table 27 - Affinity of Fab:VHH-Fc bispecific antibodies towards Fc receptors. Affinity to

[0717] CD64 and FcRn were determined using BLI.

[0718] One of the most important features bispecific antibodies must fulfil is the capability to engage both antigens at the same time. For that, the distance of the binding moieties to one another is of importance. To verify whether the simultaneous engagement of both antigens using the engineered hinges is possible, the inventors performed a sandwich-like BLI assay in which one antigen was immobilized, followed by the association of the bispecific antibody and subsequent association of the second antigen. Through this assay, the inventors clearly showed that the engineered hinge region mediates sufficient flexibility that both antigens can be simultaneously engaged (data not shown).

[0719] Herein, the inventors demonstrated that even in more complex bispecific antibodies, in which the two different binding moieties (a Fab and a VHH) were asymmetrically fused to the engineered hinges, no negative impact on the overall stability of the antibody or impairment of binding were observed. Simultaneous binding to the antigens of the bispecific was also confirmed. This further underlined the modularity of the engineered and heterologous hinge regions presented herein and further opens the possibility of this technology to be used in the construction of bispecific ADCs with tailor-made DARs.

[0720] Example 20 - Bispecific VHH:VHH-Fc antibodies comprising heterologous hinge regions After the encouraging results of Fab:VHH based bispecific antibodies, the inventors strived to further test the heterologous hinge platform using two different VHHs in a bispecific approach. Therefore, VHHs targeting antigen 1 , antigen 3 or antigen 6 were fused via an lgG1 - or lgG3- derived hinge (either D3 or D11) to the lgG1 Fc exhibiting the well-known ‘knobs into holes’ mutations in order to generate bispecific antibodies. These molecules expressed well and were analyzed for their biophysical properties (Table 28).

[0721] Table 28 - Biophysical characterization of VHH:VHH-Fc bispecific antibodies. Thermal denaturation values determined by NanoDSF are depicted for the bispecific antibodies.

[0722] While in these antibodies differences in purity and stability were revealed, no clear trend towards an lgG1- or lgG3-hinge was observed. However, most antibodies exhibited high thermal stability as well as sufficient purity after single step purification. Consequently, these molecules were further investigated for their binding affinities towards their respective antigens (Table 29).

[0723] Table 29 - Affinity of VHH:VHH-Fc bispecific antibodies. Affinities to both antigens were determined using BLI.

[0724] No differences in binding based on the utilized hinge designs were observed. Further binding profiles towards Fc receptors were determined, revealing no hinge-dependent differences in these bispecific antibodies (Table 30).

[0725] Table 30 - Affinity of VHH:VHH-Fc bispecific antibodies towards Fc receptors. Affinity to

[0726] CD64 and FcRn were determined using BLI.

[0727] To verify that these antibodies were able to mediate simultaneous binding to both antigens, a sandwich-like BLI assay was conducted. After immobilization of the first antigen and subsequent association of the bispecific antibody, the second antibody was applied. The association of the second antibody is dependent on the binding of the bispecific antibody to both antigens at the same time. Simultaneous binding to both antigens by the bispecifics was confirmed (data not shown). The inventors demonstrated that the heterologous hinges are able to mediate such a binding mode. As the hinge region is responsible for the spatial orientation of the binding moieties, this unexpected finding further supports the heterologous hinge platform presented by the inventors herein for bispecific ADC development.

[0728] Example 21 - ADC generation using bispecific VHH:VHH-Fc fusions comprising heterologous hinge regions

[0729] The bispecific antibodies exhibiting a D3 hinge region, capable of a DAR 8, were tested in conjugation experiments using a Topoisomerase I inhibitor payload in conjunction with a thiolreactive linker moiety. In all cases, the antibodies were successfully conjugated to the toxins achieving the anticipated DAR values (Table 31). Table 31 - Depiction of the anticipated and achieved DARs of the tested bispecific VHH- Fc fusions as determined by mass spectrometry.

[0730] This further highlights the suitability of the heterologous hinge approach for the design of bispecific ADCs with tailor-made DARs.

[0731] Example 22 - lgG2 and lgG4 Fes in combination with lgG1 or lgG3 hinges

[0732] To this point, the inventors tested lgG3-derived hinges, using the IgG 1 -derived counterpart as reference, in combination with lgG1 Fes. To broaden the application of heterologous hinge regions, next the inventors analyzed the lgG3-derived hinge design 3 (D3) in the context of lgG2 and lgG4 Fc regions. To allow for comparison with prior data, the A1 B3 binder was used again.

[0733] The antibodies were successfully produced and subsequently tested for a variety of biophysical parameters (Table 32).

[0734] Table 32 - Biophysical characterization of lgG3 hinge-lgG2 / 4 Fc paired constructs.

[0735] Thermal denaturation values were determined by NanoDSF. High purities after single step protein A purification were achieved. The determined melting temperatures were highly similar to the prior tested lgG1 Fc fused with either the lgG1- or lgG3-derived hinge. Analogously, binding experiments revealed no change in binding behavior upon switching the Fc isotype (Table 33).

[0736] Table 33 - Affinity of VHH-Fc exhibiting lgG2 or lgG4 Fc. Affinity to the primary antigen of the VHH-Fc and the FcRn as determined using BLI and binding to antigen-positive cancer cells.

[0737] Further, cell binding experiments confirmed a single digit nanomolar EC50 value, comparable to prior data.

[0738] In the antibody field, Fc isotypes are often chosen based on the anticipated mode of action. E.g. lgG2 and lgG4 Fes are naturally silenced and elicit less ADCC and CDC activity compared to their IgG 1 counterpart. This example underlines that, with the constructs of the present invention, the Fc isotype can be chosen solely on the anticipated effector functions and can be combined with a hinge region in order to achieve the DAR that is envisioned for the final ADC. This modularity does not come with any negative implications on purity or antigen binding.

[0739] Example 23 - Characterization of VHH-Fcs capable of a wide range of DARs (2-12) using lgG1-, lgG2-, lgG3- and lgG4-derived hinge regions

[0740] The inventors proved that the lgG3-derived hinges tested to this point (D3, D9, D10, D11) can be utilized in a modular manner. These hinges were truncated in a way to exhibit a predetermined number of inter-chain disulfide to allow for the anticipated DAR. E.g. for D3 the hinge exhibits a DAR of 8 by utilizing the four inter-heavy-chain disulfides. In the next steps, the inventors strived to demonstrate the modularity of this concept by utilizing modified hinge region from IgG 1 -, lgG2-, lgG3- and lgG4-derived hinge sequences to facilitate a wide range of DARs from as low as 2 to up to 12. To achieve this very unique feat, the inventors applied their hinge truncation and PTM modification approach to multiple hinges.

[0741] For the anticipated DAR of 2, the inventors used the lgG1 hinge, which exhibits two inter- heavy-chain disulfides, as a start point and exchanged each of the cysteine solitary with either a Serine (or Alanine) (D4, D5).

[0742] A DAR 4 version of a VHH-Fc ADC is state-of-the-art and utilizes the lgG1 hinge region. However, the investors investigated complementary approaches using a modified lgG2 hinge (D6), a modified lgG3 hinge (D7) or a modified lgG4 hinge (D8). In D6 the two most N-terminal Cys residues were replaced, as those are involved in the different disulfide isoforms described for lgG2 antibodies. In D7 the lgG3 hinge was truncated to comprise the anticipated two interchain disulfides and one additional mutation to circumvent posttranslational modifications, e.g. O-glycosylation. D8 carried the well-known S228P mutation which circumvents Fab-arm exchange in lgG4 antibodies.

[0743] When utilizing a human hinge region, higher DARs can be achieved by utilizing modified lgG3 hinges. By using a variety of truncated and PTM-optimized versions, the inventors constructed D9 (capable of DAR 6), D10 (capable of DAR 10), D11 (capable of DAR 12).

[0744] Antibodies, exhibiting these hinge designs in combination with the A1 B3 binder, were produced and purified. Surprisingly, as for the D3 design before, no hinge-dependent effects were observed for any antibody in terms of overall stability or monomer content after purification (Table 34)

[0745] Table 34 - Biophysical characterization of VHH-Fc antibodies exhibiting a variety of engineered and heterologous hinge regions. Thermal denaturation values determined by NanoDSF are depicted. Further, the inventors unexpectedly also observed no difference in binding affinity to the antigen 1 or even CD64 (Table 35).

[0746] Table 35 -Affinity of VHH-Fc exhibiting a variety of engineered and heterologous hinge regions. Affinity to the primary antigen of the VHH-Fc and of CD64 were determined using BLI.

[0747] Cell binding experiments using antigen 1 positive tumor cells revealed EC50 values of approximately 1 nM. While slight differences were observed, no clear trend in dependency of the length or isotype of the hinge sequence could be made (Table 36).

[0748] Table 36 - Cell binding of VHH-Fc exhibiting a variety of engineered and heterologous hinge regions. Target positive tumor cells were stained with different concentrations of VHH- Fcs and EC50 values were determined. In the next step, intact mass spectrometric measurements of the non-deglycosylated antibodies were performed. As expected for IgG-Fc containing antibodies, different glycosylation patterns were observed. The delta mass in Da of these antibodies to the calculated masses are depicted in Table 37.

[0749] Table 37 - Mass error of intact molecules in relation to the calculated mass of VHH-Fc exhibiting a variety of engineered and heterologous hinge regions.

[0750] The antibodies formed the anticipated dimers and low delta masses suggest that no modification occurred.

[0751] Herein, the inventors tested the D4 and D5 versions (SEQ ID NOs: 8 and 9) wherein X = S. These hinges are lgG1-derived and are consequently not heterologous hinges in the presented context. In these two designs additional O-glycosylations within the hinge region were observed (data not shown). By replacing the X to A in D5 (SEQ ID NO: 9), this modification was avoided (data not shown). No modifications in the hinges comprising the designs D6-D11 were observed.

[0752] Taken together, these experiments surprisingly demonstrated that the hinge region of an antibody can be exchanged to a truncated or otherwise modified sequence heterologous to the Fc and still yield a fully functional fusion protein. These hinge modifications do not influence cell or protein binding, nor does it affect overall stability or purity of the antibody.

[0753] Example 24 - ADC generation using VHH-Fcs capable of DAR 2-12 using lgG1-, lgG2-, lgG3- and lgG4-derived hinge regions As the inventors were able to produce antibodies exhibiting a variety of heterogeneous hinge regions, they strived to investigate whether the produced variants would be suitable for ADC generation. Consequently, a Topoisomerase I inhibitor payload was conjugated to the partially reduced antibodies via thiol-reactive chemistry as described before. The DAR for the resulting ADCs were verified by mass spectrometry (Table 38).

[0754] Table 38 - Depiction of the anticipated and achieved DARs of the tested VHH-Fc fusions exhibiting a variety of engineered and heterologous hinge regions as determined by mass spectrometry.

[0755] With high homogeneity, the inventors were able to produce ADCs with a variety of DARs. From low DARs such as 2 to very high DARs such as 12 the inventors showed that the heterogeneous hinge approach provides a reliable conjugation profile. This proves that anticipated DARs are easily achieved in the present invention, by means of state-of-the-art conjugation methods.

[0756] Example 25 - Characterization of ADCs exhibiting lgG1 lgG2-, lgG3- and lgG4-derived hinge regions

[0757] Next the inventors characterized the prior generated ADCs for their biophysical profiles. The fluorescence of the toxin made exact melting temperature determination complicated due to interference with the intrinsic fluorescence of the protein which is measured during heat- induced unfolding. Consequently approximate values were used for certain datapoints (marked as * in Table 39) Table 39 - Biophysical characterization of VHH-Fc ADCs exhibiting a variety of DARs. Thermal denaturation values determined by NanoDSF and purity assessment was done by SEC.

[0758] Interestingly, even higher DAR ADCs generated by using the technology presented herein kept their unchanged thermal stability and purity in comparison to the state-of-the-art lgG1 - hinge tested in Example 9. Further, these results indicate that even higher DAR (e.g. DAR10, DAR12) ADCs exhibit no significantly elevated aggregation.

[0759] Affinity measurements of the ADCs to antigen 1, CD64 and to FcRn also showed no DAR dependent effect (Table 40).

[0760] Table 40 - Affinity of VHH-Fc ADCs exhibiting a variety of DARs towards antigen 1 and Fc receptors. Affinity to antigen 1 , CD64 and FcRn were determined using BLI.

[0761] Analogously, cell binding profiles were quite similar among the tested ADCs with EC50 values of around 1-2 nM (Table 41). This once again underlines that the hinge and DARs tested herein do not influence the binding properties of the ADC.

[0762] Table 41 - Cell binding of VHH-Fc ADCs exhibiting a variety of DARs. Target positive tumor cells were stained with different concentrations of VHH-Fc ADCs and EC50 values were determined.

[0763] In summary, ADCs with a wide range of DARs were generated by utilizing heterologous hinge regions. The DAR anticipated for these ADCs was predetermined by the sequence of the chosen hinge. The inventors not only showed that these DARs were reached in a homogeneous manner, but they also further proved that the antibodies behaved very comparably in biophysical properties which are crucial for development of therapeutics. Among those parameters were monomer content and binding capabilities.

[0764] Example 26 - Cytotoxicity assay of ADCs exhibiting lgG1-, lgG2-, lgG3- and lgG4- derived hinge regions and a variety of DARs.

[0765] Lastly, the inventors aimed to verify the cytotoxicity of the ADCs generated in the previous example. Again, they tested antigen 1 expressing HEK cells, as well as target negative HEK cells, with a range of ADC concentrations for dose-dependent cytotoxicity. For comparison, the A1 B3-D3-lgG1-DAR8 ADC from Example 11 was tested again. Further, an unconjugated VHH-Fc, namely A1 B3-D3-lgG1 was tested as a negative control. A non-binding DAR 8 ADC was further included as an unspecific control (Figure 10).

[0766] The inventors observed a clear cytotoxicity towards target positive cells. Further, it was obvious that high DARs mediated lower IC50 values and lower bottom values. The latter is defined as the minimal remaining viability that can be observed (Figure 11)

[0767] From these results it was evident that higher DARs resulted in lower IC50 values and also lower bottom values. T o verify that this effect is not mediated solely by using higher DAR ADCs but the higher DAR in combination with high specificity, the experiment was repeated on target negative cells (Figure 12). In this experiment, irrespective of the DAR, no killing in relevant concentrations was observed and only very weak effects at extremely high concentrations were visible.

[0768] With these results the inventors for the first time showed clear differences between the ADCs, namely that higher DARs mediate higher potency in cytotoxic experiments. However, based on the addressed antigen and the anticipated indication one might want to strive for lower DARs in order to circumvent on-target off-tumor toxicities. In other cases where highly tumor- selective antigens are addressed, a high DAR might result in a more efficacious drug. In any case, an optimal DAR needs to be chosen for every ADC. Herein, the inventors demonstrate a straightforward and novel engineering approach where the antibodies can be adapted with heterologous hinge / Fc region pairings to allow to reach any anticipated DAR. As all other properties of the antibodies remain substantially unaffected with the approach of the present invention, including stability, aggregation behavior and binding affinities, the decision on the DAR can be solely made on the biological background, without being restricted to the state- of-the-art hinges and the associated DARs mediated by those hinges, while also achieving a highly homogeneous ADC product.

[0769] Discussion

[0770] The present inventors recognized limitations in the art regarding providing ADCs where the DAR can be selected for, and still provide an ADC with acceptable in vivo properties (e.g. stability) and obtainable in a substantially homogenous population. In response, the present inventors came up with a straightforward invention that allows to achieve tailor-made DAR values without compromising on any other property of the antibody or ADC. The present inventors have generated an antibody format that enables the control (or predetermination) of the DAR of an ADC by designing engineered IgG hinge regions comprising a predetermined number of cysteine residues. This entirely novel class of antibodies comprising engineered IgG hinge regions provides a functional antibody format that was produced as efficiently as a conventional antibody format. The approach of the present inventors can also solve concerns of immunogenicity (by utilizing human sequences) and concerns associated with free thiol groups (by using paired cysteines).

[0771] It was further demonstrated that modifications to avoid O-glycosylation within the engineered IgG hinges were well tolerated. The VHH-Fc constructs comprising such modifications were functional, and would be expected to provide a homogenous ADC product suitable for large- scale manufacture for clinical use.

[0772] FACS experiments showed that all of the tested VHH-Fcs comprising engineered IgG hinge regions were able to bind to target positive cells. This underlines the full functionality of the VHH-Fcs.

[0773] Thus, this work provides new antibodies that can be designed to have the desired DAR, that provide a homogenous ADC product and that have preserved or elevated ADC properties, including stability, cell binding, internalization compared to conventional antibodies.

[0774] Further, the inventors demonstrated the antibodies exhibiting the engineered lgG3 hinges are stable in human serum (Example 7). This was unexpected as, since the sixties, antibody hinge regions are considered liable for proteolytic cleavage. In the serum stability studies herein it was demonstrated that surprisingly the lgG3-derived hinge containing antibodies are stable for up to two weeks in serum without observing proteolytic cleavage. This is a prerequisite for development as a therapeutic molecule and was an unexpected finding and contradicts what was expected based on literature.

[0775] By utilizing standard methodologies, namely the partial reduction of the antibody by TCEP followed by conjugation of the linker payload molecule via thiol-reactive moieties, the inventors demonstrated successful ADC generation (Example 8). And surprisingly, the anticipated DARs were exactly achieved in a highly homogeneous manner. Therefore, the inventors did not only show for the first time a VHH-Fc ADC with a DAR of over 4 mediated by the hinge is possible, but also the general proof of concept of the heterologous hinge technology. The subsequent examples (Examples 9-13) showed that the DAR 8 molecule behaved very similarly to the state-of-the-art DAR 4 ADC while mediating more efficient cell killing. Subsequently, the inventors showed that this is not antigen specific and repeated the most critical analysis with a plethora of VHHs targeting antigen 2 and 3 (Examples 15-18) which clearly proved the modularity of the approach. This modularity was further underlined as the inventors demonstrated that the heterologous hinge technology is compatible with Fab:VHH and VHH:VHH bispecifics (Examples 19-21) and also with Fc regions from other isotypes (Example 22). Especially given the results achieved with the Fab:VHH bispecifics, it can be assumed that bispecific Fab:Fab molecules as well as monospecific IgGs are compatible with the herein presented technology as well.

[0776] After the inventors established that the hinge region of an antibody can be exchanged to a heterologous sequence which allows for a precise DAR value in a homogeneous manner, the inventors went one step further and used a plethora of hinge sequences from all four human IgG isotypes to generate ADCs exhibiting DARs as low as 2 to as high as 12. In all measured parameters the antibodies behaved identical except in cytotoxicity experiments, where a higher DAR mediated a more efficient cell killing (Examples 23-26). It must be noted that the design 11 (D11) which mediated a DAR of 12 is also a truncated and PTM modified version of the lgG3 hinge. Furthermore, using the technology described in this application ADCs with DARs of 14, 16, 18 and so forth are easily achievable as well by amending the hinge sequence accordingly.

[0777] Very surprisingly, in all experiments conducted by the inventors, no differences in cell or protein binding were observed when different hinge regions were combined with different VHH or Fab moieties. It has previously been reported, that different hinges and antibody isotypes may affect the binding properties of an antibody (Chiu et al. Antibodies (Basel) 2019 8(4):55 doi: 10.3390 / antib8040055; Roux et al. J Immunol 1998 161(8):4083-90, PMID: 9780179). This however was not observed in any cell-free or cell-based binding assay, independent on whether the ADC or its unconjugated parental molecule was examined.

[0778] Further, it was very surprising that no differences in stability were observed when a VHH was combined with multiple different hinge regions. Chiu et al., 2019 argued in their review article that human IgG hinges vary significantly in number of residues and disulfide bridges and that the choice of these parameters “contributes to the overall stability of the antibody’ (id). Unexpectedly, however, the inventors observed that thermal stability is nearly exclusively mediated by the variable regions, as the antibodies exhibiting an lgG4- or lgG3-derived hinge had very similar melting temperatures to their lgG2- or lgG1-derived counterparts (Table 34). While conjugation of the antibody to a toxin reduced the thermal stability for all antibodies, surprisingly the inventors did not observe hinge mediated effects, allowing them to choose the hinge region solely based on the anticipated DAR without compromising of thermal stability.

[0779] By utilizing a heterologous hinge technology, the inventors avoid the half-life impairing effect of the lgG3 Fc as demonstrated in Example 13. And further no proteolytic cleavage was observed in any of the antibodies or ADCs described herein. Scientists skilled in the art would expect that the literature-described problems with substantial antibody hinge engineering would hinder a heterologous hinge approach to providing ADCs. However, the inventors surprisingly found the antibodies described herein behave very favourably. Mass spectrometric analysis of a number of antibodies containing heterologous hinge regions (Example 5, Example 15, Example 23) revealed no cysteinylation, trisulfide formation, glutathionylation or any other expected but undesired modifications. Affinities to CD64, the Fc gamma receptor 1 , were unaltered for a wide range of tested molecules and cleavage effects were not observed in vitro (Example 10) nor in vitro (Example 13). Further, in PK experiments, the antibody with the heterologous hinge region behaved indistinguishably from the antibody exhibiting a heterologous hinge region (Example 13). Taken together, it is very surprising and unexpected that hinge regions do behave so favourable in a heterologous setting.

[0780] In summary, the literature clearly suggested that hinge sequences are associated with a number of problems and that the transfer of them results in heterogeneous mixtures of disulfide-isoforms. Surprisingly the inventors found (truncated) hinge sequences and a way to incorporate them into an antibody to avoid these known obstacles.

[0781] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

CLAIMS1. A polypeptide comprising an immunoglobulin fragment crystallisable (Fc) region, at least one variable domain N-terminal of the Fc region and an engineered IgG hinge region between the Fc region and the at least one variable domain, and wherein the engineered IgG hinge region is heterologous to the Fc region and / or is mutated to provide a predetermined number of cysteine residues.

2. An antibody comprising a first polypeptide comprising an Fc region, at least one variable domain N-terminal of the Fc region and an engineered IgG hinge region between the Fc region and the at least one variable domain, and wherein the engineered IgG hinge region is heterologous to the Fc region and / or is mutated to provide a predetermined number of cysteine residues.

3. An antibody-drug conjugate (ADC), wherein the antibody is conjugated to the drug by cysteine-based site-specific conjugation, wherein the antibody comprises a first polypeptide comprising an Fc region, at least one variable domain N-terminal of the Fc region and an engineered IgG hinge region between the Fc region and the at least one variable domain, and wherein the engineered IgG hinge region is heterologous to the Fc region and / or is mutated to provide a predetermined number of cysteine residues.

4. The antibody or ADC according to claim 2 or 3, wherein the antibody further comprises a second polypeptide comprising an Fc region and an engineered IgG hinge region N-terminal of the Fc region, wherein the engineered IgG hinge region is heterologous to the Fc region of the second polypeptide and / or is mutated to provide a predetermined number of cysteine residues, and wherein the engineered IgG hinge region of the first polypeptide and the engineered IgG hinge region of the second polypeptide are complementary engineered IgG hinge regions.

5. The antibody or ADC according to claim4, wherein the second polypeptide further comprises at last one variable domain N-terminal of the engineered IgG hinge region.

6. An antibody-drug conjugate (ADC), wherein the antibody is conjugated to the drug by cysteine-based site-specific conjugation,wherein the antibody comprises a first polypeptide and a second polypeptide, wherein each of the first polypeptide and the second polypeptide comprises an immunoglobulin fragment crystallisable (Fc) region, at least one variable domain which is a VHH N-terminal of the Fc region and an engineered IgG hinge region between the Fc region and the at least one VHH, wherein the engineered IgG hinge region of the first polypeptide and the engineered IgG hinge region of the second polypeptide are complementary engineered IgG hinge regions, and wherein the engineered IgG hinge region is heterologous to the Fc region and / or is mutated to provide a predetermined number of cysteine residues.

7. The polypeptide, antibody or ADC according to any one of the preceding claims, wherein the engineered IgG hinge region of each polypeptide is heterologous to the Fc region of said polypeptide.

8. The polypeptide, antibody or ADC according to any one of the preceding claims, wherein the engineered IgG hinge region of the first polypeptide or the engineered IgG hinge region of each of the first polypeptide and the second polypeptide comprises at least one cysteine residue, preferably wherein the engineered IgG hinge region comprises two, three, four, five, six, seven, eight or nine cysteine residues.

9. The polypeptide, antibody or ADC according to any one of the preceding claims, wherein the engineered IgG hinge region of the first polypeptide comprises at least one additional cysteine residue compared to the native hinge region of the Fc region of the first polypeptide, optionally wherein the engineered IgG hinge region of the second polypeptide comprises at least one additional cysteine residue compared to the native hinge region of the Fc region of the second polypeptide.

10. The polypeptide, antibody or ADC according to any one of the preceding claims, wherein the engineered IgG hinge region of the first polypeptide comprises at least one fewer cysteine residue compared to the native hinge region of the Fc region of the first polypeptide, optionally wherein the engineered IgG hinge region of the second polypeptide comprises at least one fewer cysteine residue compared to the native hinge region of the Fc region of the second polypeptide.

11. The polypeptide, antibody or ADC according to any one of the preceding claims, wherein the engineered IgG hinge region is an lgG1 hinge region, an lgG2 hinge region, an lgG3 hinge region or an lgG4 hinge region.

12. The polypeptide, antibody or ADC according to any one of the preceding claims, wherein the engineered IgG hinge region has:(i) an amino acid sequence as set forth in SEQ ID NO: 1 , or is a fragment thereof and / or a variant thereof comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 1;(ii) an amino acid sequence as set forth in SEQ ID NO: 2, or is a fragment thereof and / or a variant thereof comprising or an amino acid sequence having at least 70% identity to SEQ ID NO: 2;(iii) an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4, or is a fragment thereof and / or a variant thereof comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 3 or to SEQ ID NO: 4;(iv) an amino acid sequence as set forth in SEQ ID NO: 6 or SEQ ID NO: 7, or is a fragment thereof and / or a variant thereof comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 6 or to SEQ ID NO: 7; or(v) an amino acid sequence as set forth in SEQ ID NO: 5, or is a fragment thereof and / or a variant thereof comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 5.

13. The polypeptide, antibody or ADC according to any one of the preceding claims, wherein the engineered IgG hinge region has an amino acid sequence as set forth in any one of SEQ ID NOs: 8-118, preferably an amino acid sequence as set forth in SEQ ID NO: 8, 9, 12, 13, 18, 51, 67, 74, 84, 100, 113 or 118.

14. The polypeptide, antibody or ADC according to any one of the preceding claims, wherein the engineered IgG hinge region of the first polypeptide and / or second polypeptide comprises at least one amino acid modification to avoid O-glycosylation compared to the native IgG hinge region of the Fc region of the first polypeptide and / or second polypeptide.

15. The polypeptide, antibody or ADC according to any one of the preceding claims, wherein the engineered IgG hinge region is or is derived from a human IgG hinge region.

16. The ADC according to any one of claims 3 to 15, wherein the antibody is conjugated to the drug by cysteine-based site-specific conjugation through all of the cysteine residues within the engineered IgG hinge region.

17. The ADC according to any one of claims 3 to 16, wherein the ADC has a drug-to- antibody-ratio (DAR) of from about 2 to about 12, preferably a DAR of from about 4 to about 8.

18. The ADC according to any one of claims 3 to 17, wherein the ADC has a drug-to- antibody-ratio (DAR) of about 2, 4, 6, 8, 10, or 12, preferably a DAR of about 8.

19. A method of producing an antibody-drug conjugate (ADC) having a predetermined drug-to-antibody ratio (DAR), comprising the steps of:(i) providing an antibody as defined in any one of claims 2 to 15;(ii) reducing the antibody with a reducing agent; and(iii) conjugating the reduced antibody with the drug.

20. A method of producing an antibody-drug conjugate (ADC) having a predetermined drug-to-antibody ratio (DAR), comprising the steps of:(i) (a) providing at least one polynucleotide sequence encoding an antibody comprising a first polypeptide comprising an immunoglobulin fragment crystallisable (Fc) region and at least one variable domain N-terminal of the Fc region;(b) modifying the at least one polynucleotide sequence to encode an engineered IgG hinge region between the Fc region and the at least one variable domain of the first polypeptide, wherein the engineered IgG hinge region is heterologous to the Fc region and / or is mutated to provide a predetermined number of cysteine residues;(c) introducing the at least one polynucleotide sequence into a cell;(d) culturing the cell under conditions suitable for the expression of the at least one polynucleotide sequence;(e) isolating the antibody expressed by the cell;(ii) reducing the antibody with a reducing agent; and(iii) conjugating the reduced antibody with the drug.

21. A method of producing an antibody-drug conjugate (ADC) having a predetermined drug-to-antibody ratio (DAR), comprising the steps of:(i) (a) providing at least one polynucleotide sequence encoding an antibody wherein the antibody comprises a first polypeptide and a second polypeptide, wherein each of the first polypeptide and the second polypeptide comprises an immunoglobulinfragment crystallisable (Fc) region and at least one variable domain which is a VHH N-terminal of the Fc region;(b) modifying the at least one polynucleotide sequence to encode an engineered IgG hinge region between the Fc region and the at least one variable domain of each of the first polypeptide and the second polypeptide, wherein the engineered IgG hinge region of the first polypeptide and the engineered IgG hinge region of the second polypeptide are complementary engineered IgG hinge regions, and wherein the engineered IgG hinge region is heterologous to the Fc region and / or is mutated to provide a predetermined number of cysteine residues;(c) introducing the at least one polynucleotide sequence into a cell;(d) culturing the cell under conditions suitable for the expression of the at least one polynucleotide sequence;(e) isolating the antibody expressed by the cell;(ii) reducing the antibody with a reducing agent; and(iii) conjugating the reduced antibody with the drug.

22. The method according to any one of claims 19 to 21 , wherein the step of reducing the antibody in step (ii) is a partial reduction of the antibody to reduce the inter-chain disulphide bonds of the antibody.

23. The method according to any one of claims 19 to 22, wherein:(a) the drug is in the form of a linker-drug conjugate comprising a thiol-reactive group; and / or(b) wherein step (iii) is performed using a thiol-reactive coupling strategy.

24. The method according to any one of claims 19 to 23, wherein the method provides homogenous ADCs.

25. The method according to any one of claims 20 or 22 to 24, wherein the antibody further comprises a second polypeptide comprising an Fc region, and wherein step (i) (b) further comprises modifying the at least one polynucleotide sequence to encode an engineered IgG hinge region N-terminal of the Fc region of the second polypeptide, wherein the engineered IgG hinge region is heterologous to the Fc region of the second polypeptide and / or is mutated to provide a predetermined number of cysteine residues, and wherein the engineered IgG hinge region of the first polypeptide and the engineered IgG hinge region of the second polypeptide are complementary engineered IgG hinge regions.

26. The method according to any one of claims 19 to 25, wherein:(a) all of the cysteine residues within the engineered IgG hinge region form stable interchain disulphide bonds prior to step (ii);(b) in step (iii) the antibody is conjugated to the drug by cysteine-based site-specific conjugation through all of the cysteine residues within the engineered IgG hinge region;(c) the ADC has a drug-to-antibody-ratio (DAR) of from about 2 to about 12, preferably a DAR of from about 4 to about 8; and / or(d) the ADC has a drug-to-antibody-ratio (DAR) of about 2, 4, 6, 8, 10, or 12, preferably a DAR of about 8.

27. An ADC obtained or obtainable by the method according to any one of claims 19 to 26.

28. A pharmaceutical composition comprising the ADC according to any one of claims 3 to 18 or 27 and a pharmaceutically acceptable carrier, excipient and / or diluent.

29. The ADC according to any one of claims 3 to 18 or 27, or the pharmaceutical composition according to claim 28, for use in therapy or for use in a diagnostic method.

30. The ADC according to any one of claims 3 to 18 or 27, or the pharmaceutical composition according to claim 28, for use in the treatment of a disease or condition selected from the group consisting of cancer, autoimmune disease, infection, infectious diseases, cardiovascular diseases and liver metabolic disorders.

31. One or more nucleic acid sequence(s) capable of expressing the polypeptide or antibody of any one of claims 1 to 18 or 27.

32. A nucleic acid particle comprising the nucleic acid sequence(s) according to claim 31 .

33. A cell comprising the construct, nucleic acid sequences or nucleic acid particle according to any one of claims 1 to 18, 27, 31 or 32.

34. A method of making an ADC, comprising: i) expressing the nucleic acid sequence(s) or nucleic acid particle of claim 31 or 32 to produce an antibody; ii) reducing the antibody with a reducing agent; and iii) conjugating the reduced antibody with the drug.

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