Antibody-drug conjugates with personalized customized drug antibodies

Abstract

The present invention provides an antibody-drug conjugate (ADC), wherein the antibody is conjugated to the drug via cysteine-based site-specific conjugation, wherein the antibody comprises a first polypeptide comprising an immunoglobulin crystallizable fragment (Fc) region, at least one variable domain at the N-terminus of the Fc region, and 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 and / or mutated with respect to the Fc region to provide a predetermined number of cysteine ​​residues.

Description

Technical Field

[0001] This invention relates to antibody-drug conjugates (ADCs) having a personalized drug-to-antibody ratio (DAR). Specifically, the ADCs of this invention comprise engineered IgG hinge regions providing a predetermined number of cysteine ​​residues. This enables the use of site-specific cysteine ​​conjugation methods to generate ADCs with the desired DAR and homogeneous ADC products. The invention also relates to methods for generating said ADCs and the therapeutic use of said ADCs. Background Technology

[0002] Antibody-based therapies have proven revolutionary in the treatment of diseases such as cancer. The traditional use of monoclonal antibodies (mAbs) has certain limitations, including lack of efficacy in certain indications or low tumor expression of the antigens targeted by the mAbs. This has led to the development of next-generation antibody-based therapies, including bispecific antibodies (bsAbs), mRNA-encoded antibodies, and antibody-drug conjugates (ADCs).

[0003] An ADC is a conjugate of a mAb, bsAb, or antigen-binding antibody fragment (which may be specific to cancer-associated antigens) with a chemical linker sequence and a drug (often called a "payload"). The payload is typically a cytotoxic molecule, including small molecules, peptide toxins, and radionuclides. By generating an ADC, the payload benefits from a long in vivo half-life and targeted tumor targeting, thus avoiding off-target activity due to the conjugated antibody or antigen-binding fragment. Furthermore, the chemical linker between the antibody and the payload can be designed in a cleavable manner, so that after the ADC is internalized, tumor- or endosome-specific proteases can release the payload, enabling cell-specific activity. The payload then freely mediates toxicity via various pathways, including inhibition of topoisomerases, inhibition of microtubule polymerization, or DNA damage.

[0004] DAR is a key characteristic used to measure the quality of an ADC because it can significantly affect ADC efficiency. Higher DAR has been shown to mediate higher ADC efficiency. Measuring DAR is necessary because a low DAR value may indicate reduced efficiency, while a relatively high DAR value may negatively impact safety. The optimal DAR value for a given ADC varies depending on several factors such as payload and target tissue.

[0005] Various strategies exist for conjugating antibodies to linker-load 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 sorting enzyme tags), or conjugation using introduced non-natural amino acids or directly to N297 glycan structures. However, conjugation via lysine side chains yields heterogeneous products with varying drug-antibody ratios (DARs). Enzyme-mediated conjugation methods also have limitations, including additional preparation steps for enzyme generation and removal after the conjugation step, as any residual bacterial enzyme will be immunogenic. Furthermore, for sorting enzymes, peptide tags must be added to the antibody, which can alter the physicochemical properties of the protein and may also be immunogenic.

[0006] While some first-generation ADCs have achieved success, their complex heterogeneity has been associated with several problems, including suboptimal therapeutic indices, 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 generation of site-specific ADCs not only precisely controls the average DAR but also precisely controls the number of unique conjugate sites. Compared to the generation of first-generation ADCs, which produced thousands to millions of different structures, second-generation ADCs produce only a few unique structures, making them easier to optimize, develop, and fabricate.

[0007] One of the major obstacles in the ADC field is creating ADCs that accurately exhibit the expected DAR. Standard IgG1 antibodies exhibit two inter-heavy chain disulfide bonds and two inter-heavy chain disulfide bonds. Therefore, reducing these disulfides to two cysteine ​​residues each results in the largest DAR 8. However, when a lower DAR is expected, the resulting ADC will exhibit a very uneven DAR distribution. Fabre et al., 2020 ( Figure 1(b DOI: 10.1158 / 1078-0432.CCR-19-2238) provides a good example. Here, an IgG1 antibody was conjugated to an ADC exhibiting a DAR of 3.5. However, because IgG1 has eight free cysteine ​​residues after reduction, the authors produced a mixture of DAR 1, DAR 2, DAR 3, DAR 4, DAR 5, DAR 6, DAR 7, and DAR 8. Although their relative amounts yielded a theoretical average DAR of 3.5, the ADC was actually quite heterogeneous as a mixture of different ADCs. Since different DAR types can exhibit different PK profiles or cytotoxic effects, a more homogeneous product would be advantageous. The results presented by Jones et al., 2020 (DOI: 10.1080 / 19420862.2019.1682895) highlight the heterogeneity in prior art ADCs and clearly demonstrate that ADCs have DAR significantly lower than the achievable maximum.

[0008] Furthermore, existing technologies are riddled with other problems related to heterogeneity in antibody products. Glaser et al., 2005, described a hinge modification method for CH2-deficient antibodies, which can exist in two isoforms: a desired isoform A, in which hinge cysteine ​​residues form interchain disulfide bonds; and an undesirable isoform B, in which intrachain 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 / jbc.M508739200). Glaser and colleagues aimed to address this problem by inserting an IgG3 hinge motif into the IgG1 hinge of this antibody. This sequence is referred to as G1 / G3:PAP. While this isoform-mixed hinge increased the presence of isoform A to over 98%, "mass spectrometry analysis suggested the possible presence of at least two and up to five interchain disulfide bonds." This means that Glaser and colleagues failed to use the IgG3 hinge motif to create a homogeneous antibody product in which all five interchain disulfides form in the desired interchain manner, instead producing a heterogeneous product containing a mixture of different hinge types. As for the formation of isoform A, only one of the five interchain disulfides needs to be present, and Glaser et al. achieved their primary objective. However, this clearly demonstrates that hinge sequences cannot be used in a simple plug-and-play manner to create well-performing and homogeneous antibody products.

[0009] Therefore, in standard IgG1 antibodies, scientists need to choose between high homogeneity and a DAR of approximately 8 or lower, where a lower DAR may better suit the target biology but results in more heterogeneous ADC products. Furthermore, for certain antigens and payloads, a DAR exceeding 8 is conceivable, but this is not achievable with unmodified IgG1 antibodies and cysteine-targeted conjugation methods.

[0010] Numerous methods have been developed to generate site-specific ADCs. Conjugation strategies include site-specific coupling via cysteine ​​residues, based on the incorporation of thiol reactive groups onto the linker-load molecule. One such method, the Thiomab method, relies on the artificial introduction of cysteine ​​residues into the heavy chain (HC) and / or light chain (LC) of the antibody, containing a toxin-linker containing maleimide (the portion capable of covalently conjugating to the thiol group of cysteine) conjugated to the target antibody. However, free (unpaired) cysteine ​​residues and complex conjugation strategies can negatively impact the chemistry, manufacturing, and control (CMC) development process of antibodies (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, the artificial introduction of cysteine ​​residues can occur at immunogenic sites within the antibody (e.g., in conserved regions of the antibody backbone) and alter the antibody's immunogenicity profile.

[0011] Furthermore, with the ThioMab technique, the introduced cysteine ​​is always coupled to glutathione or the amino acid cysteine ​​and requires activation prior to coupling. This is achieved through antibody reduction. This, in turn, leads to the opening of the naturally occurring interchain disulfide, which must subsequently close again in an oxidation reaction. Only after reduction and oxidation can conjugation with the linker-load can take place. This additional oxidation step leads to a more complex process for generating ADCs (e.g., Juntula et al., Nature Biotechnology 2008 26:925-32). Figure 1a; https: / / doi.org / 10.1038 / nbt.1480. Furthermore, unpaired cysteine ​​residues are highly undesirable in antibodies because they can mediate numerous barriers 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 cysteine ​​residues are introduced into conserved regions of the antibody backbone, immunogenicity issues may arise.

[0012] Given the limitations associated with introducing cysteine ​​residues into the HC and / or LC of an antibody, alternative conjugation strategies rely on cysteine ​​residues naturally present within the antibody. For example, IgG1 antibodies exhibit four interchain disulfide bonds, two between HCs and one between each HC and its respective LC. Therefore, it is possible to reduce the interchain disulfide to expose eight free cysteine ​​residues, which can then be conjugated to the linker-load. Using such a method, a homogeneous product with eight DARs can be generated. However, antibody fragments such as VHH are limited in the number of their disulfide bridges. For example, a conventional VHH-Fc exhibits two interchain disulfide bridges between the hinge regions of the two HCs. Therefore, after reduction and subsequent conjugation to the linker-load conjugate via a thiol-reaction coupling strategy (e.g., maleimide-based conjugation), a homogeneous product with four DARs can be generated. However, the DAR of ADC products is limited to the number of naturally occurring cysteine ​​residues within the antibody that form interchain disulfides, depending on the presence of naturally occurring cysteine ​​residues within the antibody.

[0013] Single-domain antibodies (sdAbs), such as VHHs, have gained interest in the scientific community due to their small size and high versatility as fusion partners. Their small size (~15 kDa) allows for deep tissue and tumor penetration. However, they also typically exhibit a very short half-life, typically within minutes. Thus, VHHs are often fused with long-lived fusion partners, such as serum albumin-binding moieties (e.g., peptides or VHHs) or IgG1-derived crystallizable fragment (Fc) moieties. VHH-Fc fusions can mediate effector function in a manner similar to conventional IgG, while being smaller and less structurally complex, yet still possessing a similar half-life to conventional IgG. However, the use of VHHs in the context of ADCs is still in its early stages. Furthermore, these antibody styles have inherent limitations in their use as ADCs.

[0014] Specifically, for VHH-based ADCs, the DAR problem manifests in a slightly different form. To increase half-life, these molecules exhibit IgG1 Fc for most of the time, which extends half-life by binding to FcRn. The fusion of VHH with Fc is achieved by utilizing IgG1-derived hinges. Examples of these molecules include rimteravimab, simridarlimab, Ozekibart, erfonrilimab, letolizumab, envafolimab, and porustobart. Since all of them only exhibit two heavy chain disulfides, VHH-Fc-based ADCs designed following this prior art protein can only exhibit a maximum DAR of 4.

[0015] Since the 1960s, it has been believed that the hinge region of an antibody is easily cleaved by proteolytic hydrolysis (Turner & Bennich Biochem J 1968 107(2):171-8 doi: 10.1042 / bj1070171). The cleavage can be mediated by many proteases (Ryan et al., Mol Immunol 2008 45(7):1837-46 doi: 10.1016 / j.molimm.2007.10.043), and the IgG3 hinge, in particular, is considered readily cleaved by proteolytic hydrolysis (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 cleaving the antibody hinge region 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] Furthermore, scientists have particularly avoided using IgG3 antibodies for ADC development, partly due to the IgG3 hinge region. Hoffmann et al. state that IgG3 "has been avoided for ADC development to date because of its lower half-life in serum compared to other classes (e.g., 7 days, compared to 21 days for IgG1, 2, and 4), its long hinge region which is prone to proteolysis, and also evidence of potential immunogenicity" (Hoffmann et al., Oncoimmunology 2018 7(3):e1395127 doi: 10.1080 / 2162402X.2017.1395127).

[0017] Typically, scientists avoid modifying the hinge region of antibodies. This is underscored by the fact that while many articles describe modifications of both variable and constant domains, a limited number of articles describing hinge modification experiments investigate unrelated matters, such as fragmented resistance and recognition by pre-existing anti-hinge antibodies, or describe the complete removal of the hinge sequence from the antibody. Although there are reports of removing cysteine ​​residues from the IgG1 hinge to reduce DAR in combination with homologous IgG1 Fc (Cho et al., Large molecule therapeutics 2018 17(10):2176-2186 doi:10.1158 / 1535-7163.MCT-17-0982), there are no descriptions in the art of hinge modification techniques to increase DAR above the threshold of 8 for IgG or the threshold of 4 for VHH-Fc.

[0018] Due to the inherent instability of the IgG hinge, it is further taught in the art to avoid using heterogeneous hinge regions. Moritz and Stracke state that “the hinge region is readily cleaved and involves pathways leading to thioether bond formation, cysteine ​​racemization, and iso-Asp (Asp, aspartic acid) formation. Disulfide or thionyl groups are found to readily undergo reductive cleavage, trisulfide formation, cysteylation, glutathioneization, disulfide bridging to further light chains, and disulfide conjugation. Regarding efficacy, disulfide cleavage, hinge cleavage, disulfide bridging to further light chains, and cysteylation are found to affect antigen binding and crystallizable fragment (Fc) effector functionality. Smaller fragments may be cleared more quickly by the kidneys, while clearance of larger fragments appears to depend on their neonatal Fc receptor (FcRn) functionality, which in turn may be hindered by disulfide bond cleavage” (Moritz & Stricke Electrophoresis 2017 38(6):769-785 doi: 10.1002 / elps.201600425).

[0019] US 2016 / 129129 A1 discloses an antibody-drug conjugate comprising an IgG4 Fc domain and a homologous IgG4 hinge region. In this antibody, the cysteine-containing sequence is deleted. Avoidance of N-glycosylation is also disclosed. This document does not disclose more substantial modifications, such as pairing via a heterologous Fc region-hinge region.

[0020] US 2009 / 117100 A1 discloses antibody-drug conjugates modified to include unpaired / free cysteine ​​residues for drug conjugation. Similar techniques are described in US 7855275 B2, US 7723485 B2, US 2019 / 300559, and CA 3178093A1. The modifications in these documents do not involve substantial modification of the hinge region or drug conjugation with paired cysteine ​​residues (which form interchain disulfide bonds between hinge regions).

[0021] US 2016 / 176964 A1 discloses a similar technique in which one of a pair of cysteine ​​residues is mutated, leaving an unpaired cysteine ​​residue for conjugation. This document does not disclose substantial modification of the hinge region or heterologous Fc-hinge region pairing.

[0022] EP 4001303 A1 discloses the modification of antibody-drug conjugates in the context of amine linker conjugation.

[0023] WO 2015 / 095972 A1 discloses an antibody-drug conjugate modification in which a linker extension containing a cysteine ​​conjugate site is introduced onto the C-terminus of the antibody light chain. This modification does not involve substantial modification of the hinge region.

[0024] There is a need for new and effective ADC therapeutics, as well as methods for producing said therapeutics. An obstacle to this is the inherent unpredictability of novel modifications to the polypeptide sequence of the antibody's constant structural domain, which can lead to unforeseen adverse / detrimental effects on the protein structure. Current methods in the art have explored specific point mutations or deletions, but have not yet considered the possibility of modifying entire regions of the polypeptide sequence for ADC purposes. Invention Overview

[0026] This invention is based, at least in part, on the inventors’ development of antibody-drug conjugates (ADCs) with particularly advantageous and surprising properties.

[0027] The desired outcome is the ability to control the DAR of an ADC, for example, to produce an ADC optimized for a specific therapeutic use by balancing the efficacy and safety of the product. The inventors of this application have developed an engineered IgG-derived hinge region that links an Fc region to a variable domain at the N-terminus of the Fc region, wherein the hinge region advantageously contains a predetermined number of cysteine ​​residues. This enables the production of homogeneous ADC products with the desired DAR via a site-specific cysteine-based conjugation strategy. Furthermore, by utilizing the engineered hinge region based on a naturally occurring IgG hinge region, immunogenicity is reduced. The inventors of this application have also demonstrated that modifications to avoid O-glycosylation are tolerated within the engineered IgG hinge region. This is advantageous because O-glycosylation can lead to CMC challenges in subsequent ADCs, for example, by increasing antibody heterogeneity.

[0028] The inventors of this application have surprisingly demonstrated that ADCs comprising engineered IgG hinge regions exhibit retained or enhanced ADC properties, including stability, cell binding, internalization, antibody-dependent cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC). Specifically, the inventors of this application have achieved this by using heterologous isotype pairing between the Fc region and the hinge region, i.e., modifying the antibody polypeptide sequence to include a hinge region derived from an isotype different from that of the Fc region (e.g., IgG1, 2, 3, or 4). In this document, the inventors of this application have surprisingly demonstrated that this method results in providing stable and efficient ADC constructs in which DAR can be controlled via the selection of the heterologous hinge region sequence used. Furthermore, the inventors of this application have surprisingly discovered that, prior to conjugation, all cysteine ​​residues within the engineered IgG hinge region form stable interchain disulfide bonds, i.e., between corresponding cysteine ​​residues in the engineered IgG hinge regions of the first and second polypeptides of the antibody.

[0029] Therefore, in a first aspect, the present invention provides antibody-drug conjugates (ADCs).

[0030] The antibody is conjugated to the drug via cysteine-based site-specific conjugation.

[0031] The antibody comprises a first polypeptide containing an immunoglobulin crystallizable fragment (Fc) region, at least one variable domain at the N-terminus of the Fc region, and an engineered IgG hinge region between the Fc region and the at least one variable domain.

[0032] The engineered IgG hinge region is heterologous to the Fc region and / or mutated to provide a predetermined number of cysteine ​​residues.

[0033] In a preferred embodiment, the engineered IgG hinge region is heterologous to the Fc region and optionally mutated to provide a predetermined number of cysteine ​​residues.

[0034] In a further aspect, the present invention provides a method for generating an antibody-drug conjugate (ADC) having a predetermined drug-antibody ratio (DAR), comprising the following steps:

[0035] (i) (a) Provides at least one polynucleotide sequence encoding an antibody, the antibody comprising a first polypeptide comprising an immunoglobulin crystallizable fragment (Fc) region and at least one variable domain at the N-terminus of the Fc region;

[0036] (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 mutated to provide a predetermined number of cysteine ​​residues.

[0037] (c) Introducing the at least one polynucleotide sequence into the cell;

[0038] (d) The cells are cultured under conditions suitable for expressing the at least one polynucleotide sequence;

[0039] (e) Isolate the antibodies expressed by the cells;

[0040] (ii) Reduce the antibody with a reducing agent; and

[0041] (iii) The reduced antibody is conjugated with the drug.

[0042] In some embodiments, the at least one variable domain is selected from the group consisting of: single-stranded variable fragments (scFv); Fab; Fab'; F(ab)'2; Fv; single-domain antibody (sdAb); VHH; single-stranded variable domain; designed ankyrin repeat sequence protein (DARPin); and aptamers.

[0043] In some embodiments, the antibody is selected from the group consisting of: full-length immunoglobulin, scFv-Fc, Fab-Fc, Fv-Fc, sdAb-Fc, or VHH-Fc.

[0044] In some implementations, the antibody is VHH-Fc.

[0045] In some embodiments, the antibody further comprises a second polypeptide containing an Fc region, and step (i) (b) further comprises modifying the at least one polynucleotide sequence to encode an engineered IgG hinge region at the N-terminus of the Fc region of the second polypeptide, wherein the engineered IgG hinge region is heterologous to and / or mutated for the Fc region of the second polypeptide 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.

[0046] In some embodiments, the second polypeptide further comprises at least one variable domain at the N-terminus of the engineered IgG hinge region.

[0047] In a further aspect, the present invention provides antibody-drug conjugates (ADCs).

[0048] The antibody is conjugated to the drug via cysteine-based site-specific conjugation.

[0049] The antibody comprises a first polypeptide and a second polypeptide, wherein each of the first polypeptide and the second polypeptide comprises an immunoglobulin crystallizable fragment (Fc) region, at least one variable domain at the N-terminus of the Fc region as a VHH, and an engineered IgG hinge region between the Fc region and the at least one VHH.

[0050] 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

[0051] The engineered IgG hinge region is heterologous to the Fc region and / or mutated to provide a predetermined number of cysteine ​​residues.

[0052] In a further aspect, the present invention provides a method for generating an antibody-drug conjugate (ADC) having a predetermined drug-antibody ratio (DAR), comprising the following steps:

[0053] (i) Provide antibodies as defined herein;

[0054] (ii) Reduce the antibody with a reducing agent; and

[0055] (iii) The reduced antibody is conjugated with the drug.

[0056] In a further aspect, the present invention provides a method for generating an antibody-drug conjugate (ADC) having a predetermined drug-antibody ratio (DAR), comprising the following steps:

[0057] (i) (a) Provides 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 crystallizable fragment (Fc) region and at least one of the N-terminus of the Fc region as a variable domain of VHH.

[0058] (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 in 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 mutated to provide a predetermined number of cysteine ​​residues;

[0059] (c) Introducing the at least one polynucleotide sequence into the cell;

[0060] (d) The cells are cultured under conditions suitable for expressing the at least one polynucleotide sequence;

[0061] (e) Isolate the antibodies expressed by the cells;

[0062] (ii) Reduce the antibody with a reducing agent; and

[0063] (iii) The reduced antibody is conjugated with the drug.

[0064] 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 contains at least two cysteine ​​residues, preferably wherein the engineered IgG hinge region contains three, four, five, six, seven, eight or nine cysteine ​​residues.

[0065] In some embodiments, the engineered IgG hinge region of the first polypeptide additionally contains at least one cysteine ​​residue compared to the natural hinge region of the Fc region of the first polypeptide, preferably two, three, four, five, six, seven, eight or nine cysteine ​​residues compared to the natural hinge region of the Fc region of the first polypeptide.

[0066] In some embodiments, the engineered IgG hinge region of the first polypeptide contains at least one less cysteine ​​residue than the natural hinge region of the Fc region of the first polypeptide, and preferably contains two, three, four, five, six, seven, eight, or nine less cysteine ​​residues than the natural hinge region of the Fc region of the first polypeptide.

[0067] In some embodiments, the engineered IgG hinge region of the second polypeptide additionally contains at least one cysteine ​​residue compared to the natural hinge region of the Fc region of the second polypeptide, preferably two, three, four, five, six, seven, eight or nine cysteine ​​residues compared to the natural hinge region of the Fc region of the second polypeptide.

[0068] In some embodiments, the engineered IgG hinge region of the second polypeptide contains at least one less cysteine ​​residue than the natural hinge region of the Fc region of the second polypeptide, and preferably contains two, three, four, five, six, seven, eight, or nine less cysteine ​​residues than the natural hinge region of the Fc region of the second polypeptide.

[0069] In some embodiments, the engineered IgG hinge region comprises a sequence having at least 70% identity with the sequence shown in any one of SEQ ID NO: 1, 2, 3, 4 or 5.

[0070] In some implementations, the engineered IgG hinge region is an IgG1 hinge region.

[0071] In some embodiments, the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 1, or a fragment thereof and / or a variant thereof, the latter containing an amino acid sequence having at least 70% identity with SEQ ID NO: 1.

[0072] In some implementations, the engineered IgG hinge region is an IgG2 hinge region.

[0073] In some embodiments, the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 2, or a fragment thereof and / or a variant thereof, the latter containing an amino acid sequence having at least 70% identity with SEQ ID NO: 2.

[0074] In some implementations, the engineered IgG hinge region is an IgG3 hinge region.

[0075] In some embodiments, the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, or fragments thereof and / or variants thereof, the latter containing an amino acid sequence having at least 70% identity with SEQ ID NO: 3 or SEQ ID NO: 4.

[0076] In some embodiments, the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7, or fragments thereof and / or variants thereof, the latter containing an amino acid sequence having at least 70% identity with SEQ ID NO: 6 or SEQ ID NO: 7.

[0077] In some implementations, the engineered IgG hinge region is an IgG4 hinge region.

[0078] In some embodiments, the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 5, or a fragment thereof and / or a variant thereof, the latter containing an amino acid sequence having at least 70% identity with SEQ ID NO: 5.

[0079] In some embodiments, the engineered IgG hinge region of the first and / or second peptide contains at least one amino acid modification to avoid O-glycosylation, compared to the natural hinge region of the Fc region of the first and / or second peptide.

[0080] In some embodiments, the at least one amino acid modification used to avoid O-glycosylation is an amino acid substitution.

[0081] In some embodiments, the engineered IgG hinge region is an IgG3 hinge region, and the at least one amino acid modification is selected from S. 241JJ X, T 241MM The group consisting of X and its combinations, wherein the numbering follows the Kabat numbering scheme.

[0082] In some embodiments, the engineered IgG hinge region is an IgG3 hinge region, and the at least one amino acid modification is selected from S. 241F X, T 241I The group consisting of X and its combinations, wherein the numbering follows the Kabat numbering scheme.

[0083] In some embodiments, the engineered IgG hinge region is an IgG3 hinge region, and the at least one amino acid modification is selected from S. 241U X, T 241XThe group consisting of X and combinations thereof, wherein the numbering follows the Kabat numbering scheme. In some embodiments, the engineered IgG hinge region has an amino acid sequence shown in any one of SEQ ID NO: 8-118, preferably an amino acid sequence shown in SEQ ID NO: 8, 9, 12, 13, 18, 51, 67, 74, 84, 100, 112, 113 or 118, preferably an amino acid sequence shown in SEQ ID NO: 18, 51, 67, 74, 84, 100, 112, 113 or 118.

[0084] In some implementations, the engineered IgG hinge region is a human IgG hinge region or is derived from a human IgG hinge region.

[0085] In some embodiments, the antibody is selected from the group consisting of: human antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies, monoclonal antibodies, and polyclonal antibodies.

[0086] In some embodiments, at least one variable domain of the first polypeptide, the second polypeptide, or both the first polypeptide and the second polypeptide is specific to the cancer antigen.

[0087] In some embodiments, the antibody is conjugated to the drug via cysteine-based site-specific conjugation, through all cysteine ​​residues within the hinge region of the engineered IgG.

[0088] In some embodiments, the ADC has a drug-antibody ratio (DAR) of about 2 to about 12, preferably about 4 to about 8.

[0089] In some embodiments, the ADC has a drug-antibody ratio (DAR) of approximately 2, 4, 6, 8, 10, or 12, preferably approximately 8.

[0090] In some embodiments, the drug is selected from the group consisting of: cytotoxic drugs, antimicrobial agents, or immunomodulators.

[0091] In some implementations, the step of reducing the antibody in step (ii) is to partially reduce the antibody to reduce the interchain disulfide bonds of the antibody.

[0092] In some embodiments, the reducing agent is dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP).

[0093] In some embodiments, the drug is in the form of a linker-drug conjugate containing a thiol reactive group.

[0094] In some embodiments, the thiol reactive group is selected from the group consisting of: maleimide, bromoacetamide, disulfide, α-haloacetamide, α-halocarbonyl, vinyl sulfone, heteroaryl sulfone, thiosulfonate (ester), electron-deficient aryl halide, ethynylphosphonamide (ester), vinylphosphonite (ester), palladium oxidative addition complex.

[0095] In some implementations, step (iii) is performed using a thiol-reactive coupling strategy.

[0096] In some embodiments, the thiol-reactive coupling strategy is based on maleimide conjugation.

[0097] In some implementations, the method of the present invention provides a homogeneous ADC.

[0098] In some implementations, prior to step (ii), all cysteine ​​residues within the engineered IgG hinge region form stable interchain disulfide bonds.

[0099] In some embodiments, in step (iii), the antibody is conjugated to the drug via cysteine-based site-specific conjugation, through all cysteine ​​residues within the hinge region of the engineered IgG.

[0100] In a further aspect, the present invention provides an ADC that is obtained or obtainable by the method of the present invention.

[0101] In a further aspect, the present invention provides a pharmaceutical composition comprising an ADC according to the invention, and a pharmaceutically acceptable carrier, excipient, and / or diluent.

[0102] In a further aspect, the present invention provides the ADC of the present invention or the pharmaceutical composition of the present invention for use in therapy.

[0103] In a further aspect, the present invention provides the ADC of the present invention or the pharmaceutical composition of the present invention for use in the treatment of diseases or conditions selected from the group consisting of: cancer, autoimmune diseases, infections, infectious diseases, cardiovascular diseases, and liver metabolic disorders. Attached Figure Description

[0104] Figure 1Schematic diagram of cysteine-mediated conjugation of partially reduced antibodies. A. Cysteine-mediated conjugation of partially reduced IgG1 antibody results in an ADC with a DAR of 8. B. Cysteine-mediated conjugation of partially reduced VHH-Fc fusion results in an ADC with a DAR of 4. Because VHH-Fc does not exhibit a light chain, there are no two interchain disulfides present compared to conventional IgG1 antibodies, resulting in a maximum DAR of 4. C. Cysteine-mediated conjugation of a partially reduced, hinge-modified VHH-Fc fusion results in an ADC with a DAR of 8. By modifying the total number of four interchain disulfides in the hinge region, this VHH-Fc design allows for an increased DAR compared to conventional VHH-Fc fusions.

[0105] Figure 2 Internalization of VHH-Fc fusion compound. The internalization of VHH-Fc into target-positive tumor cells was investigated using a FACS-based assay.

[0106] Figure 3 Serum stability of VHH-Fc fusion protein. d0 = day 0 (0h), d1 = day 1 (24h), d2 = day 2 (48h), d3 = day 3 (72h), d7 = day 7 (168h), d14 = day 14 (336h).

[0107] Figure 4 Serum stability of ADCs with IgG1-derived (A) or IgG3-derived (B) hinge regions is shown. The concentrations of partial or complete ADCs are plotted against incubation time at 37°C in human serum or at 4°C in PBS.

[0108] Figure 5 Cytotoxicity of antigen 1 positive and negative cells was assessed, with the DAR4 ADC containing an IgG1-derived hinge tested in comparison to the DAR8 ADC containing an IgG3-derived hinge. Cytotoxicity evaluation was based on the ADCs A) A1B3, B) A1B4, and C) A1B5VHH. In all cases, both DAR4 and DAR8 ADCs were tested at different concentrations on target-positive and target-negative cells.

[0109] Figure 6 : The pharmacokinetic profiles of VHH-Fc fusions exhibiting the hinge region of IgG1 or IgG3-derived compounds.

[0110] Figure 7 In vivo stability of DAR4 and DAR8 ADCs.

[0111] Figure 8 : Cytotoxicity of antigen 3 positive cells, wherein the ADC containing an IgG3-derived hinge with 8 DAR is tested. (AG) Decreased viability of antigen 3 positive cells. (HI) Viability of antigen 1 positive cells when incubated with control antibody or ADC.

[0112] Figure 9 : Cytotoxicity of antigen 3 negative cells, wherein the ADC containing an IgG3-derived hinge with DAR of 8 is tested. (AG) Decreased viability of antigen 3 negative cells. (HI) Viability of antigen 3 positive cells when incubated with control antibody or ADC.

[0113] Figure 10 Cytotoxic cell killing of antigen-1 positive cells using an ADC with a wide range of DARs. (AI) Reduction of the viability of target-positive cells in a concentration- and DAR-dependent manner. (JL) Viability of antigen-1 positive cells when incubated with a control antibody or ADC.

[0114] Figure 11 The values ​​are expressed in nM as IC50 and baseline. The IC50 value indicates the concentration of the ADC at which 50% survivability is achieved. The baseline value is the minimum survivability, expressed as a percentage, achieved by the tested ADC.

[0115] Figure 12 : Cytotoxic cell killing of antigen 1 negative cells using an ADC with a wide range of DAR. AI) Reduced viability of target negative cells. JL) Viability of antigen 1 negative cells when incubated with a control antibody or ADC. Invention Details

[0117] Conventional ADC conjugation methods rely on the nonspecific / random coupling of the drug-linker with lysine (approximately 40 residues / IgG1) within the antibody or with naturally occurring cysteine ​​(approximately 8 residues / IgG1) forming interchain disulfides. These methods often result in an inhomogeneous characterization profile of ADCs with DAR varying between 2 and 4, leading to CMC challenges.

[0118] To overcome these drawbacks, next-generation site-specific antibody-drug conjugation methods have been developed. To date, twelve ADCs have been FDA-approved for the treatment of various cancer types, eight of which utilize cysteine-based site-specific conjugation strategies to form their products. ADCs generated using next-generation cysteine-based site-specific antibody-drug conjugation methods exhibit high homogeneity, which is advantageous. However, existing cysteine-based site-specific antibody-drug conjugation methods (e.g., the Thiomab method) require genetic modification of the antibody backbone to include free cysteine ​​residues for conjugation, leading to CMC challenges (Kostova V et al., Pharmaceuticals, 2021, 14: 442) and potentially increasing the immunogenicity of the product. For example, cysteine ​​mutations are formed as a mixed disulfide with cysteine ​​or glutathione, resulting in heterogeneous ADCs after generation. Furthermore, in the Thiomab method, the reduction of the engineered antibody used for conjugation leads to the partial reduction of interchain disulfides and cysteine ​​residues genetically modified into the antibody backbone. This makes it necessary to re-oxidize the interchain disulfide after the reduction step and before the free cysteine ​​in the genetically modified antibody can be conjugated, thus complicating the CMC process.

[0119] The inventors of this application have developed a method for generating ADCs that uses cysteine-based site-specific conjugation without incorporating artificial cysteine ​​residues into the antibody backbone (potentially, conserved regions of the antibody backbone). Therefore, the immunogenicity profile of the product remains unchanged, and time-intensive genetic modification is not required. Furthermore, the method for generating ADCs presented herein is compatible with established cysteine-based site-specific conjugation strategies (which have been used in FDA-approved ADCs).

[0120] Specifically, the inventors of this application have modified the IgG hinge region within the antibody to include a predetermined number of cysteine ​​residues. As described herein, altering the number of cysteine ​​residues present within the hinge region of the antibody allows control of the DAR of the ADC. It is desirable to be able to control the DAR of the ADC, for example, to produce an ADC optimized for a specific therapeutic use by balancing the efficacy and safety of the product. Modifying the IgG hinge region within the antibody to include a predetermined number of cysteine ​​residues enables the production of homogeneous ADC products with the desired DAR via a cysteine-based site-specific conjugation strategy. Furthermore, by utilizing an engineered hinge region based on a naturally occurring IgG hinge region, immunogenicity is reduced.

[0121] The inventors of this application have also demonstrated that modifications to avoid O-glycosylation are tolerated within the engineered IgG hinge region. Furthermore, the inventors have surprisingly discovered that, prior to conjugation, all cysteine ​​residues within the engineered IgG hinge region form stable interchain disulfide bonds, i.e., between corresponding cysteine ​​residues in the engineered IgG hinge regions of the first and second peptides of the antibody. This is advantageous because both O-glycosylation and free cysteine ​​will lead to CMC challenges in subsequent ADCs, for example, by increasing antibody heterogeneity.

[0122] The inventors of this application have surprisingly demonstrated that ADCs containing engineered IgG hinge regions exhibit retained or enhanced ADC properties, including stability, cell binding, internalization, antibody-dependent cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC).

[0123] Antibody-drug conjugates

[0124] This invention generally relates to antibody-drug conjugates (ADCs).

[0125] ADCs are a class of targeted therapeutic agents that improve the selectivity and activity (e.g., cytotoxic activity) of drugs, such as cancer drugs, by targeting specific targets, such as cancer cells.

[0126] Typically, an ADC comprises three main components: (i) an antibody (e.g., a monoclonal antibody) conjugated to (ii) a linker conjugated to (iii) a cargo or payload (e.g., a cytotoxic or chemotherapeutic agent).

[0127] In a first aspect, the present invention provides antibody-drug conjugates (ADCs).

[0128] The antibody is conjugated to the drug via cysteine-based site-specific conjugation.

[0129] The antibody comprises a first polypeptide containing an immunoglobulin crystallizable fragment (Fc) region, at least one variable domain at the N-terminus of the Fc region, and an engineered IgG hinge region between the Fc region and the at least one variable domain.

[0130] The engineered IgG hinge region is heterologous to the Fc region and / or mutated to provide a predetermined number of cysteine ​​residues.

[0131] In some embodiments, the antibody further comprises a second polypeptide comprising an Fc region and an engineered IgG hinge region at the N-terminus of the Fc region, wherein the engineered IgG hinge region is heterologous to and / or mutated with respect to the Fc region of the second polypeptide to provide a predetermined number of cysteine ​​residues, and wherein the engineered IgG hinge regions of the first polypeptide and the engineered IgG hinge regions of the second polypeptide are complementary engineered IgG hinge regions.

[0132] In one embodiment, the second polypeptide does not contain a variable domain.

[0133] In some embodiments, the second polypeptide further comprises at least one variable domain at the N-terminus of the engineered IgG hinge region.

[0134] In some implementations, the antibody is VHH-Fc.

[0135] Therefore, in a further aspect, the present invention provides antibody-drug conjugates (ADCs).

[0136] The antibody is conjugated to the drug via cysteine-based site-specific conjugation.

[0137] The antibody comprises a first polypeptide and a second polypeptide.

[0138] Each of the first polypeptide and the second polypeptide includes an immunoglobulin crystallizable fragment (Fc) region, at least one variable domain at the N-terminus of the Fc region as a VHH, and an engineered IgG hinge region between the Fc region and the at least one VHH.

[0139] 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

[0140] The engineered IgG hinge region is heterologous to the Fc region and / or mutated to provide a predetermined number of cysteine ​​residues.

[0141] Cysteine-based site-specific conjugation strategies are known in the art. For example, such strategies have been reviewed by Kostova et al. and Walsh et al. (Kostova V et al., Pharmaceuticals, 2021, 14: 442; and Walsh et al., Chem. Soc. Rev., 2021, 50, 1305-1353). Suitably, cysteine-based site-specific conjugation can be performed using any suitable method known in the art. Suitably, cysteine-based site-specific conjugation can be performed as described herein (see Examples). Therefore, the use of cysteine-based site-specific conjugation to generate ADCs according to the present invention is within the capabilities of those skilled in the art.

[0142] In some embodiments, the antibody is conjugated to the drug via a cysteine-based site-specific conjugation, through a cysteine ​​residue within the hinge region of the engineered IgG.

[0143] In some embodiments, the antibody is conjugated to the drug via cysteine-based site-specific conjugation, through all cysteine ​​residues within the hinge region of the engineered IgG.

[0144] In some implementations, it will generally be understood that each cysteine ​​residue is conjugated to a different drug molecule, i.e., via a 1:1 cysteine ​​residue binding to the drug molecule.

[0145] In some embodiments, the antibodies of the present invention are not derived from any Unmatched Cysteine ​​residues are conjugated to the drug or payload. In some embodiments, "unpaired cysteine ​​residues" are generally understood to be cysteine ​​residues not present at the same or equivalent positions in the two polypeptide chains of the antibody of the present invention. In some embodiments, the antibody of the present invention is conjugated to the drug or payload via only one or more pairs of cysteine ​​residues. In some embodiments, the antibody of the present invention is conjugated to the drug or payload via one or more cysteine ​​residues contained in the hinge region, which would otherwise be capable of forming interchain disulfide bridges between the hinge regions.

[0146] In some embodiments, the ADC has a drug-to-antibody ratio (DAR) of about 2 to about 22, such as about 2 to about 20, about 2 to about 12, about 4 to about 10, or about 6 to about 8.

[0147] In some embodiments, the ADC has a drug-antibody ratio (DAR) of about 2 to about 12, preferably about 4 to about 8.

[0148] In some embodiments, the ADC has a drug-to-antibody ratio (DAR) of approximately 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22. Suitably, the ADC has a DAR of approximately 4, 6, or 8.

[0149] In some embodiments, the ADC has a drug-antibody ratio (DAR) of approximately 2, 4, 6, 8, 10, or 12, preferably approximately 8.

[0150] It should be understood that, in the context of ADCs, the term "drug-to-antibody ratio (DAR)" refers to the average number of drug molecules linked (i.e., conjugated) to a single antibody. Therefore, "DAR" can be considered as the average number of linked payload (e.g., cytotoxic drug) molecules per antibody. The drug molecule / antibody molecule ratio can be characterized using conventional methods such as UV / Vis spectroscopy, mass spectrometry, ELISA assays, and HPLC.

[0151] In the context of this invention, it will generally be understood that the term "DAR" as used herein refers to... hinge The drug / load is linked to the region (i.e., the heterologous / engineered hinge region of the present invention). Therefore, in the context of the present invention and disclosure, any reference to “DAR” herein does not account for the possible contribution of other non-hinge region sequences / cysteine ​​residues to the DAR. To avoid confusion, such contributions to the total DAR are entirely within the scope of the present invention but may not be counted by any reference to the number of “DAR” herein. As an example, the ADC of the present invention having 4 DARs comprises a drug / load linked to four cysteine ​​residues in its engineered hinge region and may contain any number (or zero) of further drug / loads linked to non-hinge region sequences. Therefore, in some embodiments, in the context of the present invention, the DAR number can be calculated by multiplying the number of cysteine ​​residues present in a hinge region sequence by two.

[0152] However, in some other implementations, "DAR" is the total DAR, which includes all drug / load molecules connected to the ADC.

[0153] In some embodiments, the ADC / antibody / peptide of the present invention does not form surface-exposed inter-heavy-chain disulfide bridges outside the hinge region. "Inter-heavy-chain" refers to a disulfide bridge between any two immunoglobulin heavy polypeptide chains present in the antibody. "Surface-exposed" refers to a disulfide bridge that can be reduced and contributes to DAR. In some embodiments, the ADC / antibody / peptide of the present invention does not contain any disulfide bridges or cysteine ​​residues capable of forming disulfide bridges outside a) the hinge region and optionally b) the residues involved in any KiH modification. In some embodiments, this is particularly applicable to the constructs / ADCs of the present invention that do not contain Fab.

[0154] DAR is a key characteristic used to measure the quality of an ADC because it can significantly affect ADC efficacy and the CMC challenges associated with the ADC. It will also be understood that DAR can affect the safety and therapeutic efficacy of an ADC. In particular, DAR values ​​affect drug efficacy, as low drug loading reduces potency, while high drug loading can negatively impact pharmacokinetics and toxicity.

[0155] Therefore, it is advantageous to be able to control the DAR of an ADC product in order to provide an optimized ADC product with desired properties, including good therapeutic efficacy and safety. "Controlling the DAR" means that the ADC product can be designed (e.g., modified) to have the desired DAR. Thus, personalized ADC products with predetermined DAR can be produced. Suitablely, the ADC product can also be homogeneous.

[0156] As used herein, the terms "homogeneous ADC" and "homogeneous ADC product" refer to ADCs having substantially identical DAR. Suitably, the ADC of the present invention and the ADC produced by the method of the present invention have identical DAR, i.e., the same DAR, wherein the DAR is an integer value. Suitably, the ADC of the present invention and the ADC produced by the method of the present invention can be fully conjugated, i.e., all cysteine ​​residues that form stable interchain disulfide bonds within the antibody (before reduction and conjugation) can be conjugated with the drug. Therefore, the ADC of the present invention and the ADC produced by the method of the present invention may lack unconjugated or incompletely conjugated antibodies (i.e., lack antibodies with free cysteine ​​residues). Suitably, the DAR value may correspond to the number of cysteine ​​residues within the antibody that form stable interchain disulfide bonds within the antibody (before reduction and conjugation). The cysteine ​​residues that form stable interchain disulfide bonds within the antibody (before reduction and conjugation) may be located within the hinge region (e.g., the engineered IgG hinge region of the present invention) and within the LC (if present). Therefore, by providing an engineered IgG hinge region containing a predetermined number of cysteine ​​residues, the DAR value can be controlled for any given antibody pattern (e.g., antibody patterns containing LC, such as IgG antibodies, and antibody patterns not containing LC, such as VHH-Fc antibodies).

[0157] Therefore, altering the number of cysteine ​​residues present within the hinge region of the antibody allows control of the DAR of the ADC. Thus, the DAR is directly related to the predetermined number of cysteine ​​residues within the engineered IgG hinge region of the first polypeptide and (if present) the engineered IgG hinge region of the second polypeptide. Therefore, the method of the present invention provides an ADC with a predetermined DAR by using the engineered IgG hinge region as defined herein.

[0158] In one implementation, the DAR value corresponds to the total number of cysteine ​​residues within the antibody that form stable interchain disulfide bonds before reduction and conjugation into the drug.

[0159] In one embodiment, the DAR value corresponds to the total number of cysteine ​​residues in the engineered IgG hinge region of the first polypeptide and (if present) the engineered IgG hinge region of the second polypeptide.

[0160] As an example Figure 1 Provides a schematic overview of ADC generation via cysteine-based site-specific conjugation, based on conventional IgG1 antibodies ( Figure 1 A) Conventional VHH-Fc fusion compounds ( Figure 1 B) and an exemplary antibody according to the invention containing the engineered IgG hinge region described herein ( Figure 1 C).

[0161] Conventional IgG1 antibodies exhibit a total of four interchain disulfide bonds: two between HCs and one between each HC and its respective LC. Therefore, it is possible to reduce the interchain disulfide to expose eight free cysteine ​​residues, which can then be conjugated to the linker-load. Using such a method, a homogeneous product with eight DARs can be generated (…). Figure 1 A).

[0162] However, antibody fragments such as VHH are limited in the number of their disulfide bridges. For example, conventional VHH-Fc fusions (where each polypeptide chain contains a VHH, a truncated IgG1 hinge, and an IgG1 Fc) exhibit two interchain disulfide bridges between the two HCs. Since LC is absent in this antibody pattern, there are no interchain disulfide bridges between the HCs and their respective LCs, and a truncated IgG1 hinge region (SEQ ID NO: 12) or a modified IgG1 hinge region (SEQ ID NO: 13) containing two cysteine ​​residues / chains is used (as opposed to the two cysteine ​​residues / chains with respect to the full-length IgG1 hinge region). Therefore, it is possible to reduce the interchain disulfide to expose four free cysteines, which can then be conjugated to the linker-load. Thus, after reduction and subsequent conjugation to the linker-load conjugate via a thiol-reaction coupling strategy (e.g., maleimide-based conjugation), a homogeneous product with four DARs can be generated. Figure 1 B).

[0163] Conversely, by incorporating a total of four interchain disulfides into the hinge region of the engineered IgG according to the invention, the exemplary engineered VHH-Fc according to the invention exhibits four interchain disulfide bridges between the two HCs. Since LCs are absent in this antibody pattern, there are no interchain disulfide bridges between the HCs and their respective LCs. Therefore, it is possible to reduce the interchain disulfides of this exemplary engineered VHH-Fc according to the invention to expose eight free cysteine ​​residues, which can then be conjugated to the linker-load. Thus, after reduction and subsequent conjugation to the linker-load conjugate via a thiol-reaction coupling strategy (e.g., maleimide-based conjugation), a homogeneous product with eight DARs can be generated (…). Figure 1 C).

[0164] Therefore, this invention enables the generation of ADCs with predetermined DAR. Figure 1 In the scenario of the exemplary ADC provided in C, the predetermined DAR is eight. The invention is not limited to this exemplary ADC – the invention enables the generation of ADCs with any predetermined DAR, wherein the predetermined DAR is an integer, the integer being 2 or a multiple thereof.

[0165] The antibody and drug components of the antibody-drug conjugates of the present invention are linked together (i.e. conjugated) via linkers as defined herein.

[0166] Such linkers typically have a chemically reactive group at each end. These linkers can form a covalent attachment between two molecules (e.g., an antibody and a drug). Therefore, the antibody and the drug can be covalently linked to the linker. Suitably, one region of the linker can bind to the antibody, and another region of the linker can bind to the drug.

[0167] In some embodiments, the linker may be a cleavable linker. In some embodiments, the linker may be a cleavable linker based on a maleimide tetrapeptide.

[0168] In some embodiments, the linker comprises a thiol reactive group selected from the group consisting of: maleimide, bromoacetamide, disulfide, α-haloacetamide, α-halocarbonyl, vinyl sulfone, heteroaryl sulfone, thiosulfonate (ester), electron-deficient aryl halide, ethynylphosphonamide (ester), vinylphosphonite (ester), palladium oxidative addition complex, disulfone, water-soluble allyl sulfone, thiol-alkynyl bioconjugation having a terminal alkyne or cyclooctyne, dibromo-maleimide (DBM) and dithio-maleimide (DTM), heterothiobromo-maleimide (TBM), dibromopyrazine dione, divinylpyrimidine, and DiPODS (two oxadiazolylmethyl sulfone moieties linked by a phenyl group).

[0169] In some embodiments, the linker comprises a thiol reactive group selected from the group consisting of: maleimide, bromoacetamide, disulfide, α-haloacetamide, α-halocarbonyl, vinyl sulfone, heteroaryl sulfone, thiosulfonate (ester), electron-deficient aryl halide, ethynylphosphonamide (ester), vinylphosphonite (ester), and palladium oxidative addition complex.

[0170] The linkers used in the cysteine-based site-specific conjugation strategy according to the present invention are known in the art. Any suitable linker known in the art can be used for application according to the present invention.

[0171] Not wishing to be bound by theory, the antibody-drug conjugates according to the present invention may have (but are not limited to) one or more of the following characteristics:

[0172] High payload efficiency;

[0173] High drug-antibody ratio;

[0174] Stable connector - payload;

[0175] Tumor-selective, severable connective tissue;

[0176] ADCC activity; and / or

[0177] ADCC activity and bystander antitumor effect.

[0178] In some embodiments, the antibody or ADC of the present invention is stable in serum. In some embodiments, "stable" means not cleaved by proteolytic hydrolysis. In some embodiments, the serum is human serum. In some embodiments, the stability is maintained at 37°C for at least two weeks. In some embodiments, the antibody or ADC of the present invention is stable in human serum at 37°C for at least two weeks.

[0179] In some embodiments, the present invention provides compositions comprising the ADC of the present invention having high DAR homogeneity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or approximately 100% DAR homogeneity). In embodiments, it should be understood that DAR homogeneity is the percentage of ADCs having the same number of DARs (e.g., the same number of conjugated drug molecules).

[0180] In some embodiments, the ADC of the present invention specifically binds to the target antigen and has maintained affinity for its target antigen. Regarding "maintained affinity," it should be understood that the ADC of the present invention has unchanged or substantially unchanged affinity for the target antigen when compared with the same antibody construct that does not contain the conjugated drug molecule.

[0181] In some embodiments, the ADC of the present invention has a maintained affinity for CD64. "Maintained affinity" should be understood to mean that, when compared with the same antibody construct that does not contain the conjugated drug molecule, the ADC of the present invention has an unchanged or substantially unchanged affinity for CD64.

[0182] In some embodiments, the ADC of the present invention has maintained thermal stability. "Maintained thermal stability" should be understood as meaning that the ADC of the present invention has unchanged or substantially unchanged thermal stability when compared to the same antibody construct that does not contain the conjugated drug molecule.

[0183] Engineered IgG hinge region

[0184] As discussed above, the present invention allows control of the DAR of an ADC by altering the number of cysteine ​​residues present within the hinge region of the antibody. It is not desirable to be bound by theory, as even though interchain disulfides between the hinge regions of the antibody stabilize the IgG molecule prior to conjugation, they are not essential for the structural integrity of the antibody. Therefore, it is possible to conjugate the antibody to the linker-drug via all cysteine ​​residues within the engineered IgG hinge region without compromising the structural integrity of the antibody. The IgG hinge region can be any IgG hinge region as defined according to the present invention, such as IgG1, IgG2, IgG3, IgG4, or IgG pseudogene (e.g., IGHGP) hinge regions, or any variant, derivative, fragment, or truncated form thereof according to the present invention. The IgG hinge region of the present invention can be of any species, ortholog, or paralog. Thus, hinge regions encompassing all species, including all animals, humans, rodents, and other orthologs, but preferably human IgG hinge regions or IgG hinge regions derived therefrom.

[0185] In one embodiment, the engineered IgG hinge region of the first peptide is heterologous to the Fc region of the first peptide. Thus, the engineered IgG hinge region heterologous to the Fc region of the first peptide provides a predetermined number of cysteine ​​residues.

[0186] 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. Thus, the engineered IgG hinge region mutated as described herein provides a predetermined number of cysteine ​​residues.

[0187] In one embodiment, the engineered IgG hinge region of the first peptide is heterologous to the Fc region of the first peptide and mutated to provide a predetermined number of cysteine ​​residues. Thus, an engineered IgG hinge region heterologous to the Fc region of the first peptide and mutated as described herein provides a predetermined number of cysteine ​​residues.

[0188] In one embodiment, the engineered IgG hinge region of the second peptide is heterologous to the Fc region of the second peptide. Thus, using an engineered IgG hinge region heterologous to the Fc region of the second peptide provides a predetermined number of cysteine ​​residues.

[0189] 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. Thus, the engineered IgG hinge region mutated as described herein provides a predetermined number of cysteine ​​residues.

[0190] In one embodiment, the engineered IgG hinge region of the second peptide is heterologous to the Fc region of the second peptide and mutated to provide a predetermined number of cysteine ​​residues. Thus, a predetermined number of cysteine ​​residues are provided using an engineered IgG hinge region that is heterologous to the Fc region of the second peptide and mutated as described herein.

[0191] As used herein, the term "IgG hinge region" refers to the region of a natural IgG chain that is represented as a hinge region. For example, for human IgG isotypes, the IgG hinge region may be represented according to the Kabat numbering scheme.

[0192] 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 peptide or the second peptide, mutated as described herein to provide a predetermined number of cysteine ​​residues, or heterologous to the Fc region of the first peptide or the second peptide and mutated as described herein to provide a predetermined number of cysteine ​​residues.

[0193] As used herein, the term "heterologous to the Fc region" refers to an engineered IgG hinge region that is or is based on a hinge region of an immunoglobulin isotype different from the Fc region of the polypeptide or a variant of the Fc region of the polypeptide (described herein). For example, the IgG2 hinge, IgG3 hinge, and IgG4 hinge are each heterologous to the IgG1 Fc region or a variant thereof; the IgG1 hinge, IgG3 hinge, and IgG4 hinge are each heterologous to the IgG2 Fc region or a variant thereof; and so on. The heterologous hinge region may further be mutated as described herein.

[0194] In embodiments, it will generally be understood that the two hinge regions in the antibody / ADC of the present invention have the same IgG isotype. In some embodiments, the two hinge regions contain the same number and relative positions of cysteine ​​residues. In some embodiments, the two hinge regions contain the same sequence.

[0195] In some embodiments, the peptide / antibody / ADC of the present invention is defined as a pair comprising isotypes of hinge and Fc regions that do not exist in nature. In addition to this non-natural pairing of isotypes of hinge and Fc regions, the hinge and / or Fc regions may also undergo additional modifications, substitutions, etc.

[0196] In some embodiments, the Fc region is an IgG1 Fc region, a derivative thereof, or a fragment thereof, and the hinge region is selected from the IgG2, IgG3, or IgG4 hinge regions. In some embodiments, the Fc region is an IgG2 Fc region, a derivative thereof, or a fragment thereof, and the hinge region is selected from the IgG1, IgG3, or IgG4 hinge regions. In some embodiments, the Fc region is an IgG3 Fc region, a derivative thereof, or a fragment thereof, and the hinge region is selected from the IgG1, IgG2, or IgG4 hinge regions. In some embodiments, the Fc region is an IgG4 Fc region, a derivative thereof, or a fragment thereof, and the hinge region is selected from the IgG1, IgG2, or IgG3 hinge regions.

[0197] In some embodiments, the Fc region is an IgG1 Fc region, and the hinge region is selected from IgG2, IgG3, or IgG4 hinge regions. In some embodiments, the Fc region is an IgG2 Fc region, and the hinge region is selected from IgG1, IgG3, or IgG4 hinge regions. In some embodiments, the Fc region is an IgG3 Fc region, and the hinge region is selected from IgG1, IgG2, or IgG4 hinge regions. In some embodiments, the Fc region is an IgG4 Fc region, and the hinge region is selected from IgG1, IgG2, or IgG3 hinge regions. In these embodiments, it will generally be understood that the two hinge regions have the same IgG isotype.

[0198] In some embodiments, the hinge region is an IgG1 hinge region, a derivative thereof, or a fragment thereof, and the Fc region is not an IgG1 Fc region, a derivative thereof, or a fragment thereof. In some embodiments, the hinge region is an IgG2 hinge region, a derivative thereof, or a fragment thereof, and the Fc region is not an IgG2 Fc region, a derivative thereof, or a fragment thereof. In some embodiments, the hinge region is an IgG3 hinge region, a derivative thereof, or a fragment thereof, and the Fc region is not an IgG3 Fc region, a derivative thereof, or a fragment thereof. In some embodiments, the hinge region is an IgG4 hinge region, a derivative thereof, or a fragment thereof, and the Fc region is not an IgG4 Fc region, a derivative thereof, or a fragment thereof.

[0199] As used herein, the terms "homologous to the Fc region" and "native hinge region of the Fc region" refer to a hinge region (e.g., an engineered IgG hinge region) that is an immunoglobulin isotype hinge region identical to the Fc region of the polypeptide or a variant of the Fc region of the polypeptide (described herein). For example, the IgG1 hinge is homologous to the IgG1 Fc region or a variant thereof; the IgG2 hinge is homologous to the IgG2 Fc region or a variant thereof; and so on. The homologous hinge regions are mutated as described herein to form the engineered IgG hinge region according to the invention.

[0200] In some embodiments, as will generally be understood herein, modifying the hinge region of the peptide / antibody / ADC of the present invention means that its amino acid sequence is a sequence not found in nature. Suitably, in some embodiments, the peptide / antibody / ADC of the present invention comprises a non-naturally occurring sequence. Suitably, in some embodiments, the peptide / antibody / ADC of the present invention comprises a sequence not found in nature. Suitably, in some embodiments, the peptide / antibody / ADC of the present invention comprises a synthetic sequence.

[0201] The natural IgG hinge region can be mutated by adding, deleting, and / or substituting at least one residue present in the naturally occurring hinge region to provide an engineered IgG hinge region with a preselected number of cysteine ​​residues according to the invention. Suitably, the nucleotide sequence encoding the natural IgG hinge region can be mutated by adding, deleting, and / or substituting at least one nucleotide present in the sequence encoding the naturally occurring hinge region to encode an engineered IgG hinge region with a preselected number of cysteine ​​residues according to the invention.

[0202] In some implementations, the natural IgG hinge region is mutated by addition to provide a predetermined number of cysteine ​​residues.

[0203] In some implementations, the native IgG hinge region is mutated by deletion to provide a predetermined number of cysteine ​​residues. Therefore, the engineered IgG hinge region can be a truncated native IgG hinge region. The native IgG hinge region can be truncated to provide a predetermined number of cysteine ​​residues.

[0204] In some implementations, the natural IgG hinge region is mutated by substitution to provide a predetermined number of cysteine ​​residues.

[0205] In some implementations, the natural IgG hinge region is mutated by addition and deletion to provide a predetermined number of cysteine ​​residues.

[0206] In some implementations, the natural IgG hinge region is mutated by addition and substitution to provide a predetermined number of cysteine ​​residues.

[0207] In some implementations, the natural IgG hinge region is mutated by deletion and substitution to provide a predetermined number of cysteine ​​residues.

[0208] Using conventional techniques in molecular biology to mutate the hinge region of natural IgG to provide a predetermined number of cysteine ​​residues is within the capabilities of a person skilled in the art. Generally, suitable conventional methods include directed mutagenesis, gene synthesis, and recombinant DNA / RNA techniques.

[0209] As used herein, the term "complementary engineered IgG hinge region" means that the engineered IgG hinge regions of the first and second peptides have the same length and contain cysteine ​​residues at the same positions (according to the Kabat numbering scheme) within the hinge regions. The location of the cysteine ​​residues at the same positions within the hinge regions of the first and second peptides allows the cysteine ​​residues within the engineered IgG hinge regions to form stable interchain disulfide bonds within the antibody before conjugation to the payload. Therefore, the engineered IgG hinge regions of the first and second peptides each contain the same number of cysteine ​​residues, i.e., a predetermined number of cysteine ​​residues in the engineered IgG hinge region of the first peptide is the same as a predetermined number of cysteine ​​residues in the engineered IgG hinge region of the second peptide. Suitably, the engineered IgG hinge regions of the first and second peptides may be identical.

[0210] In a preferred embodiment, the engineered IgG hinge region of the first and / or second polypeptide contains a predetermined number of cysteine ​​residues.

[0211] In some embodiments, the engineered IgG hinge region of the first polypeptide contains at least one cysteine ​​residue. Suitably, the engineered IgG hinge region contains two, three, four, five, six, seven, eight, nine, ten, or eleven cysteine ​​residues.

[0212] The engineered IgG hinge regions of the first polypeptide and the second polypeptide each contain at least one cysteine ​​residue. In some embodiments, each of the engineered IgG hinge regions contains at least two, three, four, five, six, seven, eight, nine, ten, or eleven cysteine ​​residues. Therefore, the predetermined number of cysteine ​​residues can be one, two, three, four, five, six, seven, eight, nine, ten, or eleven cysteine ​​residues.

[0213] In one embodiment, each engineered IgG hinge region contains at least two cysteine ​​residues. In one embodiment, each engineered IgG hinge region contains at least three cysteine ​​residues. In one embodiment, each engineered IgG hinge region contains at least four cysteine ​​residues. In one embodiment, each engineered IgG hinge region contains at least five cysteine ​​residues. In one embodiment, each engineered IgG hinge region contains at least six cysteine ​​residues. In one embodiment, each engineered IgG hinge region contains at least seven cysteine ​​residues. In one embodiment, each engineered IgG hinge region contains at least eight cysteine ​​residues. In one embodiment, each engineered IgG hinge region contains at least nine cysteine ​​residues. In one embodiment, each engineered IgG hinge region contains at least ten cysteine ​​residues. In one embodiment, each engineered IgG hinge region contains at least eleven cysteine ​​residues.

[0214] The predetermined number of cysteine ​​residues is determined by the desired DAR. For example, if the desired DAR (also referred to as the "predetermined DAR") is eight, and the antibody does not contain any LC, then the predetermined number of cysteine ​​residues in the engineered IgG hinge region of each of the first and second peptides is four. As a further example, if the desired DAR (also referred to as the "predetermined DAR") is ten, and the antibody does not contain any LC, then the predetermined number of cysteine ​​residues in the engineered IgG hinge region of each of the first and second peptides is five.

[0215] In some embodiments, the engineered IgG hinge region of the first polypeptide additionally contains at least one 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 additionally contains two, three, four, five, six, seven, eight, or nine cysteine ​​residues compared to the native hinge region of the Fc region of the first polypeptide.

[0216] In some embodiments, the engineered IgG hinge region of the first polypeptide contains at least one less cysteine ​​residue than the native hinge region of the Fc region of the first polypeptide. Suitablely, the engineered IgG hinge region of the first polypeptide contains at least two, three, four, five, six, seven, eight, or nine cysteine ​​residues than the native hinge region of the Fc region of the first polypeptide.

[0217] In some embodiments, the engineered IgG hinge region of the second polypeptide additionally contains at least one 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 additionally contains two, three, four, five, six, seven, eight, or nine cysteine ​​residues compared to the native hinge region of the Fc region of the second polypeptide.

[0218] In some embodiments, the engineered IgG hinge region of the second polypeptide contains at least one less cysteine ​​residue than the native hinge region of the Fc region of the second polypeptide. Suitably, the engineered IgG hinge region of the second polypeptide contains at least two, three, four, five, six, seven, eight, or nine cysteine ​​residues than the native hinge region of the Fc region of the second polypeptide.

[0219] In some embodiments, all cysteine ​​residues within the engineered IgG hinge region of the first and second peptides form stable interchain disulfide bonds prior to drug conjugation. In other words, the antibody does not contain any free cysteine ​​residues within the engineered IgG hinge region of the first and second peptides. Therefore, all cysteine ​​residues within the engineered IgG hinge region of the first peptide can form stable interchain disulfide bonds with corresponding cysteine ​​residues (i.e., cysteine ​​residues at the same position according to the Kabat numbering system) within the engineered IgG hinge region of the second peptide. For illustrative examples of complete disulfide bond formation in antibody profiles without LC, see [link to illustrative examples]. Figure 1 B or Figure 1The left-hand plate of C. Alternatively, all cysteine ​​residues within the engineered IgG hinge region of the first polypeptide may form stable interchain disulfide bonds with corresponding cysteine ​​residues (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 (if present). For illustrative examples of complete disulfide bond formation in antibody patterns containing LC, see [reference needed]. Figure 1 The left-hand plate of A. Free (unpaired) cysteine ​​can negatively impact the chemistry, manufacturing, and control (CMC) development process of antibodies. These CMC challenges include, but are not limited to, antibody heterogeneity, low stability, aggregation, low solubility, and reduced potency. Therefore, the ADC of the present invention has the advantage of avoiding these challenges associated with free cysteine.

[0220] Suitably, the engineered IgG hinge regions of the first and second peptides can be mutated as described herein, so that they do not contain any free cysteine ​​residues prior to drug conjugation. In this respect, the natural human IgG1 hinge region contains cysteine ​​residues that form stable interchain disulfide bonds with LC. For use with antibody patterns that do not contain LC (e.g., VHH-Fc), the natural human IgG1 hinge region can therefore be mutated as described herein to eliminate cysteine ​​residues that do not form stable interchain disulfide bonds in the absence of LC (e.g., by deletion or substitution).

[0221] As described herein, the engineered IgG hinge region may be heterologous to the Fc region of the first peptide or the second peptide. Therefore, the engineered IgG hinge region may be a native IgG1, IgG2, IgG3, or IgG4 hinge region, provided that the engineered IgG hinge region is heterologous to the Fc region of the first peptide or the second peptide. For example, if the Fc region of the first or second peptide is an IgG2 Fc region, then the engineered IgG hinge region may be a native IgG1 hinge region.

[0222] Therefore, in some embodiments, the engineered IgG hinge region has a sequence shown in any one of SEQ ID NO: 1, 2, 3, 4 or 5.

[0223] The engineered IgG hinge region can be mutated by truncating the native IgG hinge region to provide a predetermined number of cysteine ​​residues. Therefore, the engineered IgG hinge region can be a fragment of the native IgG1, IgG2, IgG3, or IgG4 hinge region, providing a predetermined number of cysteine ​​residues. The engineered IgG hinge region can be heterologous to the Fc region of the first peptide or the second peptide, and can be mutated by truncating the native IgG hinge region heterologous to the Fc region of the first peptide or the second peptide to provide a predetermined number of cysteine ​​residues.

[0224] Therefore, in some embodiments, the engineered IgG hinge region is a fragment having the amino acid sequence shown in any one of SEQ ID NO: 1, 2, 3, 4, or 5. The fragment may have a length of about 4 to about 60 amino acids (suitably, about 5 to about 55, about 10 to about 50, about 15 to about 45, about 20 to about 40, or about 25 to about 35). 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).

[0225] The engineered IgG hinge region can be mutated by substituting one or more amino acids from the natural IgG hinge region to provide a predetermined number of cysteine ​​residues. For example, the natural IgG hinge region can be mutated by substituting one or more cysteine ​​residues with amino acid residues other than cysteine ​​to reduce the number of cysteine ​​residues in the resulting engineered IgG hinge region. Alternatively or additionally, the natural IgG hinge region can be mutated by substituting one or more non-cysteine ​​amino acid residues with cysteine ​​residues to increase the number of cysteine ​​residues in the resulting engineered IgG hinge region.

[0226] The engineered IgG hinge region may be heterologous to the Fc region of the first peptide or the second peptide, and may be mutated by substitution of one or more amino acids of the native IgG hinge region heterologous to the Fc region of the first peptide or the second peptide to provide a predetermined number of cysteine ​​residues.

[0227] Therefore, in some embodiments, the engineered IgG hinge region has an amino acid sequence comprising or composed of the following amino acid sequences: 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 with any one of SEQ ID NO: 1, 2, 3, 4 or 5.

[0228] The engineered IgG hinge region can be mutated by truncating the natural IgG hinge region and by substituting one or more amino acids in the natural IgG hinge region to provide a predetermined number of cysteine ​​residues.

[0229] The engineered IgG hinge region may be heterologous to the Fc region of the first peptide or the second peptide, and is mutated by truncating the native IgG hinge region heterologous to the Fc region of the first peptide or the second peptide and by substituting one or more amino acids of the native IgG hinge region heterologous to the Fc region of the first peptide or the second peptide to provide a predetermined number of cysteine ​​residues.

[0230] Therefore, in some embodiments, the engineered IgG hinge region is a fragment or variant of any one of SEQ ID NO: 1, 2, 3, 4 or 5, which comprises or consists of the following amino acid sequence: 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 with any one of SEQ ID NO: 1, 2, 3, 4 or 5.

[0231] In some embodiments, the engineered IgG hinge region is an IgG1 hinge region. Suitably, the engineered IgG hinge region is an IgG1 hinge region that has been mutated as described herein.

[0232] An illustrative human IgG1 sequence is shown below:

[0233] EPKSCDKTHTCPPCP (SEQ ID NO: 1)

[0234] In some embodiments, the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 1, or fragments thereof and / or variants thereof, the latter 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 with 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 with SEQ ID NO: 1.

[0235] In some embodiments, the engineered IgG hinge region is an IgG2 hinge region. Suitably, the engineered IgG hinge region is an IgG2 hinge region that has been mutated as described herein.

[0236] An illustrative human IgG2 sequence is shown below:

[0237] ERKCCVECPPCP (SEQ ID NO: 2)

[0238] In some embodiments, the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 2, or fragments thereof and / or variants thereof, the latter 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 with 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 with SEQ ID NO: 2.

[0239] In some embodiments, the engineered IgG hinge region is an IgG3 hinge region. Suitably, the engineered IgG hinge region is an IgG3 hinge region that has been mutated as described herein.

[0240] An illustrative human IgG3 hinge region sequence is shown below:

[0241] ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP (SEQ IDNO: 3)

[0242] A further illustrative example of the human IgG3 hinge region sequence is as follows:

[0243] ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCP (SEQ ID NO: 4)

[0244] In some embodiments, the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, or fragments thereof and / or variants thereof, the latter 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 with SEQ ID NO: 3 or 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 with SEQ ID NO: 3 or SEQ ID NO: 4.

[0245] Human IGHG3 The 01 sequence (SEQ ID NO: 3) is arranged with four exons. The amino acid sequences translated from exons 2, 3, and 4 are identical. Any exon sequence can be used in the practice of this invention. Therefore, the fragment of SEQ ID NO: 3 can correspond to the sequence of exon 1 or the sequence of exons 2, 3, or 4.

[0246] An illustrative example of the human IgG3 hinge region exon 1 sequence (translated into amino acids) is as follows:

[0247] ELKTPLGDTTHTCPRCP (SEQ ID NO: 6)

[0248] An illustrative example of the human IgG3 hinge region exon 2, exon 3, or exon 4 sequence (translated into amino acids) is as follows:

[0249] EPKSCDTPPCPRCP (SEQ ID NO: 7)

[0250] In some embodiments, the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7, or fragments thereof and / or variants thereof, the latter 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 with SEQ ID NO: 6 or 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 with SEQ ID NO: 6 or SEQ ID NO: 7.

[0251] In some embodiments, the engineered IgG hinge region is an IgG4 hinge region. Suitably, the engineered IgG hinge region is an IgG4 hinge region that has been mutated as described herein.

[0252] An illustrative human IgG4 hinge sequence is shown below:

[0253] ESKYGPPCPSCP (SEQ ID NO: 5)

[0254] In some embodiments, the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 5, or fragments thereof and / or variants thereof, the latter 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 with 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 with SEQ ID NO: 5.

[0255] It is known that threonine and serine residues within the hinge of human IgG3 are readily O-glycosylated. Serine residues in the human IgG3 hinge, as well as threonine and serine residues in the hinge regions of other IgG isotypes, can also undergo O-glycosylation. Unwilling to be bound by theory, O-glycosylation can lead to controllable molecular weighting (CMC) challenges related to ADCs, for example, by increasing product heterogeneity. Therefore, it is desirable to avoid O-glycosylation in antibody hinge regions for ADC applications.

[0256] Following the teachings disclosed herein, modifying the hinge region of native IgG to avoid O-glycosylation using conventional techniques in molecular biology is within the capabilities of those skilled in the art. Generally, suitable and conventional methods include directed mutagenesis, gene synthesis, and recombinant DNA / RNA techniques.

[0257] In some embodiments, the engineered IgG hinge region of the first and / or second polypeptide contains at least one amino acid modification to avoid O-glycosylation, relative to the native IgG hinge region of the Fc region of the first and / or second polypeptide. Suitably, the at least one amino acid modification may be the deletion or substitution of a serine and / or threonine residue. Suitably, the at least one amino acid modification may be the deletion or substitution of at least one serine residue. Suitably, the at least one amino acid modification may be the deletion or substitution of all serine residues within the native IgG hinge region. Suitably, the at least one amino acid modification may be the deletion or substitution of at least one threonine residue. Suitably, the at least one amino acid modification may be the deletion or substitution of all threonine residues within the native IgG hinge region. Suitably, the at least one amino acid modification may be the deletion or substitution of at least one serine residue and at least one threonine residue. Suitably, the at least one amino acid modification may be the deletion or substitution of all serine and threonine residues within the native IgG hinge region.

[0258] In some embodiments, the at least one amino acid modification used to avoid O-glycosylation is an amino acid substitution. In some embodiments, the amino acid substitution is a substitution with an amino acid residue that is not serine or threonine. In some embodiments, the amino acid substitution is a substitution with alanine. In some embodiments, the amino acid substitution is a serine-to-alanine substitution and / or a threonine-to-alanine substitution, as applicable.

[0259] In some embodiments, the engineered IgG hinge region is an IgG3 hinge region, and the at least one amino acid modification is selected from S. 241F X, T 241I The group consisting of X and combinations thereof, wherein the numbering follows the Kabat numbering scheme, and wherein X is any amino acid other than C, S, or T.

[0260] In some embodiments, the engineered IgG hinge region is an IgG3 hinge region, and the at least one amino acid modification is selected from S. 241U X, T 241X The group consisting of X and combinations thereof, wherein the numbering follows the Kabat numbering scheme, and wherein X is any amino acid other than C, S, or T.

[0261] In some embodiments, the engineered IgG hinge region is an IgG3 hinge region, and the at least one amino acid modification is selected from S. 241JJ X, T 241MM The group consisting of X and combinations thereof, wherein the numbering follows the Kabat numbering scheme, and wherein X is any amino acid other than C, S, or T.

[0262] In one embodiment, the engineered IgG hinge region is an IgG3 hinge region, and the at least one amino acid modification is S. 241JJ X, wherein the number follows the Kabat numbering scheme, and X is any amino acid other than C, S, or T.

[0263] In some embodiments, the engineered IgG hinge region is an IgG3 hinge region, and the at least one amino acid modification is T. 241MM X, where the number follows the Kabat numbering scheme, and X is any amino acid other than C or T.

[0264] In some embodiments, the engineered IgG hinge region is an IgG3 hinge region, and the at least one amino acid modification is S. 241JJ X and T 241MM X, wherein the number follows the Kabat numbering scheme, and X is any amino acid other than C, S, or T.

[0265] In some embodiments, the engineered IgG hinge region is an IgG3 hinge region, and the at least one amino acid modification is selected from S. 241F X, T 241I X, S 241U X, T 241X X, S 241JJ X, T 241MM The group consisting of X and combinations thereof, wherein the numbering follows the Kabat numbering scheme, and wherein X is any amino acid other than C, S, or T.

[0266] The following Table 1 details illustrative engineered IgG hinge region sequences that can be used in the practice of this invention.

[0267] Table 1 - Illustrative engineered IgG hinge region sequences. Predetermined DARs (referred to as "DARs" in this table) for ADCs are also provided, wherein the first polypeptide and the second polypeptide each contain the hinge sequence.

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282] In some embodiments, the engineered IgG hinge region has an amino acid sequence shown in any one of SEQ ID NO: 8-118, or a fragment thereof and / or a variant thereof, the latter 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 8-118. Suitable fragments of SEQ ID NO: 8-118 used in accordance with the present invention are described in Table 1.

[0283] In some embodiments, the engineered IgG hinge region has an amino acid sequence shown in any one of SEQ ID NO: 8-118, preferably an amino acid sequence shown in any one of SEQ ID NO: 8, 9, 12, 13, 18, 51, 67, 74, 84, 100, 113 or 118, more preferably an amino acid sequence shown in any one of SEQ ID NO: 8, 9, 18, 51, 67, 74, 84, 100, 113 or 118.

[0284] In some embodiments, the engineered IgG hinge region is an amino acid sequence shown in any one of SEQ ID NO: 8-118, preferably an amino acid sequence shown in any one of SEQ ID NO: 8, 9, 12, 13, 18, 51, 67, 74, 84, 100, 113 or 118, more preferably a fragment of an amino acid sequence shown in any one of SEQ ID NO: 8, 9, 18, 51, 67, 74, 84, 100, 113 or 118.

[0285] In some embodiments, the engineered IgG hinge region comprises or consists of the following amino acid sequences: amino acid sequences that are identical to any one of SEQ ID NO: 8-118, preferably any one of SEQ ID NO: 8, 9, 12, 13, 18, 51, 67, 74, 84, 100, 113 or 118, more preferably amino acid sequences that have 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%) the amino acid sequences shown in any one of SEQ ID NO: 8, 9, 18, 51, 67, 74, 84, 100, 113 or 118.

[0286] In some embodiments, the engineered IgG hinge region is an amino acid sequence shown in any one of SEQ ID NO: 8-118, preferably an amino acid sequence shown in any one of SEQ ID NO: 8, 9, 12, 13, 18, 51, 67, 74, 84, 100, 113 or 118, more preferably a fragment or variant of an amino acid sequence shown in any one of SEQ ID NO: 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 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 with it.

[0287] In some embodiments, the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 8, or a fragment thereof and / or a variant thereof, the latter comprising or consisting of the following 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 with it.

[0288] In some embodiments, the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 9, or a fragment thereof and / or a variant thereof, the latter comprising or consisting of the following 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 with it.

[0289] In some embodiments, the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 12, or a fragment thereof and / or a variant thereof, the latter comprising or consisting of the following 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 with it.

[0290] In some embodiments, the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 13, or a fragment thereof and / or a variant thereof, the latter comprising or consisting of the following 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 with it.

[0291] In some embodiments, the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 18, or a fragment thereof and / or a variant thereof, the latter comprising or consisting of the following 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 with it.

[0292] In some embodiments, the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 51, or a fragment thereof and / or a variant thereof, the latter comprising or consisting of the following 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 with it.

[0293] In some embodiments, the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 67, or a fragment thereof and / or a variant thereof, the latter comprising or consisting of the following 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 with it.

[0294] In some embodiments, the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 74, or a fragment thereof and / or a variant thereof, the latter comprising or consisting of the following 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 with it.

[0295] In some embodiments, the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 84, or a fragment thereof and / or a variant thereof, the latter comprising or consisting of the following 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 with it.

[0296] In some embodiments, the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 100, or a fragment thereof and / or a variant thereof, the latter comprising or consisting of the following 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 with it.

[0297] In some embodiments, the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 113, or a fragment thereof and / or a variant thereof, the latter comprising or consisting of the following 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 with it.

[0298] In some embodiments, the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 118, or a fragment thereof and / or a variant thereof, the latter comprising or consisting of the following 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 with it.

[0299] In some implementations, the engineered IgG hinge region is or is derived from human, rodent, camel, rabbit, sheep, goat or chicken IgG hinge regions.

[0300] 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 has lower immunogenicity compared to non-human IgG hinge region sequences.

[0301] As used herein, the terms “derived from” or “based on” a natural (e.g., human) IgG hinge region refer to a hinge region that has been mutated as described herein to provide a predetermined number of cysteine ​​residues.

[0302] Antibody

[0303] As used herein, the term "antibody" may refer to a protein or polypeptide having an antigen-binding domain comprising at least one complementarity-determining region (CDR).

[0304] The term "complementarity-determining region" or "CDR" typically refers to one of the six hypervariable regions within the variable region of an antibody, such as the three CDRs / variable domains / VHH, the six CDRs in the standard VHH-Fc fusion construct, and the twelve CDRs in the standard antibody construct.

[0305] The "complementarity-determining region" or "CDR" of an antibody, related to its antigen-binding domain, refers to a hypervariable region or highly variable loop in the variable region of the antibody's heavy or light chain, which primarily facilitates antigen binding. CDRs can interact with the antigen conformation and largely determine binding to the antigen (although some structural regions are known to be involved in binding). Both the heavy and light chain variable regions contain three CDRs (heavy chain CDRs 1, 2, and 3, and light chain CDRs 1, 2, and 3, numbered from the amino terminus to the carboxyl terminus).

[0306] The techniques used to prepare and use various antibody-based constructs and fragments are well known in the art.

[0307] As used herein, “antigen binding site” or “antigen binding domain” means a protein or polypeptide containing at least one complementarity-determining region (CDR). An antigen binding site may contain three CDRs, which is equivalent to a single-domain antibody (sdAb) domain (e.g., a VHH domain).

[0308] As used herein, the term “constant immunoglobulin domain” can refer to a constant domain of an immunoglobulin, such as the CH3 domain.

[0309] In a conventional full-length antibody (e.g., an IgG antibody) containing four polypeptides (two light chains and two heavy chains), the Fc region forms a homodimer of the CH2-CH3 domain of each heavy chain polypeptide.

[0310] As used herein, the term "crystallizable fragment (Fc) region" can refer to the Fc region of an immunoglobulin containing a CH2-CH3 domain, or any fragment, truncated form, derivative, or variant thereof. A truncated form may include at least one constant immunoglobulin domain or a fragment thereof. In some embodiments, the Fc region contains or is composed of a CH2 domain. In some embodiments, the Fc region contains or is composed of a CH3 domain. In some embodiments, the Fc region contains or is composed of both CH2 and CH3 domains. Preferably, the Fc region of the present invention is a full-length or substantially full-length Fc region. The Fc regions of the present invention include those that have undergone known / standard / conventional modifications. The Fc regions of the present invention are not particularly species-specific, although humans are preferred. Therefore, Fc regions encompassing all species include all animals, humans, rodents, and other orthologs. Fc regions or Fc region-like sequences encoded by paralogs are also included. Suitably, in embodiments of the present invention, the Fc region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD Fc region. Suitably, in embodiments of the present invention, the Fc region may be an IgG1, IgG2, IgG3, or IgG4 Fc region. In a preferred embodiment, the Fc region is an IgG1 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, for example, an Fc region inactivated by mutation. In some embodiments, "inactivated" means that the Fc region lacks some or all of the effector functions of one or more of the native Fc region.

[0311] sdAbs are naturally found IgG molecules in animals such as camels (IgG-derived VHHs, such as IGG2 and IgG3 VHHs) and sharks (IgNAR-type sbAbs). Camelid sdAbs lack a light chain and the first constant domain (CH1) of the heavy chain of conventional IgG. Therefore, the antigen-binding fragment of sdAbs contains only a single variable domain, often referred to as the variable heavy chain domain of the heavy chain (VHH domain) (also referred to herein as the "single-domain variable heavy chain immunoglobulin" domain). Thus, the VHH contains 3 CDRs.

[0312] As used herein, the term “variable immunoglobulin domain” or “variable domain” can refer to a variable domain of an immunoglobulin, such as the VHH domain.

[0313] The immunoglobulin domains used in this invention are not limited to specific sequences and may include any suitable known immunoglobulin domains.

[0314] 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: single-chain variable fragments (scFv); Fab; Fab'; F(ab)'2; Fv; single-domain antibody (sdAb); VHH; single-chain variable domain; designed ankyrin repeat sequence protein (DARPin); and aptamers.

[0315] In some embodiments where Fab is present in the first and / or second polypeptide, an additional upper hinge region is present in the polypeptide, for example at the N-terminus of an engineered hinge region thereon. It will generally be understood that this additional upper hinge region allows disulfide bonds with the light chain of the Fab. For the avoidance of ambiguity, and for the purposes of this invention, the disulfide bond / cysteine ​​involved therein is not included in the DAR or cysteine ​​residues of the engineered hinge region.

[0316] 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: single-chain variable fragments (scFv); Fab; Fv; single-domain antibodies (sdAb); VHH; single-chain variable domains; and designed ankyrin repeat sequence proteins (DARPin).

[0317] 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 at the N-terminus of the Fc region of the first polypeptide may be a VHH, and the variable domain at the N-terminus of the Fc region of the second polypeptide may also be a VHH.

[0318] In one embodiment, the at least one variable domain of the first polypeptide has a different type than the at least one variable domain of the second polypeptide. For example, the variable domain at the N-terminus of the Fc region of the first polypeptide may be scFv, and the variable domain at the N-terminus of the Fc region of the second polypeptide may be VHH. Suitably, the antibody can be generated using CrossMAb technology (Schaefer et al., PNAS, 2011, 108: 11187-92; Takahashi et al., Cell, 1982, 29: 671-679; Grubert et al., ACS Omega 2022, 7, 4, 3671-3679; and Klein et al., mAbs, 2016, 8: 1010-1020).

[0319] In some embodiments, the antibody is selected from the group consisting of: full-length immunoglobulin, scFv-Fc, Fab-Fc, Fab'-Fc, F(ab)'2-Fc, Fv-Fc, sdAb-Fc, or VHH-Fc.

[0320] In some embodiments, the antibody is selected from the group consisting of: full-length immunoglobulin, scFv-Fc, Fab-Fc, Fv-Fc, sdAb-Fc, or VHH-Fc.

[0321] In some preferred embodiments, the antibody is VHH-Fc. In some preferred embodiments, the antibody is Fab-Fc. In some preferred embodiments, the antibody is a bispecific antibody. In some preferred embodiments, the bispecific antibody comprises 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.

[0322] In some embodiments, the antibody is selected from the group consisting of: human antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies, monoclonal antibodies, and polyclonal antibodies.

[0323] The term "chimeric antibody" generally refers to an antibody obtained by fusing the variable region of a non-human antibody with the constant region of a human antibody, which can reduce the immune response induced by the non-human antibody. The non-human antibody can be, for example, a mouse, camel, rabbit, sheep, goat, or chicken antibody. As an example, to establish a chimeric antibody, a hybridoma secreting a specific monoclonal antibody can be constructed, and the variable region gene can be cloned from mouse hybridoma cells; then, the constant region gene of a human antibody can be cloned as needed, and the mouse variable region gene and the human constant region gene can be linked to form a chimeric gene; the chimeric gene is then inserted into an expression vector, in which the chimeric antibody molecule can be expressed in a eukaryotic or prokaryotic system.

[0324] The term "humanized antibody," also known as a CDR-grafted antibody, generally refers to an antibody produced by grafting a mouse CDR sequence onto a human antibody variable region framework, i.e., an antibody produced within a different type of human germline antibody framework sequence. Therefore, humanized mouse antibodies are more likely to elicit an immune response in humans. Such framework sequences can be obtained from public DNA databases or publicly available literature (which includes germline antibody gene sequences). For example, germline DNA sequences of the human heavy and light chain variable region genes can be obtained from the VBase human germline sequence database. Furthermore, antibody hinge and constant domain sequences can be derived from the IMGT database, if desired.

[0325] The terms "fully humanized antibody," "fully human antibody," or "fully human antibody," which can also be called "fully humanized monoclonal antibody," can contain both humanized variable and constant regions to eliminate immunogenicity and toxic side effects. The development of monoclonal antibodies has gone through four stages: 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. Related technologies for preparing fully human antibodies include: human hybridoma technology, EBV-transformed B-lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology, and single-cell B-cell antibody preparation technology, etc.

[0326] "Monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that the antibodies constituting the group are identical, except for the possibility of naturally occurring mutations present in small amounts. Monoclonal antibodies are highly specific and target a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically consist of a large number of antibodies targeting different epitopes (or specific to different epitopes). The modifier "monoclonal" indicates the characteristic of antibodies obtained from a substantially homogeneous group of antibodies and should not be interpreted as meaning that the antibody was produced by any particular method.

[0327] In some implementations, the sequence of the antibody can be defined using a Kabat number (Kabat EA et al., (1991)).

[0328] Antibodies can be obtained using techniques including immunizing animals with target antigens and isolating antibodies from serum. Monoclonal antibodies can be prepared using the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0329] In one embodiment, the antibody used according to the present invention may be monovalent, bivalent, trivalent, tetravalent, or pentavalent, depending on the number of antigen-binding domains (e.g., VHH domains) present. Similarly, in one embodiment, the antibody used according to the present invention may be monospecific or multispecific, such as bispecific, trispecific, tetraspecific, etc., depending on the number of different antigen-binding domains (e.g., VHH domains) present.

[0330] Any suitable multispecific or multivalent antibody pattern known in the art can be used in the practice of this invention. Suitably, the bispecific antibody may be IgG-scFv, IgG-sdAb, IgG-VHH, scFv-Fc-scFv, KiH-IgG, κλ-body, KiH-Fc-Fab / scFv.

[0331] The variable domains (e.g., VHH domains) used in this invention are not particularly limited and may contain any antigen-binding site specific to the selected target protein. Methods for determining the binding specificity of an antibody to a specific antigen are known in the art and include, but are not limited to, biolayer interferometry (BLI), surface plasmon resonance (SPR) analysis (e.g., using a BIAcore instrument), ELISA, Western blotting, in situ hybridization, immunohistochemistry, flow cytometry, Förster resonance energy transfer (FRET), phage display libraries, yeast two-hybrid screening, 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, and isothermal titration calorimetry.

[0332] Methods for providing variable domains for specific targets, such as sdAbs containing VHH domains, are known in the art (see Caussinus et al., Nat Struct Mol Biol; 2011, 19(1), 117-121; and Fulcher et al., Open Biol, 2016, 6(10), pii 160255). Furthermore, methods for isolating antigen-specific VHHs from immune or semi-synthetic libraries using phage, yeast, or ribosome display have been established in the art (see Muyldermans, J Biotechnol. 2001 Jun, 74(4):277-302; and Dufner et al., Trends Biotechnol. 2006 Nov, 24(11):523-9).

[0333] As an example, VHH can be obtained by immunizing, for example, a dromedary camel, camel, llama, or alpaca with the desired antigen, followed by isolating the mRNA encoding VHH. The single-domain shark variable domain (VNAR) of neoantigen receptor antibodies is also known and suitable as a candidate sdAb for the VHH domain according to the present invention. Therefore, as a further example, VNAR can be obtained by immunizing a shark with the desired antigen, followed by isolating the mRNA encoding VNAR. A library of VHH or VNAR can then be generated using reverse transcription and PCR. Standard screening techniques such as phage display and ribosome display can be used to identify suitable clones that bind to the target antigen.

[0334] Once the most robust clones are identified, their DNA sequences can be optimized, for example, to improve their stability against enzymes. Humanization can also be performed.

[0335] VHH and VNAR can be expressed in cells using conventional vectors (e.g., those described in this paper).

[0336] The ability of an antibody to specifically bind to its target can be determined using methods known in the art. For example, binding can be measured by, for instance, biofilm interferometry (BLI), surface plasmon resonance (SPR) analysis (using a BIAcore instrument), Western blotting, flow cytometry, in situ hybridization, and / or microscopy. Suitably, binding affinity can be measured by, for instance, biofilm interferometry (BLI), surface plasmon resonance (SPR) analysis (using a BIAcore instrument), and / or flow cytometry. Suitably, binding affinity can be measured as described herein (see Example 3).

[0337] In one embodiment, the variable domains of the first polypeptide and the second polypeptide have the same binding specificity.

[0338] In one embodiment, the variable domains of the first polypeptide and the second polypeptide have different binding specificities.

[0339] In one embodiment, the variable domain (e.g., a VHH domain) of the first polypeptide is specific for a first target, and the variable domain (e.g., a VHH domain) of the second polypeptide is specific for a second target. In a preferred embodiment, the first and second targets are different. Therefore, in one embodiment, the antibody comprising the first and second polypeptides is a heterodimer. In an alternative embodiment, the variable domain (e.g., a VHH domain) of the first polypeptide is specific for the same target as the variable domain (e.g., a VHH domain) of the second polypeptide. Therefore, in one embodiment, the antibody comprising the first and second polypeptides is a homodimer.

[0340] In one embodiment, the first polypeptide further comprises at least one binding portion or an associate of the binding portion at the C-terminus of the Fc region. In one embodiment, the at least one binding portion or associate is located immediately at the C-terminus of the Fc region. Suitably, the first polypeptide further comprises a binding portion at the C-terminus of the Fc region. Suitably, the first polypeptide further comprises an associate of the binding portion at the C-terminus of the Fc region.

[0341] In one embodiment, the second polypeptide further comprises at least one binding portion or an associate of the binding portion at the C-terminus of the Fc region. In one embodiment, the at least one binding portion or associate is located immediately at the C-terminus of the Fc region. Suitably, the second polypeptide further comprises a binding portion at the C-terminus of the Fc region. Suitably, the second polypeptide further comprises an associate of the binding portion at the C-terminus of the Fc region.

[0342] In one embodiment, the first polypeptide further comprises a binding portion or an association of the binding portion at the C-terminus of the Fc region of the first polypeptide, and the second polypeptide further comprises a binding portion or an association of the binding portion at the C-terminus of the Fc region of the second polypeptide. In one embodiment, each of the binding portions or associations is immediately located at the C-terminus of the Fc region of its respective polypeptide sequence. Suitably, each of the first polypeptide and the second polypeptide further comprises a binding portion at the C-terminus of the Fc region of its respective polypeptide. Suitably, each of the first polypeptide and the second polypeptide further comprises an association of the binding portion at the C-terminus of the Fc region of its respective polypeptide. Suitably, the first polypeptide further comprises a binding portion at the C-terminus of the Fc region of the first polypeptide, and the second polypeptide further comprises an association of the binding portion at the C-terminus of the Fc region of the second polypeptide. Suitably, the first polypeptide further comprises an association of the binding portion at the C-terminus of the Fc region of the first polypeptide, and the second polypeptide further comprises a binding portion at the C-terminus of the Fc region of the second polypeptide.

[0343] "At the N-terminus of the Fc region" means that the variable domain or engineered IgG hinge region is located at the N-terminus of the Fc region by any means known in the art. In one embodiment, the engineered IgG hinge region is directly fused to the N-terminus of the Fc region, i.e., the engineered IgG hinge region may be adjacent to the Fc region. However, in one embodiment, the engineered IgG hinge region is connected to the N-terminus of the Fc region via a connection sequence described herein. The variable domain is connected to the N-terminus of the Fc region via an engineered IgG hinge sequence described herein.

[0344] "At the N-terminus of the engineered IgG hinge region" means that the variable domain is located at the N-terminus of the engineered IgG hinge region by any means known in the art. In one embodiment, the variable domain is directly fused to the N-terminus of the engineered IgG hinge region, i.e., the variable domain may be adjacent to the engineered IgG hinge region. However, in one embodiment, the variable domain is connected to the N-terminus of the engineered IgG hinge region via a connection sequence described herein.

[0345] "At the C-terminus of the Fc region" means that the bonding portion or its associated element is located at the C-terminus of the Fc region by any means known in the art. In one embodiment, the bonding portion or its associated element is directly fused to the C-terminus of the Fc region, i.e., the bonding portion or its associated element may be adjacent to the Fc region. However, in one embodiment, the bonding portion or its associated element is connected to the C-terminus of the Fc region via a connection sequence described herein.

[0346] The linker sequence of the first and / or second polypeptide may contain one or more linkers (e.g., glycine-serine (GS) linkers), such as those widely known in the art. In one embodiment, the linker sequence contains a GS linker. In one embodiment, the linker sequence consists of a GS linker. In one embodiment, the linker sequence is GS. In a preferred embodiment, the linker sequence does not contain cysteine ​​residues. Therefore, the linker sequences of the first and second polypeptides may not form any interchain disulfide bonds. Preferably, the linker sequences of the first and second polypeptides do not form interdomain disulfide bonds.

[0347] The binding moiety of the first and / or second polypeptide can be any suitable binding entity capable of specifically binding to a target (e.g., a target polypeptide sequence). Many binding moieties are known in the art, including those based on antigen-binding sites of antibodies, antibody mimics, and T-cell receptors. For example, the binding moieties may comprise: a variable immunoglobulin domain; an antigen-binding site of an antibody; a single-chain variable fragment (scFv); Fab; Fv; a single-domain antibody (sdAb); VHH; a single-chain variable domain (which may be a VH or VL chain with 3 CDRs); or an artificial single-binding agent, such as Darpin (a designed ankyrin repeat sequence protein). In a preferred embodiment, the binding moieties are antigen-binding fragments of the antibody. In a preferred embodiment, the antigen-binding fragment of the antibody is a VHH.

[0348] The association of the binding moiety can be any motif specifically recognized by the binding moiety. Numerous associations for binding moiety are provided in the art. In one embodiment, the association is an antigen, epitope, or polypeptide tag specifically recognized by an antibody or an antigen-binding fragment of an antibody. In a preferred embodiment, the association is a polypeptide tag. In another preferred embodiment, the association is a polypeptide tag specifically recognized by an antigen-binding fragment of an antibody.

[0349] In one embodiment, the binding portion located at the C-terminus of the Fc region of the first peptide and / or the second peptide is preferably a VHH domain and is specific to the third target. In one embodiment, the third target is different from the first and second targets. In one embodiment, the binding portion located at the C-terminus of the Fc region of each of the first and second peptides is preferably a VHH domain and is specific to the third and fourth targets. In one embodiment, each of the first, second, third, and fourth targets is different.

[0350] In one embodiment, the association of the binding moiety located at the C-terminus of the Fc region of the first peptide and / or the second peptide is not specifically recognized by either the variable domain (e.g., VHH domain) of the first and second peptides or the binding moiety (if present).

[0351] In one embodiment, "specifically" or "specifically recognizing" the binding portion or variable domain (e.g., VHH) of the target means binding to the target with an affinity equivalent to that of a functional antibody fragment. In one embodiment, the target is a target antigen.

[0352] In embodiments where the first and / or second polypeptide comprises multiple variable domains and / or binding moieties (e.g., VHH domains), each variable domain and / or binding moieties (e.g., VHH domains) in the same polypeptide can be specific to the same target. In embodiments where the first and / or second polypeptide comprises multiple variable domains and / or binding moieties (e.g., VHH domains), each variable domain and / or binding moieties (e.g., VHH domains) in the same polypeptide can be specific to different targets.

[0353] Any suitable Fc region known in the art can be used in the practice of this invention. In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein, thereby producing an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions. For example, Fc modifications for modulated effector functions are known in the art. Fc modifications include, but are not limited to, silencing effector functions, enhancing effector functions, and prolonging or shortening half-life. Liu et al. provide a review of Fc modifications (Liu et al., Antibodies, 2020, 9: 64).

[0354] In one embodiment, the first and second polypeptides comprise modifications that enhance the formation of a dimer (preferably a heterodimer comprising a monomer of the first polypeptide and a monomer of the second polypeptide). In one embodiment, the modification may be one of those modifications widely known in the field of antibody technology for (hetero)dimerization purposes between antibody chains (e.g., "knob-into-hole" (KiH) modifications). In a typical embodiment, the modification is present in the CH3 domain of the first and second polypeptides.

[0355] 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 contain modifications that enhance the formation of a dimer comprising the first and second polypeptides. In embodiments where the first and second polypeptides each comprise more than one CH3 domain, all CH3 domains may contain such modifications.

[0356] As used herein, the term "enhancing the formation of dimers comprising the first and second polypeptides" includes promoting the dimerization of the first and second polypeptides. Therefore, in one embodiment, the modification promotes the dimerization of the first and second polypeptides. In one embodiment, the modification promotes heterodimerization of the first and second polypeptides. Heterodimerization means that the first polypeptide is different from the second polypeptide, and the first polypeptide will dimerize with the second polypeptide, but the first polypeptide will not dimerize with the second polypeptide, and the second polypeptide will not dimerize with the second polypeptide. In one embodiment, the modification promotes homodimerization of the first and second polypeptides. Homodimerization means that the first polypeptide is the same as the second polypeptide, and the first polypeptide will dimerize with the second polypeptide.

[0357] Various means of promoting the dimerization of two structural domains (e.g., homodimerization or heterodimerization) are known in the art. In one embodiment, the two structural domains may include modifications that enhance dimer formation, such as increasing the affinity of the two structural domains, promoting and / or enabling the formation of one or more disulfide bonds, reducing steric hindrance to dimerization, promoting electrostatic and / or hydrophilic / hydrophobic interactions between the two structural domains, or any combination thereof.

[0358] In practice, any suitable technique may be used to promote dimerization of the Fc region (e.g., the CH3 domain) (i.e., dimerization of at least one of the CH3 domains of the first polypeptide and at least one of the CH3 domains of the second polypeptide).

[0359] Preferably, the modification that enhances the formation of (hetero)dimers between Fc regions (e.g., CH3 domains) is the KiH technique (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 a modified pair of CH3 domains (CH3 heterodimers) undergoing heterodimerization, wherein an asymmetric hydrophobic mutation is introduced between the homodimer CH3 domains. KiH involves introducing such mutations that create a protrusion (“pestle”) at the interface of the first CH3 domain and a corresponding cavity (“mortar”) at the interface of the second CH3 domain, such that the protrusion can be located in the cavity to promote heterodimer assembly and inhibit homodimer formation. Therefore, the KiH variant is thermodynamically favorable for the formation of heterodimers rather than homodimers.

[0360] As a further example, the present invention may employ: chain exchange modified domain (SEED) CH3 dimers (Davis et al., Protein Eng Des Sel., 2010, 23:195-202); electrostatic guidance using DD-KK variants with asymmetric electrostatic interactions (Gunasekaran et al., J Biol Chem., 2010, 285: 19637-46); Zymeworks' Azymetric technology ( https: / / www.zymeworks.com / technologies / azymetric / ); Bispecific binding via antibody based on T-cell receptor (BEAT) (Skegro et al.) , J Biol Chem., 2017,292: 9745-9759); Fast-Ig and ART-Ig ( https: / / www.chugai-pharm.co.jp / english / profile / rd / technologies.html DEKK dimerization technology ( 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); CH3 interface for computational design, such as the 7.8.60 design (Leaver-Fay et al.) ,Structure, 2016, 24: 641-651); the EW-RVT variant, which is designed to replace conservative electrostatic interactions with asymmetric hydrophobic interactions and to add asymmetric long-range electrostatic interactions at the edges of the heterodimer CH3 interface (Choi et al., Mol Cancer Ther., 2013, 12: 2748-59); and the K370E used in the “A107” variant. CH3A -E357N CH3B The mutation replaces the electrostatic interactions of the homodimer with hydrogen bonds that stabilize the heterodimer.

[0361] Any suitable KiH modification known in the art can be used in the practice of this invention. For example, T366W (“mortar”) modification and T366S, L368A and Y407V (“mortar”) modifications can be used (according to EU residue numbers (Edelman et al., Proc Natl Acad Sci US A., 1969, 63: 78-85)).

[0362] The variable domains (e.g., VHH domains) used in this invention are not particularly limited and can contain any antigen-binding site specific to the selected target protein. The selected target protein can be a cancer antigen, an immune cell marker, or a non-human cell antigen.

[0363] In some embodiments, at least one variable domain of the first polypeptide, the second polypeptide, or both the first polypeptide and the second polypeptide is specific to the cancer antigen.

[0364] Various tumor-associated antigens (TAAs) are known in the art. In one embodiment, the variable domain of the present invention (e.g., the VHH domain) is capable of specifically binding to a TAA.

[0365] In some embodiments, at least one variable domain of the first polypeptide, the second polypeptide, or both the first polypeptide and the second polypeptide is specific for the immune cell marker. Various immune cell markers are known in the art. For example, anti-CD163 ADCs targeting macrophages are known in the art (Liu et al., Expert Opinion on Biological Therapy, 2016, 16: 591-593). As a further example, ADCs targeting CXCR4 are known in the art. CXCR4 is highly expressed on T cells, B cells, monocytes, and hematopoietic stem cells, and minimally to noly expressed on non-hematopoietic cells (Liu et al., Expert Opinion on Biological Therapy, 2016, 16: 591-593).

[0366] Furthermore, ADCs targeting non-human cells are known in the art. For example, ADCs targeting intracellular Streptococcus aureus (Streptococcus virosa) have been reported. Streptococcus aureus Antibody-antibiotic conjugates (AACs) (Liu et al., Expert Opinion on Biological Therapy, 2016, 16: 591-593).

[0367] drug

[0368] The drug conjugated to the antibody may also be referred to as "cargo" or "load". The loads used in this invention are not particularly limited and may include any therapeutic or diagnostic reagent that can be conjugated to the antibody. In addition to the use of synthetic cytotoxins, conjugations of other loads to site-specific antibodies have been reported. These other loads include non-cytotoxic compounds such as proteins / peptides, polysaccharides, lipids, and nucleic acids.

[0369] Suitably, the payload may be a small molecule, oligonucleotide (e.g., RNA or DNA), peptide, dye (e.g., IRDye 700 DX), cytotoxic drug, chemotherapeutic agent, antimicrobial agent (e.g., antibiotic), protein degrader, enzyme inhibitor, protein ligand, or immunomodulator. 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, belotecone, deruxtecan, ethanotecan, or camptothecin. Suitablely, in some embodiments, the payload may be a topoisomerase II inhibitor, such as etoposide, doxorubicin, or epirubicin.

[0370] In some embodiments, the drug is selected from the group consisting of: cytotoxic drugs, chemotherapeutic drugs, antimicrobial agents (e.g., antibiotics) or immunomodulators.

[0371] The term "cytotoxic or chemotherapeutic agent" refers to a drug that reduces or eliminates the viability of cells. Suitable cytotoxic or chemotherapeutic agents are known in the art.

[0372] The term "cytotoxic drug" generally refers to a toxic drug. Suitably, the cytotoxic drug can be a chemical molecule potent enough to disrupt the normal growth of tumor cells exposed to it. The cytotoxic drug can kill tumor cells at sufficiently high concentrations. The "cytotoxic drug" can include toxins, such as small molecule toxins of bacterial, fungal, plant, or animal origin, or toxins that are enzymatically active, radioactive isotopes, toxic drugs, chemotherapeutic agents, antibiotics, and lysozymes.

[0373] For example, the cytotoxic drug may be selected from the group consisting of: auristatins, metansins, tubulysins, taxanes, galicariin, amanita toxins, pyrrolobenzodiazepines, camptothecins, and their derivatives. Suitably, the cytotoxic drug may be MMAE, MMAF, DM1, DM4, pyroxine, Dxd, SN38, ethanotecan, doxorubicin, SG3199, or paclitaxel.

[0374] Methods for generating antibody-drug conjugates

[0375] In a further aspect, the present invention provides a method for generating an antibody-drug conjugate (ADC) having a predetermined drug-antibody ratio (DAR), comprising the following steps:

[0376] (i) Provide antibodies as defined herein;

[0377] (ii) Reduce the antibody with a reducing agent; and

[0378] (iii) Conjugate the reduced antibody with the drug.

[0379] In a further aspect, the present invention provides a method for generating an antibody-drug conjugate (ADC) having a predetermined drug-antibody ratio (DAR), comprising the following steps:

[0380] (i) (a) Provides at least one polynucleotide sequence encoding an antibody, the antibody comprising a first polypeptide comprising an immunoglobulin crystallizable fragment (Fc) region and at least one variable domain at the N-terminus of the Fc region;

[0381] (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 mutated to provide a predetermined number of cysteine ​​residues.

[0382] (c) Introducing the at least one polynucleotide sequence into the cell;

[0383] (d) The cells are cultured under conditions suitable for expressing the at least one polynucleotide sequence;

[0384] (e) Isolate the antibodies expressed by the cells;

[0385] (ii) Reduce the antibody with a reducing agent; and

[0386] (iii) Conjugate the reduced antibody with the drug.

[0387] In some embodiments, the antibody further comprises a second polypeptide containing an Fc region, and step (i) (b) further comprises modifying the at least one polynucleotide sequence to encode an engineered IgG hinge region at the N-terminus of the Fc region of the second polypeptide, wherein the engineered IgG hinge region is heterologous to and / or mutated for the Fc region of the second polypeptide 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.

[0388] In some embodiments, the second polypeptide further comprises at least one variable domain at the N-terminus of the engineered IgG hinge region.

[0389] In a further aspect, the present invention provides a method for generating an antibody-drug conjugate (ADC) having a predetermined drug-antibody ratio (DAR), comprising the following steps:

[0390] (i) (a) Provides 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 crystallizable fragment (Fc) region and at least one of the N-terminus of the Fc region as a variable domain of VHH.

[0391] (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 in 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 mutated to provide a predetermined number of cysteine ​​residues;

[0392] (c) Introducing the at least one polynucleotide sequence into the cell;

[0393] (d) The cells are cultured under conditions suitable for expressing the at least one polynucleotide sequence;

[0394] (e) Isolate the antibodies expressed by the cells;

[0395] (ii) Reduce the antibody with a reducing agent; and

[0396] (iii) Conjugate the reduced antibody with the drug.

[0397] The at least one polynucleotide sequence may be modified to encode an engineered IgG hinge region by introducing a nucleic acid sequence encoding the engineered IgG hinge region into the at least one polynucleotide sequence. Alternatively, the at least one polynucleotide sequence may be modified to mutate the native IgG hinge region, thereby providing an engineered IgG hinge region according to the invention, as described herein.

[0398] Modifying the at least one polynucleotide sequence to encode the engineered IgG hinge region according to the invention using conventional techniques in molecular biology is within the capabilities of those skilled in the art. Generally, suitable conventional methods include directed mutagenesis, gene synthesis, and recombinant DNA / RNA techniques.

[0399] Using conventional molecular and cell biology techniques to introduce at least one polynucleotide sequence encoding a component of the present invention into cells is within the capabilities of those skilled in the art. For example, vectors or expression cassettes may be used.

[0400] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. According to the present invention, and as an example, some vectors used in recombinant nucleic acid technology allow entities, such as segments of nucleic acids (e.g., heterologous DNA segments, such as heterologous cDNA segments), to be transferred to target cells and expressed by the target cells. The vector can facilitate the integration of nucleotide sequences encoding antibodies of the present invention to maintain the nucleotide sequences encoding the antibodies of the present invention and their expression within target cells. The expression cassettes described herein comprise nucleic acid regions containing sequences capable of being transcribed.

[0401] The cysteine-based site-specific conjugation can be performed using any suitable method known in the art and described herein.

[0402] In some embodiments, the step of reducing the antibody in step (ii) is the antibody itself. Suitably, a cysteine-based site-specific conjugation strategy involves partially reducing the antibody to reduce the interchain disulfide bonds of the antibody. This directed reduction of the antibody allows control over the DAR of the product, i.e., by selectively reducing the interchain disulfide bonds of the antibody, the conjugation of the antibody to the linker-drug will occur via free cysteine ​​residues generated in the partial reduction step. This contributes to the production of homogeneous ADC products with consistent DAR.

[0403] In some implementations, the step of reducing the antibody in step (ii) is to partially reduce the antibody to reduce the interchain disulfide bonds of the antibody.

[0404] In some embodiments, the reducing agent is dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP).

[0405] In some embodiments, the drug is in the form of a linker-drug conjugate containing a thiol reactive group. The linker may be as described herein.

[0406] In some embodiments, the linker comprises a thiol reactive group selected from the group consisting of: maleimide, bromoacetamide, disulfide, α-haloacetamide, α-halocarbonyl, vinyl sulfone, heteroaryl sulfone, thiosulfonate (ester), electron-deficient aryl halide, ethynylphosphonamide (ester), vinylphosphonite (ester), palladium oxidative addition complex, disulfone, water-soluble allyl sulfone, thiol-alkynyl bioconjugation having a terminal alkyne or cyclooctyne, dibromo-maleimide (DBM) and dithio-maleimide (DTM), heterothiobromo-maleimide (TBM), dibromopyrazine dione, divinylpyrimidine, and DiPODS (two oxadiazolylmethyl sulfone moieties linked by a phenyl group).

[0407] In some embodiments, the thiol reactive group is selected from the group consisting of: maleimide, bromoacetamide, disulfide, α-haloacetamide, α-halocarbonyl, vinyl sulfone, heteroaryl sulfone, thiosulfonate (ester), electron-deficient aryl halide, ethynylphosphonamide (ester), vinylphosphonite (ester), palladium oxidative addition complex.

[0408] In some implementations, step (iii) is performed using a thiol-reactive coupling strategy.

[0409] In some embodiments, the thiol-reactive coupling strategy is based on maleimide conjugation.

[0410] In some implementations, the method of the present invention provides a homogeneous ADC.

[0411] In some implementations, prior to step (ii), all cysteine ​​residues within the engineered IgG hinge region form stable interchain disulfide bonds.

[0412] In some embodiments, in step (iii), the antibody is conjugated to the drug via cysteine-based site-specific conjugation, through all cysteine ​​residues within the hinge region of the engineered IgG.

[0413] In a further aspect, the present invention provides an ADC that is obtained or obtainable by the method of the present invention.

[0414] Pharmaceutical Composition

[0415] In a further aspect, the present invention provides a pharmaceutical composition comprising an ADC according to the invention.

[0416] 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 peptides and / or compounds. Such formulations may, for example, be in a form suitable for intravenous infusion.

[0417] Therefore, in a further aspect, the present invention provides a pharmaceutical composition comprising an ADC according to the invention, and a pharmaceutically acceptable carrier, excipient, and / or diluent.

[0418] Therapeutic uses

[0419] In a further aspect, the present invention provides the ADC of the present invention or the pharmaceutical composition of the present invention for use in therapy.

[0420] In a further aspect, the present invention provides the ADC of the present invention or the pharmaceutical composition of the present invention for use in therapeutic or diagnostic methods.

[0421] In a further aspect, the present invention provides the use of the ADC of the present invention or the pharmaceutical composition of the present invention in the preparation of pharmaceuticals or diagnostic reagents.

[0422] In a further aspect, the present invention provides a therapeutic or diagnostic method comprising administering the ADC of the present invention or the pharmaceutical composition of the present invention to a subject.

[0423] Therapeutic methods include methods for treating diseases and methods for preventing diseases.

[0424] Methods for treating diseases relate to the therapeutic use of the ADC or pharmaceutical composition of the present invention. In this respect, the ADC or pharmaceutical composition according to the present invention can be administered to a subject with an existing disease or condition in order to alleviate, reduce or improve at least one symptom associated with said disease, and / or slow, reduce or block the progression of said disease.

[0425] Methods for preventing disease relate to the preventive use of the ADC or pharmaceutical composition of the present invention. In this respect, the ADC or pharmaceutical composition according to the present invention may be administered to a subject who has not yet contracted the disease and / or does not exhibit any symptoms of the disease, in order to inhibit or weaken the cause of the disease, or reduce or prevent the development of at least one symptom associated with the disease. The subject may be predisposed to the disease or considered to be at risk of developing the disease.

[0426] The disease to be treated and / or prevented could be cancer.

[0427] Suitablely, the cancer can be a solid tumor or a liquid tumor.

[0428] The cancers mentioned can include, for example, neuroblastoma, prostate cancer, bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell), leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, and thyroid cancer.

[0429] In a further aspect, the present invention provides the ADC of the present invention or the pharmaceutical composition of the present invention for use in the treatment of diseases or conditions selected from the group consisting of: cancer, autoimmune diseases, infections, infectious diseases, cardiovascular diseases, and liver metabolic disorders.

[0430] The therapeutic or diagnostic uses of ADCs are 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).

[0431] In some embodiments, the subjects of the medical uses and treatments according to the present invention may be mammals.

[0432] In some implementations, the subject can be a human.

[0433] In some implementations, the subject may alternatively be a non-human mammal, including, for example, a primate, monkey, dog, cat, horse, cow, sheep, pig, rabbit, rat, or mouse.

[0434] In some implementations, the subject may be a patient, such as a human patient.

[0435] In some implementations, the subject may have and / or have been diagnosed with one or more types of cancer.

[0436] In some implementations, the subject may suffer from and / or have been diagnosed with an autoimmune disease, infection, infectious disease, cardiovascular disease, or liver metabolic disorder.

[0437] Nucleic acid sequences and particles

[0438] In a further aspect, the present invention provides one or more nucleic acid sequences capable of expressing antibodies for use according to the present invention.

[0439] In a further aspect, the present invention provides one or more nucleic acid sequences capable of expressing a first polypeptide sequence as defined herein.

[0440] In a further aspect, the present invention provides one or more nucleic acid sequences capable of expressing a second polypeptide sequence as defined herein.

[0441] In a further aspect, the present invention provides one or more nucleic acid sequences capable of expressing a first polypeptide sequence and a second polypeptide sequence as defined herein.

[0442] According to this 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, it means that, if present in a suitable environment, such as within a cell, the nucleic acid can be expressed to produce the peptide or polypeptide.

[0443] Suitablely, the nucleic acid sequence capable of expressing the antibody for use according to the invention may comprise multiple nucleic acid sequences encoding components of the construct (e.g., the first polypeptide, the second polypeptide, the Fc region, the variable domain, the VHH domain, the engineered IgG hinge region, and / or the linker sequence as described herein).

[0444] Those skilled in the art will understand that, due to the degeneracy of the genetic code, many different polynucleotides and nucleic acids can encode the same polypeptide. Furthermore, it should be understood that, using conventional techniques, those skilled in the art can perform nucleotide substitutions that do not affect the sequence of the polypeptide encoded by the polynucleotides described herein, to reflect the codon usage in any particular host organism to which said polypeptide is to be expressed. Suitably, the polynucleotides of the present invention are codon-optimized to enable expression in mammalian cells (particularly those described herein).

[0445] The nucleic acids according to the present invention may comprise DNA and / or RNA. The nucleic acids may be single-stranded or double-stranded. They may also be polynucleotides comprising synthetic or modified nucleotides therein. Many different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and thiophosphate backbones with an acridine or polylysine chain added at the 3' and / or 5' ends of the molecule. It should be understood herein that the polynucleotides may be modified by any method available in the art. Such modifications may be made to enhance the in vivo activity or lifetime of the desired polynucleotide.

[0446] In a further aspect, the present invention provides a vector comprising one or more nucleic acid sequences of the present invention.

[0447] In one embodiment, the vector comprises a plurality of nucleic acid sequences encoding different components provided by the present invention. For example, in one embodiment, the vector comprises a first nucleic acid sequence encoding a first polypeptide of the present invention and a second nucleic acid sequence encoding a second polypeptide of the present invention.

[0448] In the implementation scheme, the vector may be, for example, a plasmid, or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon-based vector, or a synthetic mRNA.

[0449] In one embodiment, the vector is capable of transfecting or transducing cells.

[0450] One or more nucleic acid sequences of the present invention can 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 (e.g., polymers and lipids) and negatively charged nucleic acids involve particle formation. This leads to the complexation and spontaneous formation of nucleic acid particles containing the one or more nucleic acid sequences.

[0451] Therefore, in a further aspect, the present invention provides nucleic acid particles comprising the nucleic acid sequence according to the present invention.

[0452] Different types of nucleic acid-containing particles have been previously described as suitable for delivering nucleic acids (e.g., RNA) in particulate form (see, for example, Kaczmarek, JC et al., 2017, Genome Medicine 9, 60). For non-viral delivery vectors, the nanoparticle encapsulation of nucleic acids physically protects them from degradation and, depending on specific chemistry, can facilitate cellular uptake and endosome escape.

[0453] In the context of this disclosure, the term "particle" refers to a structured entity formed of molecules or molecular complexes, particularly particle-forming compounds. In some embodiments, the particle comprises a coating (e.g., one or more layers or sheets) made of one or more types of amphiphilic substances (e.g., amphiphilic lipids). In this context, the expression "amphiphilic substance" means that the substance has both hydrophilic and lipophilic properties. The coating may also comprise other substances (e.g., other lipids) that are not necessarily amphiphilic. In some embodiments, the term "particle" refers to a micrometer or nanometer-sized structure, such as a dense structure at the micrometer or nanometer scale. According to this disclosure, the term "particle" includes nanoparticles.

[0454] Nucleic acid particles (e.g., RNA particles and / or DNA particles) include formulations based on lipid nanoparticles (LNPs) and lipid complexes (LPX).

[0455] Typically, lipid complexes (LPX) can be obtained by mixing two aqueous phases (i.e., a phase containing nucleic acids (e.g., RNA and / or DNA) and a phase containing lipid dispersions). In some embodiments, the lipid phase comprises liposomes.

[0456] Typically, lipid nanoparticles (LNPs) can be obtained by directly mixing nucleic acids (e.g., RNA and / or DNA) in an aqueous phase with lipids in a phase containing an organic solvent (e.g., ethanol). In that case, lipids or lipid mixtures can be used for particle formation, which does not form a layered (bilayer) phase in water.

[0457] In some embodiments, the LNP comprises, or is composed of, cationic / ionizable lipids and accessory lipids (e.g., phospholipids, cholesterol, and / or polyethylene glycol (PEG) lipids). In some embodiments, in the nucleic acid LNPs (e.g., DNA LNPs) described herein, the nucleic acid (e.g., DNA) is bound to an ionizable lipid occupying a central core of the LNP. In some embodiments, PEG lipids, together with phospholipids, form the surface of the LNP. In some embodiments, the surface comprises a bilayer. In some embodiments, cholesterol and ionizable lipids (in charged and uncharged forms) may be distributed throughout the LNP.

[0458] In some embodiments, nucleic acids (e.g., RNA and / or DNA, such as mRNA) may be non-covalently associated with the particles described herein. In embodiments, the nucleic acids (e.g., RNA and / or DNA, especially mRNA) may adhere to the outer surface of the particles (surface nucleic acids) and / or may be contained within the particles (encapsulated nucleic acids (e.g., encapsulated DNA)).

[0459] In a further aspect, the present invention provides a cell comprising a nucleic acid sequence or nucleic acid particles according to the present invention.

[0460] Reagent test kit

[0461] In a further aspect, the present invention provides a kit comprising the polypeptides, antibodies, nucleic acid sequences, nucleic acid particles, or cells of the present invention.

[0462] In some embodiments, the kit further includes instructions on conjugating one or more drugs or payload molecules to the peptides or antibodies of the present invention.

[0463] In some embodiments, when present, the kit further includes instructions regarding the expression of the nucleic acid (sequence) or nucleic acid particles of the present invention.

[0464] In some embodiments, the kit further includes instructions on preparing the ADC.

[0465] General definition

[0466] This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of embodiments of this disclosure. Numerical ranges include the numbers defining the ranges. Unless otherwise stated, any nucleic acid sequence is written from left to right in a 5' to 3' orientation; amino acid sequences are written from left to right in an amino (N) to carboxyl (C) orientation.

[0467] Where a numerical range is provided, it should be understood that, unless the context explicitly specifies otherwise, each intermediate value between the upper and lower limits of that range, down to one-tenth of the unit of the lower limit, is also explicitly disclosed. Every smaller range between any stated value or intermediate value within the stated range and any other stated value or intermediate value within that stated range is covered in this disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded from the range, and each range in which any one or both limits are included is also covered in this disclosure, subject to any explicitly excluded limits within the stated range. Where a stated range includes one or both of the limits, ranges excluding any or both of those included limits are also included in this disclosure.

[0468] The term "polypeptide" is used in its conventional sense to refer to a series of amino acids, typically L-amino acids, linked together, typically by peptide bonds between the α-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 "residue" is used to refer to an amino acid in an amino acid sequence.

[0469] The term "variant" in relation to polypeptides refers to a polypeptide that has a function equivalent to the amino acid sequence described herein, but includes one or more amino acid substitutions, insertions, or deletions.

[0470] As used herein, the terms “polynucleotide,” “nucleotide,” “nucleic acid sequence,” and “nucleic acid” are intended to be synonymous with each other.

[0471] The terms “variant,” “homologous,” or “derivative” in relation to nucleotide sequences include any substitution, variation, modification, replacement, deletion, or addition to one (or more) nucleic acids of the sequence.

[0472] "Sequence identity" between two nucleic acid or protein sequences refers to the percentage of identical nucleotides between the sequences. The terms "%identical" and "%identical," or similar terms, are intended specifically to refer to the percentage of identical nucleotides or amino acids in an optimal alignment between the sequences to be compared. This percentage is purely statistical, and the differences between the two sequences may, but are not necessarily, be randomly distributed across the entire length of the sequences being compared. The comparison of two sequences is often performed by comparing the sequences relative to segments or "comparison windows" after optimal alignment in order to identify local regions of the respective sequences. The best alignment for comparison can be performed manually, or with the aid of local homology algorithms (Smith and Waterman, 1981, Ads App. Math. 2, 482), global homology algorithms (Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443), similarity search algorithms (Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444), or with computer programs using the aforementioned algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA, in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin). In some implementations, the BLASTN or BLASTP algorithm is used to determine the percentage of identity between two sequences. This algorithm is available on the website of the United States National Center for Biotechnology Information (NCBI) (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 the BLASTN algorithm on the NCBI website include: (i) an expected threshold set to 10; (ii) a word length set to 28; (iii) the maximum match in the query range set to 0; (iv) match / non-match scores set to 1, -2; (v) a gap cost set to linear; and (vi) the use of filters for low-complexity regions.In some embodiments, the algorithm parameters for the BLASTP algorithm used on the NCBI website include: (i) an expected threshold set to 10; (ii) a word length set to 3; (iii) the maximum match in the query range set to 0; (iv) a matrix set to BLOSUM62; (v) gap costs set as follows: presence: 11, extension: 1; and (vi) conditional composition score matrix adjustment.

[0473] The identity percentage is obtained by determining the number of identical positions corresponding to the sequences to be compared, dividing that number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying the result by 100.

[0474] In some embodiments, a degree of similarity or identity is given for regions that comprise at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, or approximately 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, then a degree of identity is given for at least approximately 100, at least approximately 120, at least approximately 140, at least approximately 160, at least approximately 180, or approximately 200 nucleotides (in some embodiments, consecutive nucleotides). In some embodiments, a degree of similarity or identity is given for the entire length of the reference sequence.

[0475] In some implementations, "isolated" means removed (e.g., purified) from its natural state or from an artificial composition (e.g., a composition derived from a production process). For example, nucleic acids, peptides, or polypeptides naturally present in living organisms are not "isolated," but the same nucleic acids, peptides, or polypeptides that are partially or completely separated from their natural coexisting substances are "isolated." Isolated nucleic acids, peptides, or polypeptides may exist in a substantially purified form or may exist in a non-natural environment (e.g., host cells).

[0476] As used in this paper, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence.

[0477] In the context of this disclosure, the term "transcription" refers to the process in which the genetic code in a DNA sequence is transcribed into RNA (especially mRNA). Subsequently, the RNA can be translated into peptides or polypeptides.

[0478] Regarding RNA, the terms "expression" or "translation" refer to the process in the ribosomes of the cell through which the strand of mRNA directs the assembly of amino acid sequences to prepare peptides or polypeptides.

[0479] The term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and their modified forms. The term includes genomic DNA, cDNA, mRNA, recombinant and chemically synthesized molecules. Nucleic acids can exist as single-stranded or double-stranded molecules and as linear or covalently circularly closed molecules. Nucleic acids can be isolated. According to this disclosure, the term "isolated nucleic acid" means that the nucleic acid (i) is amplified in vitro, for example via polymerase chain reaction (PCR) with respect to DNA or in vitro transcription with respect to RNA (using, for example, RNA polymerase), (ii) is recombinantly produced by cloning, (iii) is purified, for example by cutting and separation by gel electrophoresis, or (iv) is synthesized, for example by chemical synthesis.

[0480] The term "nucleoside" (abbreviated as "N" in this document) refers to compounds that can be considered as nucleotides without a phosphate group. While a nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), a nucleotide consists of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[0481] The five standard nucleosides that commonly constitute naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine, and guanosine. These five nucleosides are usually abbreviated as their single-letter codes U, A, T, C, and G, respectively. However, thymidine is more commonly written as "dT" ("d" stands for "deoxy") because it contains a 2'-deoxyfuranose moiety instead of the furanose ring present in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA), not ribonucleic acid (RNA). Conversely, uridine is found in RNA, not DNA. The remaining three nucleosides can be found in both RNA and DNA. In RNA, they would be represented as A, C, and G, while in DNA, they would be represented as dA, dC, and dG.

[0482] The modified purine (A or G) or pyrimidine (C, T or U) base moiety is preferably modified with one or more alkyl groups, more preferably with one or more C1-4 alkyl groups, and even more preferably with one or more methyl groups. Specific examples of the modified purine or pyrimidine base moiety 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-pseuuridine, and N(1)-methyl-uracil.

[0483] In this document, the term "DNA" refers to a nucleic acid molecule comprising deoxyribonucleotide residues. In a preferred embodiment, DNA comprises all or most of deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" means a nucleotide lacking a hydroxyl group at the 2'-position of the β-D-furanose ribosyl group. DNA encompasses, but is not limited to, double-stranded DNA, single-stranded DNA, isolated DNA such as partially purified DNA, substantially pure DNA, synthetic DNA, recombinant DNA, and modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alteration may refer to the addition of a non-nucleotide substance to an internal DNA nucleotide or the end of the DNA. It is also contemplated herein that the nucleotides in the DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the purposes of this disclosure, such altered DNA is considered an analogue of naturally occurring DNA. If the content of deoxyribonucleotide residues in a molecule is greater than 50% based on the total number of nucleotide residues in the molecule (e.g., 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%), then the molecule contains "a majority of deoxyribonucleotide residues". The total number of nucleotide residues in the molecule is the sum of all nucleotide residues (whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or their analogues).

[0484] DNA can be recombinant DNA and can be obtained through the cloning of nucleic acids (especially cDNA). cDNA can be obtained through reverse transcription of RNA.

[0485] The term "RNA" refers to a nucleic acid molecule comprising ribonucleotide residues. In a preferred embodiment, the RNA comprises all or most of ribonucleotide residues. As used herein, "ribonucleotide" means a nucleotide having a hydroxyl group at the 2'-position of the β-D-furanose ribosyl group. RNA encompasses, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinant RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alteration may refer to the addition of a non-nucleotide substance to an internal RNA nucleotide or the terminus of the RNA. It is also contemplated herein that the nucleotides in the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the purposes of this disclosure, such altered / modified nucleotides may be referred to as analogs of naturally occurring nucleotides, and the corresponding RNA comprising such altered / modified nucleotides (i.e., altered / modified RNA) may be referred to as analogs of naturally occurring RNA. If the content of ribonucleotide residues in a molecule is greater than 50% based on the total number of nucleotide residues in the molecule (e.g., 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%), then the molecule contains "a majority of ribonucleotide residues". The total number of nucleotide residues in the molecule is the sum of all nucleotide residues (whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or their analogues).

[0486] "RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), single-stranded RNA (ssRNA), dsRNA, repressive RNA (e.g., antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (e.g., small activating RNA), and immunostimulatory RNA (isRNA). In some embodiments, "RNA" refers to mRNA.

[0487] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly specifies otherwise.

[0488] As used herein, the term “comprising” is synonymous with “including” or “containing” and is inclusive or open-ended, and does not exclude additional undescribed members, elements, or method steps. The term “comprising” also includes the term “consisting of”.

[0489] Embodiments of the present invention

[0490] Various preferred features and embodiments of the invention will now be described with reference to the following numbered paragraphs:

[0491] 1. A polypeptide comprising an immunoglobulin crystallizable fragment (Fc) region, at least one variable domain at the N-terminus of the Fc region, and an engineered IgG hinge region between the Fc region and the at least one variable domain, and

[0492] The engineered IgG hinge region is heterologous to the Fc region and / or mutated to provide a predetermined number of cysteine ​​residues.

[0493] 2. An antibody comprising a first polypeptide, the first polypeptide comprising an Fc region, at least one variable domain at the N-terminus of the Fc region, and an engineered IgG hinge region between the Fc region and the at least one variable domain, and

[0494] The engineered IgG hinge region is heterologous to the Fc region and / or mutated to provide a predetermined number of cysteine ​​residues.

[0495] 3. Antibody-drug conjugates (ADCs).

[0496] The antibody is conjugated to the drug via cysteine-based site-specific conjugation.

[0497] The antibody comprises a first polypeptide containing an immunoglobulin crystallizable fragment (Fc) region, at least one variable domain at the N-terminus of the Fc region, and an engineered IgG hinge region between the Fc region and the at least one variable domain.

[0498] The engineered IgG hinge region is heterologous to the Fc region and / or mutated to provide a predetermined number of cysteine ​​residues.

[0499] 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: single-chain variable fragments (scFv); Fab; Fab'; F(ab)'2; Fv; single-domain antibody (sdAb); VHH; single-chain variable domain; designed ankyrin repeat sequence protein (DARPin); and aptamers.

[0500] 5. An antibody or ADC according to any one of paragraphs 2 to 4, wherein the antibody is selected from the group consisting of full-length immunoglobulin, scFv-Fc, Fab-Fc, Fv-Fc, sdAb-Fc, or VHH-Fc.

[0501] 6. The antibody or ADC according to any one of paragraphs 2 to 5, wherein the antibody is VHH-Fc.

[0502] 7. An antibody or ADC according to any one of paragraphs 2-6, wherein the antibody further comprises a second polypeptide, the second polypeptide comprising an Fc region and an engineered IgG hinge region at the N-terminus of the Fc region, wherein the engineered IgG hinge region is heterologous and / or mutated with respect to the Fc region of the second polypeptide 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.

[0503] 8. The antibody or ADC according to paragraph 7, wherein the second polypeptide further comprises at least one variable domain at the N-terminus of the engineered IgG hinge region; preferably, wherein the at least one variable domain is selected from the group consisting of: single-chain variable fragments (scFv); Fab; Fab'; F(ab)'2; Fv; single-domain antibody (sdAb); VHH; single-chain variable domain; designed ankyrin repeat sequence protein (DARPin); and aptamers; more preferably, wherein the at least one variable domain is VHH.

[0504] 9. The antibody or ADC according to paragraph 8, wherein the ADC comprises one of the following:

[0505] a) A first polypeptide containing VHH and a second polypeptide containing VHH;

[0506] b) A first polypeptide containing VHH and a second polypeptide containing Fab;

[0507] c) A first polypeptide containing Fab and a second polypeptide containing VHH; or

[0508] d) A first polypeptide containing Fab and a second polypeptide containing Fab.

[0509] 10. Antibody-drug conjugates (ADCs).

[0510] The antibody is conjugated to the drug via cysteine-based site-specific conjugation.

[0511] The antibody comprises a first polypeptide and a second polypeptide, wherein each of the first polypeptide and the second polypeptide comprises an immunoglobulin crystallizable fragment (Fc) region, at least one variable domain at the N-terminus of the Fc region as a VHH, and an engineered IgG hinge region between the Fc region and the at least one VHH.

[0512] 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

[0513] The engineered IgG hinge region is heterologous to the Fc region and / or mutated to provide a predetermined number of cysteine ​​residues.

[0514] 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 the polypeptide.

[0515] 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.

[0516] 13. The polypeptide, antibody, or ADC according to any one of the preceding paragraphs, wherein the engineered IgG hinge region of the first polypeptide additionally comprises at least one cysteine ​​residue, and preferably two, three, four, five, six, seven, eight, or nine cysteine ​​residues, compared to the natural hinge region of the Fc region of the first polypeptide.

[0517] 14. The polypeptide, antibody, or ADC according to any one of the preceding paragraphs, wherein the engineered IgG hinge region of the first polypeptide contains at least one less cysteine ​​residue than the native hinge region of the Fc region of the first polypeptide, preferably at least two, three, four, five, six, seven, eight, or nine less cysteine ​​residues than the native hinge region of the Fc region of the first polypeptide.

[0518] 15. The polypeptide, antibody, or ADC according to any one of paragraphs 7-14, wherein the engineered IgG hinge region of the second polypeptide additionally comprises at least one cysteine ​​residue, and preferably two, three, four, five, six, seven, eight, or nine cysteine ​​residues, compared to the native hinge region of the Fc region of the second polypeptide.

[0519] 16. The polypeptide, antibody, or ADC according to any one of paragraphs 7-14, wherein the engineered IgG hinge region of the second polypeptide contains at least one less cysteine ​​residue than the native hinge region of the Fc region of the second polypeptide, preferably at least two, three, four, five, six, seven, eight, or nine less cysteine ​​residues than the native hinge region of the Fc region of the second polypeptide.

[0520] 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 with the sequence shown in any one of SEQ ID NO: 1, 2, 3, 4, or 5.

[0521] 18. The polypeptide, antibody, or ADC according to any one of the preceding paragraphs, wherein the engineered IgG hinge region is an IgG1 hinge region.

[0522] 19. The polypeptide, antibody, or ADC according to any one of the preceding paragraphs, wherein the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 1, or a fragment thereof and / or a variant thereof, the latter containing an amino acid sequence having at least 70% identity with SEQ ID NO: 1.

[0523] 20. The polypeptide, antibody, or ADC according to any one of paragraphs 1-17, wherein the engineered IgG hinge region is an IgG2 hinge region.

[0524] 21. The polypeptide, antibody, or ADC according to any one of paragraphs 1-17 or 20, wherein the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 2, or a fragment thereof and / or a variant thereof, the latter containing an amino acid sequence having at least 70% identity with SEQ ID NO: 2.

[0525] 22. The polypeptide, antibody, or ADC according to any one of paragraphs 1-17, wherein the engineered IgG hinge region is an IgG3 hinge region.

[0526] 23. The polypeptide, antibody, or ADC according to any one of paragraphs 1-17 or 22, wherein the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, or a fragment thereof and / or a variant thereof, the latter containing an amino acid sequence having at least 70% identity with SEQ ID NO: 3 or SEQ ID NO: 4.

[0527] 24. The polypeptide, antibody, or ADC according to any one of paragraphs 1-17, 22, or 23, wherein the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7, or a fragment thereof and / or a variant thereof, the latter containing an amino acid sequence having at least 70% identity with SEQ ID NO: 6 or SEQ ID NO: 7.

[0528] 25. The polypeptide, antibody, or ADC according to any one of paragraphs 1-17, wherein the engineered IgG hinge region is an IgG4 hinge region.

[0529] 26. The polypeptide, antibody, or ADC according to any one of paragraphs 1-17 or 25, wherein the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 5, or a fragment thereof and / or a variant thereof, the latter containing an amino acid sequence having at least 70% identity with SEQ ID NO: 5.

[0530] 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 shown in any one of SEQ ID NO: 8-118, preferably an amino acid sequence shown in SEQ ID NO: 8, 9, 12, 13, 18, 51, 67, 74, 84, 100, 113, or 118.

[0531] 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 the second polypeptide contains at least one amino acid modification to avoid O-glycosylation, relative to the native IgG hinge region of the Fc region of the first polypeptide and / or the second polypeptide.

[0532] 29. The polypeptide, antibody, or ADC according to paragraph 28, wherein the at least one amino acid modification for avoiding O-glycosylation is an amino acid substitution.

[0533] 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 shown in any one of SEQ ID NO: 34-44, 51-61, 64-67, 70-75, 78-87, 90-111, or 113.

[0534] 31. The polypeptide, antibody, or ADC according to any one of paragraphs 28-30, wherein the engineered IgG hinge region is an IgG3 hinge region, and the at least one amino acid modification is selected from S. 241JJ X, T 241MM The group consisting of X and its combinations, wherein the numbering follows the Kabat numbering scheme.

[0535] 32. The polypeptide, antibody, or ADC according to any one of the preceding paragraphs, wherein the engineered IgG hinge region is a human IgG hinge region or is derived from a human IgG hinge region.

[0536] 33. The polypeptide, antibody, or ADC according to any one of the preceding paragraphs, wherein the antibody is selected from the group consisting of: human antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies, monoclonal antibodies, and polyclonal antibodies.

[0537] 34. The polypeptide, antibody, or ADC according to any one of the preceding paragraphs, wherein at least one variable domain of the first polypeptide, the second polypeptide, or both the first polypeptide and the second polypeptide is specific to the cancer antigen.

[0538] 35. The polypeptide, antibody, or ADC according to any one of the preceding paragraphs, wherein the antibody is conjugated to the drug via cysteine-based site-specific conjugation, through all cysteine ​​residues within the hinge region of the engineered IgG.

[0539] 36. The polypeptide, antibody, or ADC according to any one of the preceding paragraphs, wherein the ADC has a drug-antibody ratio (DAR) of about 2 to about 12, preferably a DAR of about 4 to about 8.

[0540] 37. The polypeptide, antibody, or ADC according to any one of the preceding paragraphs, wherein the ADC has a drug-antibody ratio (DAR) of about 2, 4, 6, 8, 10, or 12, preferably about 8 DAR.

[0541] 38. The polypeptide, antibody, or ADC according to any one of the preceding paragraphs, wherein the drug is selected from the group consisting of cytotoxic drugs, antimicrobial agents, or immunomodulators.

[0542] 39. A method for generating an antibody-drug conjugate (ADC) having a predetermined drug-antibody ratio (DAR), comprising the following steps:

[0543] (i) Provide an antibody as defined in any of paragraphs 2-38;

[0544] (ii) Reduce the antibody with a reducing agent; and

[0545] (iii) Conjugate the reduced antibody with the drug.

[0546] 40. A method for generating an antibody-drug conjugate (ADC) having a predetermined drug-antibody ratio (DAR), comprising the following steps:

[0547] (i) (a) Provides at least one polynucleotide sequence encoding an antibody, the antibody comprising a first polypeptide comprising an immunoglobulin crystallizable fragment (Fc) region and at least one variable domain at the N-terminus of the Fc region;

[0548] (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 mutated to provide a predetermined number of cysteine ​​residues.

[0549] (c) Introducing the at least one polynucleotide sequence into the cell;

[0550] (d) The cells are cultured under conditions suitable for expressing the at least one polynucleotide sequence;

[0551] (e) Isolate the antibodies expressed by the cells;

[0552] (ii) Reduce the antibody with a reducing agent; and

[0553] (iii) Conjugate the reduced antibody with the drug.

[0554] 41. A method for generating an antibody-drug conjugate (ADC) having a predetermined drug-antibody ratio (DAR), comprising the following steps:

[0555] (i) (a) Provides 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 crystallizable fragment (Fc) region and at least one of the N-terminus of the Fc region as a variable domain of VHH.

[0556] (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 in 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 mutated to provide a predetermined number of cysteine ​​residues;

[0557] (c) Introducing the at least one polynucleotide sequence into the cell;

[0558] (d) The cells are cultured under conditions suitable for expressing the at least one polynucleotide sequence;

[0559] (e) Isolate the antibodies expressed by the cells;

[0560] (ii) Reduce the antibody with a reducing agent; and

[0561] (iii) Conjugate the reduced antibody with the drug.

[0562] 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 interchain disulfide bonds of the antibody.

[0563] 43. The method according to any one of paragraphs 39-42, wherein the reducing agent is dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP).

[0564] 44. The method according to any one of paragraphs 39-43, wherein the drug is in the form of a linker-drug conjugate containing a thiol reactive group.

[0565] 45. According to the method described in paragraph 44, the thiol reactive group is selected from the group consisting of: maleimide, bromoacetamide, disulfide, α-haloacetamide, α-halocarbonyl, vinyl sulfone, heteroaryl sulfone, thiosulfonate (ester), electron-deficient aryl halide, ethynylphosphonamide (ester), vinylphosphonite (ester), palladium oxidative addition complex.

[0566] 46. ​​The method according to any one of paragraphs 39-45, wherein step (iii) is carried out using a mercapto-reactive coupling strategy.

[0567] 47. The method according to paragraph 46, wherein the thiol reactive coupling strategy is based on maleimide conjugation.

[0568] 48. The method according to any one of paragraphs 39-47, wherein the method provides a homogeneous ADC.

[0569] 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 at the N-terminus of the Fc region of the second polypeptide, wherein the engineered IgG hinge region is heterologous and / or mutated for the Fc region of the second polypeptide 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.

[0570] 50. The method according to paragraph 49, wherein the second polypeptide further comprises at least one variable domain at the N-terminus of the engineered IgG hinge region; preferably, wherein the at least one variable domain is selected from the group consisting of: single-chain variable fragments (scFv); Fab; Fab'; F(ab)'2; Fv; single-domain antibody (sdAb); VHH; single-chain variable domain; designed ankyrin repeat sequence protein (DARPin); and aptamers; more preferably, wherein the at least one variable domain is VHH.

[0571] 51. The method according to any one of 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.

[0572] 52. The method according to any one of paragraphs 40-51, wherein the engineered IgG hinge region of the first polypeptide additionally comprises at least one cysteine ​​residue, and preferably two, three, four, five, six, seven, eight or nine cysteine ​​residues, compared to the natural hinge region of the Fc region of the first polypeptide.

[0573] 53. The method according to any one of paragraphs 40-51, wherein the engineered IgG hinge region of the first polypeptide contains at least one less cysteine ​​residue than the natural hinge region of the Fc region of the first polypeptide, preferably at least two, three, four, five, six, seven, eight or nine cysteine ​​residues less than the natural hinge region of the Fc region of the first polypeptide.

[0574] 54. The method according to any one of paragraphs 42 or 49-53, wherein the engineered IgG hinge region of the second polypeptide additionally comprises at least one cysteine ​​residue, and preferably two, three, four, five, six, seven, eight or nine cysteine ​​residues, compared to the natural hinge region of the Fc region of the second polypeptide.

[0575] 55. The method according to any one of paragraphs 42 or 49-53, wherein the engineered IgG hinge region of the second polypeptide contains at least one less cysteine ​​residue than the natural hinge region of the Fc region of the second polypeptide, preferably at least two, three, four, five, six, seven, eight or nine cysteine ​​residues less than the natural hinge region of the Fc region of the second polypeptide.

[0576] 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 with the sequence shown in any one of SEQ ID NO: 1, 2, 3, 4 or 5.

[0577] 57. The method according to any one of paragraphs 40-56, wherein the engineered IgG hinge region is an IgG1 hinge region.

[0578] 58. The method according to any one of paragraphs 40-57, wherein the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 1, or a fragment thereof and / or a variant thereof, the latter containing an amino acid sequence having at least 70% identity with SEQ ID NO: 1.

[0579] 59. The method according to any one of paragraphs 40-56, wherein the engineered IgG hinge region is an IgG2 hinge region.

[0580] 60. The method according to any one of paragraphs 40-56 or 59, wherein the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 2, or a fragment thereof and / or a variant thereof, the latter containing an amino acid sequence having at least 70% identity with SEQ ID NO: 2.

[0581] 61. The method according to any one of paragraphs 40-56, wherein the engineered IgG hinge region is an IgG3 hinge region.

[0582] 62. The method according to any one of paragraphs 40-56 or 61, wherein the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 39 or SEQ ID NO: 40, or a fragment thereof and / or a variant thereof, the latter comprising an amino acid sequence having at least 70% identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 39 or SEQ ID NO: 40.

[0583] 63. The method according to any one of paragraphs 40-56, wherein the engineered IgG hinge region is an IgG4 hinge region.

[0584] 64. The method according to any one of paragraphs 40-56 or 63, wherein the engineered IgG hinge region has the amino acid sequence shown in SEQ ID NO: 5, or a fragment thereof and / or a variant thereof, the latter containing an amino acid sequence having at least 70% identity with SEQ ID NO: 5.

[0585] 65. The method according to any one of paragraphs 40-64, wherein the engineered IgG hinge region has an amino acid sequence shown in any one of SEQ ID NO: 8-118, preferably an amino acid sequence shown in SEQ ID NO: 8, 9, 12, 13, 18, 51, 67, 74, 84, 100, 113 or 118.

[0586] 66. The method according to any one of paragraphs 40-65, wherein the engineered IgG hinge region of the first polypeptide and / or the second polypeptide contains at least one amino acid modification to avoid O-glycosylation, relative to the natural hinge region of the Fc region of the first polypeptide and / or the second polypeptide.

[0587] 67. The method according to paragraph 66, wherein the at least one amino acid modification for avoiding O-glycosylation is an amino acid substitution.

[0588] 68. The method according to any one of paragraphs 40-67, wherein the engineered IgG hinge region has an amino acid sequence shown in any one of SEQ ID NO: 34-44, 51-61, 64-67, 70-75, 78-87, 90-111 or 113.

[0589] 69. The method according to any one of paragraphs 66-68, wherein the engineered IgG hinge region is an IgG3 hinge region, and the at least one amino acid modification is selected from S. 241JJ X, T 241MM The group consisting of X and its combinations, wherein the numbering follows the Kabat numbering scheme.

[0590] 70. The method according to any one of paragraphs 40-69, wherein prior to step (ii), all cysteine ​​residues within the hinge region of the engineered IgG form stable interchain disulfide bonds.

[0591] 71. The method according to any one of paragraphs 40-70, wherein the engineered IgG hinge region is a human IgG hinge region or is derived from a human IgG hinge region.

[0592] 72. The method according to any one of paragraphs 40 or 42-71, wherein the antibody is selected from the group consisting of: human antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies, monoclonal antibodies and polyclonal antibodies.

[0593] 73. The method according to paragraph 40 or any one of 42-72, wherein the antibody is selected from the group consisting of full-length immunoglobulin, scFv-Fc, Fab-Fc, Fv-Fc, sdAb-Fc or VHH-Fc.

[0594] 74. The method described in paragraph 73, wherein the antibody is VHH-Fc.

[0595] 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 the second polypeptide is selected from the group consisting of: single-chain variable fragments (scFv); Fab; Fab'; F(ab)'2; Fv; single-domain antibody (sdAb); VHH; single-chain variable domain; designed ankyrin repeat sequence protein (DARPin); and aptamers.

[0596] 76. The method according to any one of paragraphs 40-75, wherein at least one variable domain of the first polypeptide, the second polypeptide, or both the first polypeptide and the second polypeptide is specific to the cancer antigen.

[0597] 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 cysteine ​​residues within the hinge region of the engineered IgG.

[0598] 78. The method according to any one of paragraphs 40-77, wherein the ADC has a drug-antibody ratio (DAR) of about 2 to about 12, preferably about 4 to about 8.

[0599] 79. The method according to any one of paragraphs 40-78, wherein the ADC has a drug-antibody ratio (DAR) of about 2, 4, 6, 8, 10 or 12, preferably about 8 DAR.

[0600] 80. The method according to any one of paragraphs 40-79, wherein the drug is selected from the group consisting of cytotoxic drugs, antimicrobial agents, or immunomodulators.

[0601] 81. An ADC obtained or available by any of the methods described in paragraphs 39-80.

[0602] 82. A pharmaceutical composition comprising an ADC according to any one of paragraphs 3-38 or 81, and a pharmaceutically acceptable carrier, excipient, and / or diluent.

[0603] 83. The ADC according to any one of paragraphs 3-38 or 81, or the pharmaceutical composition according to paragraph 82, for use in a therapeutic or diagnostic method.

[0604] 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 disease, cardiovascular disease, and liver metabolic disorder.

[0605] 85. One or more nucleic acid sequences capable of expressing a polypeptide or antibody according to any one of paragraphs 1 to 38 or 81.

[0606] 86. Nucleic acid particles comprising the nucleic acid sequence as described in paragraph 85.

[0607] 87. A cell comprising a construct, nucleic acid sequence, or nucleic acid particle according to any one of paragraphs 1 to 38, 81, 85, or 86.

[0608] 88. A method for preparing an ADC, comprising:

[0609] i) Expressing nucleic acid sequences or nucleic acid particles according to paragraphs 85 or 86 to produce antibodies;

[0610] ii) Reduce the antibody with a reducing agent; and

[0611] iii) Conjugate the reduced antibody with the drug.

[0612] The publications discussed herein are provided only for their content prior to the filing date of this application. Nothing herein should be construed as an admission that such publications constitute prior art to the claims appended herein.

[0613] The invention will now be further described by way of embodiments intended to assist those skilled in the art in implementing the invention, and not intended to limit the scope of the invention in any way.

[0614] Further embodiments of the invention involving IgG3-derived hinges

[0615] Certain preferred embodiments of the present invention relate to providing an ADC comprising an IgG3 or IgG3-derived hinge region and optionally engineered or heterologous (i.e., non-IgG3 or IgG3-derived) Fc region. These embodiments can be understood in conjunction with other embodiments / details of the invention herein, and also in conjunction with the following numbered paragraphs:

[0616] A1. A polypeptide comprising an immunoglobulin crystallizable fragment (Fc) region, at least one variable domain at the N-terminus of the Fc region, and an IgG3 hinge region, a fragment thereof, or a derivative thereof between the Fc region and the at least one variable domain.

[0617] A2. An antibody comprising a first polypeptide comprising an Fc region, at least one variable domain at the N-terminus of the Fc region, and an IgG3 hinge region, a fragment thereof, or a derivative thereof between the Fc region and the at least one variable domain.

[0618] A3. Antibody-drug conjugates (ADCs).

[0619] The antibody is conjugated to the drug via cysteine-based site-specific conjugation.

[0620] The antibody comprises a first polypeptide containing an Fc region, at least one variable domain at the N-terminus of the Fc region, and an IgG3 hinge region, a fragment thereof, or a derivative thereof between the Fc region and the at least one variable domain.

[0621] A4. The polypeptide, antibody, or ADC according to any one of paragraphs A1 to A3, wherein the IgG hinge region is a heterologous engineered IgG hinge region for the Fc region and / or mutated to provide a predetermined number of cysteine ​​residues.

[0622] A5. The polypeptide, antibody, or ADC according to any one of paragraphs A1 to A4, wherein the Fc region is not an IgG3 Fc region, a variant, fragment, or derivative thereof, and optionally the Fc region is an IgG1 Fc region, an IgG2 Fc region, or an IgG4 Fc region.

[0623] A6. An 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 IgG3 hinge region, a fragment thereof, or a derivative thereof at the N-terminus of the Fc region, optionally wherein the hinge region is a heterologous engineered IgG hinge region for the Fc region of the second polypeptide and / or 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.

[0624] A7. The antibody or ADC according to paragraph A6, wherein the second polypeptide further comprises at least one variable domain at the N-terminus of the engineered IgG hinge region.

[0625] A8. Antibody-drug conjugates (ADCs).

[0626] The antibody is conjugated to the drug via cysteine-based site-specific conjugation.

[0627] The antibody comprises a first polypeptide and a second polypeptide, wherein each of the first polypeptide and the second polypeptide comprises an immunoglobulin crystallizable fragment (Fc) region, at least one of the N-terminus of the Fc region serving as a variable domain of a VHH, and an IgG3 hinge region, a derivative thereof, or a fragment thereof between the Fc region and the at least one VHH.

[0628] The IgG hinge regions of the first polypeptide and the second polypeptide are complementary IgG hinge regions, and

[0629] Optionally, the IgG hinge region is a heterologous engineered IgG hinge region for the Fc region and / or mutated to provide a predetermined number of cysteine ​​residues.

[0630] A9. The polypeptide, antibody, or ADC according to any one of the preceding paragraphs A#, wherein the engineered IgG hinge region of the first polypeptide additionally comprises at least one cysteine ​​residue relative to the native IgG3 hinge region, and optionally wherein the engineered IgG hinge region of the second polypeptide additionally comprises at least one cysteine ​​residue relative to the native IgG3 hinge region.

[0631] A10. The polypeptide, antibody, or ADC according to any one of the preceding paragraphs A#, wherein the engineered IgG hinge region of the first polypeptide contains at least one less cysteine ​​residue compared to the native IgG3 hinge region, and optionally wherein the engineered IgG hinge region of the second polypeptide contains at least one less cysteine ​​residue compared to the native IgG3 hinge region.

[0632] A11. The polypeptide, antibody, or ADC according to any one of the preceding paragraphs A#, wherein the IgG hinge region is:

[0633] (i) Having the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, or fragments thereof and / or variants thereof, the latter containing an amino acid sequence having at least 70% identity with SEQ ID NO: 3 or SEQ ID NO: 4; or

[0634] (ii) Having the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7, or a fragment thereof and / or a variant thereof, the latter containing an amino acid sequence having at least 70% identity with SEQ ID NO: 6 or SEQ ID NO: 7.

[0635] A12. The polypeptide, antibody, or ADC according to any one of the preceding paragraphs A#, wherein the IgG hinge region has an amino acid sequence shown in any one of SEQ ID NO: 28-113.

[0636] A13. The polypeptide, antibody, or ADC according to any one of the preceding paragraphs A#, wherein the IgG hinge region of the first polypeptide and / or the second polypeptide contains at least one amino acid modification to avoid O-glycosylation, relative to the native IgG3 hinge region.

[0637] A14. The polypeptide, antibody, or ADC according to any one of the preceding paragraphs A#, wherein the IgG hinge region is a human IgG3 hinge region or is derived from a human IgG3 hinge region.

[0638] A15. The ADC according to any one of paragraphs A3 to A14, wherein the antibody is conjugated to the drug via a cysteine-based site-specific conjugation, through all cysteine ​​residues within the IgG hinge region.

[0639] A16. An ADC according to any one of paragraphs A3 to A15, wherein the ADC has a drug-antibody ratio (DAR) of about 2 to about 12, preferably about 4 to about 8.

[0640] A17. An ADC according to any one of paragraphs A3 to A16, wherein the ADC has a drug-antibody ratio (DAR) of about 2, 4, 6, 8, 10 or 12, preferably about 8 DAR.

[0641] A18. The polypeptide, antibody, or ADC according to any one of paragraphs A1 to A17, wherein the variable domain at the N-terminus of the Fc region in the first and / or first and second polypeptides is VHH.

[0642] A19. The polypeptide, antibody, or ADC according to any one of paragraphs A1 to A18, wherein the antibody or ADC is stable in serum.

[0643] A20. The polypeptide, antibody, or ADC according to any one of paragraphs A1 to A19, for use in a therapeutic or diagnostic method.

[0644] Other aspects of the invention (e.g., those relating to methods, compositions, and nucleic acids) are equally applicable to the IgG3 hinge embodiments of the invention set forth in this section.

[0645] Example

[0646] Materials and methods

[0647] Expression of recombinant antibody and VHH-Fc fusion protein

[0648] Human codon-optimized antibodies and VHH-Fc sequences containing engineered and / or heterologous hinge sequences were generated through gene synthesis and cloned into the pTWIST expression vector (Twist Bioscience). For recombinant expression of the antibody and VHH-Fc fusion, Expi293 cells were transfected with ExpiFectamine (ThermoFisherScientific) using the manufacturer's experimental protocol. Culture supernatant was harvested from the Expi293 production cell line after 4–5 days, centrifuged, and the recombinant protein was used for further characterization and purification.

[0649] Toxin payload conjugated with antibody and VHH-Fc fusion protein

[0650] Add 100 mM EDTA buffer to a solution of the target antibody / VHH-Fc fusion protein (1-20 mg / ml, 1.0 equivalent) in 20 mM phosphate-buffered saline to a final concentration of 1 mM. Adjust the pH of the solution to 7.0-8.0 with 0.5 M disodium hydrogen phosphate. Then, add 25 mM of freshly prepared TCEP hydrochloride (3.0-6.0 equivalent, adjusted according to the desired DAR value) in 20 mM phosphate-buffered saline (pH 7.0-8.0) to the antibody solution and mix gently. After 60-90 minutes, add 30 mM of the linker-load (5.0-10.0 equivalent, adjusted according to the desired DAR value) in DMSO to the antibody solution. Gently mix the resulting solution and incubate at 22-37°C. Two hours later, the reaction system was purified, and the buffer was exchanged using ultrafiltration or gel filtration with 20 mM histidine buffer (pH 6.0) or another suitable buffer to provide the antibody-drug conjugate. The drug-antibody ratio (DAR) was analyzed using hydrophobic interaction chromatography or mass spectrometry.

[0651] Western blot for aggregate detection

[0652] Western blot analysis was performed using the supernatant of previously cultured Expi293 cells as the sample. The Western blot was performed using a Jess device from BioTechne.

[0653] According to the manufacturer's experimental protocol, the supernatant was diluted 1:10 and applied to the machine. Separation was performed under non-reducing conditions, and subsequent detection was conducted using an anti-human HRP antibody.

[0654] Affinity to target antigen

[0655] For affinity measurements, the Octet HTX device from Sartorius was used.

[0656] Commercially available AHC biosensors were soaked in kinetic buffer (KB) purchased from the instrument manufacturer for at least 10 minutes. After a 60-second baseline in KB, the target antibody / VHH-Fc protein (or the corresponding ADC) was loaded onto the sensor for 300 seconds or until a response threshold of 0.7–1 nm was reached. After a 120-second baseline in KB, association with antigen 1 was measured for 600 seconds using seven different concentrations of antigen (starting at 50 nM and decreasing to 0.78125 nM in a 1:1 serial dilution series). As a reference, KB was measured without antigen. Dissociation was acquired for 600 seconds in KB. The signal from the reference well was subtracted from the signals from all other biosensors, and the signals of these processed data were aligned with the baseline mean before the association step. For inter-step correction, the data were aligned with the dissociation step, and Savitzky-Golay filtering was applied to all curves. A 1:1 Langmuir binding model was used to globally fit association and dissociation.

[0657] Affinity to target antigen

[0658] For affinity measurements, the Octet HTX device from Sartorius was used.

[0659] Commercially available Ni-NTA or SAX2.0 biosensors were soaked in kinetic buffer (KB) purchased from the instrument manufacturer for at least 10 minutes. After a 60-second baseline in KB, a His-tagged or biotinylated antigen was loaded for 300 seconds or until a 1 nM threshold for response was reached. After a 120-second baseline in KB, association with the target protein or ADC containing Fc was measured for 600 seconds using seven different concentrations (starting at 50 nM and decreasing to 0.78125 nM in a 1:1 serial dilution series). For reference, KB measurements were performed without the tested item. Dissociation was acquired in KB for 600 seconds. The signal from the reference well was subtracted from the signals from all other biosensors, and the signals of these processed data were aligned with the baseline mean before the association step. For inter-step correction, the data were aligned with the dissociation step, and Savitzky-Golay filtering was applied to all curves. A 1:1 Langmuir binding model was used to globally fit association and dissociation.

[0660] Affinity for CD64

[0661] For affinity measurements, the Octet HTX device from Sartorius was used.

[0662] Commercially available Ni-NTA biosensors were soaked in kinetic buffer (KB) purchased from the instrument manufacturer for at least 10 minutes. After a 60-second baseline in KB, CD64 with a His tag was loaded for 300 seconds or until a 1 nM threshold for the response was reached. After a 120-second baseline in KB, association with the target antibody / VHH-Fc / ADC was measured for 600 seconds using seven different concentrations of the test item (starting at 50 nM and decreasing to 0.78125 nM in a 1:1 serial dilution series). For reference, KB was measured without the test item. Dissociation was acquired for 600 seconds in KB. The signal from the reference well was subtracted from the signals from all other biosensors, and the signals of these processed data were aligned with the baseline mean before the association step. For inter-step correction, the data were aligned with the dissociation step, and Savitzky-Golay filtering was applied to all curves. A 1:1 Langmuir binding model was used for global fitting of association and dissociation.

[0663] Affinity for FcRn

[0664] To determine the binding to FcRn, the SAX2.0 biosensor was immersed in KB for 10 min, followed by a 60 sec baseline in KB. The loading of biotinylated human FcRn was performed for 300 sec or until the 1.5 nm threshold of response was reached. After subsequent quenching in 100 μg / mL biocytidine solution, the baseline was measured for 120 sec in 100 mM sodium phosphate, 150 mM NaCl, 0.05% Tween-20 (pH 6.0) buffer (KB-pH 6). The Fc-containing test item was diluted in KB-pH 6 to a 1:1 serial dilution from 1600 nM to 25 nM. Association was measured for 60 sec, followed by 60 sec of dissociation in KB-pH 6. The signal from the reference well was subtracted from the signals from all other biosensors, and the signals from these processed data were aligned with the average of the second baseline. For inter-step correction, the data were aligned with the dissociation step, and Savitzky-Golay filtering was applied to all curves. A 1:1 Langmuir binding model was used to globally fit association and dissociation. Because the FcRn interaction exhibits heterogeneous binding behavior with the Fc moiety of the antibody, only the first 5 or 10 seconds of dissociation were fitted.

[0665] Dynamic light scattering (DLS) and nano-differential scanning fluorescence (NanoDSF)

[0666] Thermal stability (via NanoDSF) and size distribution (via DLS) were investigated using the Prometheus PANTA from NanoTemper. 10 μL of antibody / VHH-Fc protein or ADC sample was loaded into a capillary. After loading, the capillary was mounted into the instrument and DLS analysis was performed. To assess thermal stability, all samples were subsequently subjected to temperature increases of 1 °C / min from 25 °C to 95 °C. During this process, the intrinsic fluorescence of the protein was measured at 350 nm and 330 nm. This ratio was plotted against temperature, and the first derivative was calculated. The minimum and maximum values ​​correspond to the TM values.

[0667] Size exclusion chromatography (SEC)

[0668] Aggregation analysis was performed via SEC using an Agilent Infinity II HPLC system and a Biozen 1.8 μm dSEC-2, 200 Å LC column (300 × 4.6 mm). The flow rate was adjusted to 0.25 mL / min, resulting in a pressure of approximately 255 bar. The mobile phase consisted of 0.2 M potassium phosphate, 250 mM KCl (pH 6.2), and 5% acetonitrile. Each run took 20 minutes, excluding the 2–3 minute wash interval between analyses. 10 μL of the antibody / VHH-Fc fusion protein or ADC assay was applied and detected by absorbance at 280 nm.

[0669] EC50 measurement

[0670] Antigen-positive or negative cells were seeded into 96-well microtiter plates (round bottom) to a concentration of 2 × 10⁻⁶. 5The final density was determined, and cells were incubated at 4°C for 30 min in the presence of different concentrations of antibody / VHH-Fc fusion or ADC. The concentration range covered from 90 nM to 123 pM. Cells were then washed twice with FACS buffer (1× phosphate-buffered saline + 10 mL 0.5 M ethylenediaminetetraacetic acid + 10 mL fetal bovine serum (FBS)) and once with 1× phosphate-buffered saline, followed by incubation at 4°C for 30 min with either anti-human APC detection antibody or antitoxin AF64 detection antibody. After another washing step with FACS buffer, cells were fixed with BD Fixative and analyzed using FACS celesta (BD Biosciences). An excitation laser at 633 nm was used to detect the APC signal. Mean fluorescence intensity (MFI) values ​​were plotted against antibody / VHH-Fc or ADC concentrations, and nonlinear fitting was used to fit the resulting data points to the respective EC50 values ​​of the antibody / VHH-Fc or ADC.

[0671] FACS internalization

[0672] Antigen-positive cells were seeded into FACS tubes to a concentration of 2 × 10⁻⁶. 5 The final density was determined, and the mixture was incubated at 4°C for 30 minutes in the presence of 100 nM or 500 nM HH-Fc fusion.

[0673] Cells were then washed twice with FACS buffer and once with 1× phosphate-buffered saline, followed by resuspending in 1 mL of growth medium. Cells were incubated at 37°C or on ice for 30 min, 1 h, 2 h, or 4 h. Cells were washed twice with FACS buffer and once with PBS, followed by application of anti-human APC detection antibody for 30 min. After another washing step with FACS buffer and PBS, cells were fixed with BD Fixative and analyzed using FACS celesta (BD Biosciences). An excitation laser at 633 nm was used to detect the APC signal. The MFI values ​​of the 4°C samples were compared with their respective 37°C counterparts to visualize the differences in fluorescence intensity mediated by internalization.

[0674] Mass spectrometry of unconjugated antibodies

[0675] For complete mass determination, optionally, 10 μg of each VHH-Fc fusion / antibody was deglycosylated in the original buffer by overnight incubation with PNGase F at 37 °C. Volumes corresponding to 1 μg of (de)glycosylated protein were separated by reversed-phase chromatography on a Waters Acquity I-Class UPLC column maintained at 80 °C. The protein was eluted with a linear gradient of 15–30% acetonitrile for 12 min, followed by increases to 95% acetonitrile over 2 min. 0.1% (v / v) formic acid was used as a 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 using ProteinMetrics Inc. Byos software. The raw mass spectra were integrated over the entire elution range and deconvoluted with settings suitable for the m / z range and instrument resolution. For the annotation of peaks in the deconvolution mass spectrum, homodimers are assumed to have all possible disulfide bridges and asparagine that is converted to aspartic acid through deglycosylation.

[0676] Serum stability

[0677] To test serum stability, incubate a 0.5 mg / mL solution of the test item in PBS (control) or human serum at 4°C (control) or 37°C. At certain time points, sample and freeze until analysis of the test item is performed.

[0678] Mass spectrometry for DAR determination

[0679] Sample preparation: The antibody-drug conjugate was reduced in its original buffer by adding tris(2-carboxyethyl)phosphonic acid hydrochloride to a final concentration of 10 mM and incubating at 40 °C for 45 min. The reduced sample was then directly subjected to LC-MS measurement.

[0680] High-resolution liquid chromatography-mass spectrometry (HPLC-MS): A total mass of 1 μg of reduced ADC was separated at 80 °C on an Acquity I-Class UPLC system equipped with a 2.1 × 50 mm Acquity UPLC Protein BEH C4 column (Waters Inc.). A linear gradient of 10–50% acetonitrile in water was run for 7 min at a flow rate of 0.4 ml / min. 0.1% (v / v) formic acid was used as a modifier in all solvents. The eluted proteins were analyzed on a Waters Xevo G2-XS QTOF mass spectrometer operated in positive ion, sensitive mode. Full-scan mass spectra were acquired from 400 to 4500 m / z using Glu-fibrinogen peptide B as the lock mass.

[0681] LC-MS Data Analysis: The raw mass spectra were integrated onto the chromatographic peaks of the eluted protein and deconvoluted using Byos (ProteinMetrics Inc.). The "neutral" mass peaks from the deconvoluted mass were annotated using the average molecular weight calculated from the amino acid sequence under the following assumptions: no disulfide bridges formed; glutamine at the N-terminus of the protein was converted to pyroglutamic acid; and C-terminal lysine residues were pruned. The following variable modifications were considered for annotation: up to one glycan (G0F, G1F, or G2F); up to six effective loadings. A tolerance of 15 Da was allowed for the annotation of the deconvoluted mass. The distribution of the drug-antibody ratio was determined based on the relative intensity of the deconvoluted mass peaks.

[0682] Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays

[0683] For the CDC assay, 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 for four hours with human serum (final concentration 25%) and anti-antigen antibodies (at various concentrations varying from 50,000 ng / mL to 28.9 ng / mL). Antibodies that specifically bound to different "control targets" were used as positive controls for antigen-negative CHO-K1 cells. CDC activity was determined using the CellTiter-Glo® 2.0 assay (which measures luminescence produced due to ATP-dependent luciferin oxidation) according to the manufacturer's instructions.

[0684] For the ADCC assay, a 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 healthy donors were thawed and pre-cultured for 48 hours prior to the assay, and used as effector cells at an E:T ratio of 40:1. Target cells, control cells, and effector cells were incubated for 24 hours with anti-antigen standard antibodies and anti-antigen antibodies (at various concentrations varying from 50,000 ng / mL to 0.01 ng / mL). Antibodies that specifically bound to different "control targets" were used as positive controls for antigen-negative cells. Specific lysis was quantified by measuring luciferase activity in the remaining viable tumor cells after the addition of Promega Luciferin Reagent. Decreased luminescence indicated target cell lysis, reflecting the efficacy of the antibody in inducing ADCC.

[0685] Cytotoxicity assay

[0686] Target-positive and target-negative HEK cells were seeded in white 96-well plates at densities of 5000 and 4000 cells / well, respectively. After incubation at 37°C for 24 hours, cells were treated with a 5x serially diluted ADC. Cell viability was measured as ATP concentration at 72 hours post-treatment by adding CellTiterGlo-Reagent (Promega) and subsequently performing luminescence measurements using a Clario Star Plus plate reader. Cell viability was analyzed as a percentage of untreated cells. Dose-response curves and curve fitting were performed using GraphPad Prism (9.5.1).

[0687] In vivo stability analysis of unlabeled antibodies and ADCs in mice

[0688] The pharmacokinetic (PK) of ADCs in serum was evaluated by administering unlabeled antibodies at a dose of 10 mg / kg to female Balb / c mice (7–8 weeks old) via the tail vein. A drug solution consisting of either unlabeled antibody or ADC in phosphate-buffered saline was administered in 100 μL volumes. Blood was collected from the facial vein at 5 minutes, 1 hour, 4 hours, 8 hours, 1 day, 3 days, and 5 days post-injection.

[0689] The Gyros ELISA was used to measure unlabeled antibodies and ADCs in serum samples.

[0690] The Gyros xPand ELISA method was used to determine the concentrations of ADC and total antibody in serum. Data were generated using GyrolabGeneric TK or PK disks. Serum samples were centrifuged at 4500 rpm for four minutes at 4°C and diluted 1:10 in Reagent E in 96-well plates. Standard curves and controls were prepared by diluting the test items in Reagent E containing 10% serum from untreated mice. Standards, controls, and biotin-labeled capture reagents were transferred to 96-well plates. The same procedure was performed with fluorophore-labeled detection antibodies. Master plates were prepared according to the Gyrolab Manager instructions. Gyrolab 20HC (TK) or 1000HC (PK) disks were placed in the Gyros xPand ELISA apparatus. Design and evaluation were performed using Gyrolab Manager and Evaluator.

[0691] In these experiments, the antigen was biotinylated as a capture reagent and diluted to 0.1 mg / mL in PBS-T. An Alexa 647-labeled anti-human IgG antibody (Reagent B from the Gyrolab Generic PK Kit) was used to detect total antibody in solution, but it could not distinguish between ADC and unconjugated antibody. Therefore, an antitoxin antibody was also used to quantify ADC. This labeled antitoxin antibody was diluted to 25 nM in Rexxip F. However, by comparing the two measurements (with anti-human vs. antitoxin settings), it is possible to distinguish the total amount of antibody and ADC, and thus draw conclusions about the stability of the ADC.

[0692] Antibody and ADC Nomenclature

[0693] This document describes various antibodies, including VHH-Fc fusions, Fab:VHH-Fc bispecific antibodies, and VHH:VHH-Fc bispecific antibodies, as well as their toxin-conjugated ADC forms. For monospecific antibodies, the nomenclature includes the prefix "AxBy". "Ax" indicates the antigen, and "By" indicates the binding agent, where "x" and "y" are numbers. The subsequent infix describes the design of the hinge region, for example, where "D1" refers to design 1, and D3 refers to design 3 (Table 2). As a suffix, the isotype of the Fc used is stated, for example, regarding IgG1Fc classified as belonging to the IgG1 isotype. For ADCs rather than antibodies, an additional suffix is ​​given stating the drug-antibody ratio for the molecule in question. Therefore, the ADC "A1B3-D3-IgG1Fc-DAR8" refers to an ADC specific to antigen 1 containing a third binding agent, exhibiting a hinge region according to design 3, an IgG1-Fc moiety, and conjugation to 8 toxins / antibodies (DAR of 8). Due to its binding ability to both antigens, the bispecific antibody (bs) exhibits a prefix numbered consecutively starting from bs1.

[0694] Table 2 - Infixes involving hinge areas and converted to the corresponding Seq ID NO.

[0695]

[0696] Example 1 – Western blot analysis of cell culture supernatants from different VHH-Fc fusions

[0697] The inventors of this application have for the first time elucidated the use of an ADC containing an engineered IgG hinge region with a predetermined number of cysteine ​​residues to control DAR. Specifically, to create an ADC containing an engineered IgG hinge region, the inventors modified a VHH-Fc fusion containing an IgG1 Fc region and a heterologous hinge region. The inventors used a human IgG2-derived hinge region or a human IgG3-derived hinge region as the heterologous hinge region. The human IgG hinge region sequence was used to minimize immunogenicity. Furthermore, to provide a homogeneous ADC product, the inventors modified the IgG3-derived hinge region to replace easily O-glycosylated serine and threonine residues with alanine residues. The inventors also compared VHH-Fc containing an engineered IgG hinge with conventional VHH-Fc containing an IgG1 Fc region and a truncated IgG1 hinge. Thus, conventional VHH-Fc antibodies containing a truncated IgG1 hinge and antibodies containing an engineered IgG hinge region were produced.

[0698] The inventors of this application have developed antibodies containing five different VHH domains (A1B1-5) (each specific to the same target antigen) to study the effects of the engineered IgG hinge region, regardless of the VHH or VHH specificity used.

[0699] In ExpiHEK cells, all five VHHs that bind to the target antigen are transiently generated as VHH-Fc fusions containing either a truncated IgG1 hinge (D1), an IgG2-derived hinge region (D2), or an IgG3-derived hinge region (D3), resulting in a total of 15 constructs. Therefore, the inventors first generated five conventional VHH-Fcs containing both an IgG1 Fc region and a truncated IgG1 hinge region for comparison. Then, the inventors of this application generated 10 VHH-Fcs containing both an IgG1 Fc region and an engineered IgG hinge region: five VHH-Fcs containing an IgG2-derived hinge region and five VHH-Fcs containing an IgG3-derived hinge region.

[0700] The IgG hinge region is as follows:

[0701] Truncated IgG1 hinge: DKTHTCPPCP (SEQ ID NO: 12)

[0702] IgG2-derived hinge: ERKCCVECPPCP (SEQ ID NO: 2)

[0703] IgG3-derived hinge: PRCPEPKACDAPPPCPRCP (SEQ ID NO: 112).

[0704] The standard VHH-Fc (containing a truncated IgG1 hinge region SEQ ID NO: 12) will provide an ADC with a DAR of 4 after cysteine-based site-specific conjugation. VHH-Fc containing a modified IgG2-derived or IgG3-derived hinge region will provide an ADC with a DAR of 8 after cysteine-based site-specific conjugation. Therefore, VHH-Fc containing a modified IgG2-derived or IgG3-derived hinge region provides an antibody optimized for higher DAR.

[0705] The inventors then performed aggregation analysis using unreduced samples via Western blotting. Cell culture supernatants, each containing one of the 15 VHH-Fc fusions, were analyzed. Detection was performed using anti-human antibodies.

[0706] By utilizing IgG1 hinges and IgG3-derived hinges, all VHH-Fc were successfully generated, and they showed no signs of aggregates, dimers, or fragmented products.

[0707] In some constructs, the IgG2-derived hinge leads to the formation of aggregates or dimers. An exception is the VHH-Fc2 variant (A1B2-D2-IgG1Fc), which primarily produces the desired monomeric bands.

[0708] Thus, this paper presents constructs having hinge regions derived from each of IgG2 and IgG3, capable of forming monomers. Therefore, VHH-Fc antibodies containing engineered IgG hinge regions exhibit comparable performance to conventional VHH-Fc, regardless of VHH specificity. Furthermore, the inventors have shown that modifications to prevent O-glycosylation are tolerated in the hinge regions. As shown below, all tested constructs are functional, regardless of aggregate or dimer formation.

[0709] Example 2 – Biophysical Characterization of VHH-Fc Fusion

[0710] To further verify the VHH-Fc fusions containing engineered IgG hinge regions, the inventors of this application conducted biophysical characterization of the 15 VHH-Fc fusions described above (5 conventional VHH-Fcs and 10 VHH-Fcs containing engineered IgG hinge regions derived from IgG2 or IgG3 hinges).

[0711] Table 3 describes the thermal denaturation values ​​(determined by NanoDSF) and SEC purity for the following VHH-Fc fusions. DLS results were also determined (data not shown).

[0712] Stability analysis of each of the VHH-Fc fusions was performed using nano-differential scanning fluorescence (NanoDSF). Generally, stability appeared to be primarily mediated by the VHH candidate and less dependent on the hinge region structure. High stability with TM values ​​up to 76 °C was observed.

[0713] SEC analysis further confirmed that the monodispersity of the IgG1 hinge and IgG3-derived hinge candidates is very high, and monodispersity is also observed in some IgG2-derived variants.

[0714] Therefore, biophysical characterization confirmed the results presented above in Example 1.

[0715] Table 3 – Biophysical characterization of the VHH-Fc fusions, comparing IgG1-derived hinge regions, IgG2-derived hinge regions, and IgG3-derived hinge regions. SEC purity and thermal denaturation values ​​as determined by NanoDSF are described for the VHH-Fc fusions.

[0716]

[0717] Example 3 – Affinity of VHH-Fc Fusion

[0718] To further validate VHH-Fc, the inventors of this application conducted affinity tests on each of the 15 VHH-Fcs described above (5 conventional VHH-Fcs and 10 VHH-Fcs containing engineered IgG hinge regions derived from IgG2 hinges or IgG3 hinges).

[0719] The Octet HTX apparatus was used to determine the affinity of the 15 different IgG-derived hinged VHH-Fc fusions for the following substances: (i) the target antigen of VHH-Fc, and (ii) huCD64. A 1:1 Langmuir binding model was used to globally fit association and dissociation, and the results are shown in Table 4 below.

[0720] As shown below, all three hinge regions mediate the same binding to the antigen, highlighting that specificity and affinity remain unchanged after conversion to a different hinge sequence. All target antigen candidates bind to the target antigen and CD64 with high affinity. This suggests that ADCC and CDC activities are preserved for antibodies containing both engineered and heterologous IgG hinge regions because FcγR binding is not impaired by the engineered hinge sequence.

[0721] It appears that there are no negative impacts after adaptively adjusting the heterogeneous hinge to allow for higher DAR. Furthermore, it appears that there are no negative impacts after modifying the hinge to avoid O-glycosylation. Further, for each design, comparable results were obtained with five different VHHs, regardless of VHH specificity. CD64 binding also showed independence from the hinge region utilized. This allows for hinge design selection based solely on DAR and not on other properties.

[0722] Table 4 - Affinities of VHH-Fc fusions containing IgG1-hinge, IgG2-hinge, and IgG3-hinge for antigen 1 and CD64. BLI was used to determine the primary antigen for VHH-Fc and the affinity for CD64.

[0723]

[0724] Example 4 – Cell Binding of VHH-Fc Fusion

[0725] To further verify VHH-Fc, the inventors of this application analyzed the cell binding of VHH-Fc, which contains an IgG1 hinge and an IgG3-derived hinge region as described above.

[0726] Target-positive tumor cells were stained with VHH-Fc containing different concentrations of IgG1- and IgG3-derived hinges. Mean fluorescence intensity (MFI) values ​​were plotted against VHH-Fc concentrations, and the resulting data points were fitted using a non-linear method to obtain the respective EC50 values ​​for VHH-Fc. These values ​​are shown in Table 5 below.

[0727] These experiments revealed EC50 values ​​ranging from sub-nM to single-digit nM for most target cells. Different hinge regions mediate the same binding to antigen-positive cells. Thus, EC50 is not affected by factors such as optimizing the hinge region for higher DAR (i.e., utilizing heterologous hinge regions), modifications to avoid O-glycosylation, or VHH specificity. This further emphasizes that hinge design can be selected based solely on DAR without considering other properties.

[0728] Table 5 - Cell binding of VHH-Fc fusion products. Target-positive tumor cells were stained with different concentrations of VHH-Fc, and EC50 values ​​were measured.

[0729]

[0730] Example 5 – Internalization of VHH-Fc Fusion

[0731] To further validate VHH-Fc, the inventors of this application evaluated the internalization of VHH-Fc containing IgG1 hinges and IgG3-derived hinges as described above.

[0732] The internalization of VHH-Fc containing IgG1 hinges and IgG3-derived hinges into target-positive tumor cells was investigated using an FACS-based assay, which confirmed rapid internalization of all candidates, independent of engineered hinge regions.

[0733] Internalization was studied at four different time points (0.5, 1, 2, and 4 hours). As a control, the candidates were incubated on ice to reduce internalization.

[0734] Internalization was examined by incubation at 37°C followed by FACS analysis. All candidates clearly showed strong and rapid internalization, detectable after only 30 minutes. Figure 2 ).

[0735] from Figure 2 It is clear that all tested hinge regions mediate the same internalization into antigen-positive cells. Internalization is not adversely affected by optimizing the hinge region for a higher DAR, or by modification to avoid O-glycosylation, as the different hinge regions tested showed the same internalization for a given VHH. This again underscores that hinge design can be selected based solely on DAR without considering other properties.

[0736] Example 6 – Complete mass spectrometry data of the VHH-Fc fusion protein

[0737] Mass spectrometry was used to determine the intact mass of deglycosylated VHH-Fc containing IgG1 hinge and IgG3-derived hinge regions.

[0738] Table 6 below shows the calculated quality and the (experimental) quality of deconvolution. The overall quality is extremely close to the calculated quality in terms of value. This confirms that the disulfide bridge closes as expected and that no mismatched interchain disulfides are present. High resolution was achieved, making the presence of unpaired hinge cysteines extremely unlikely.

[0739] Table 6 - Complete quality data for the VHH-Fc fusion protein. Complete quality of the deglycosylated VHH-Fc fusion protein containing the IgG1 hinge and IgG3-derived hinge region.

[0740]

[0741] Example 7 – Serum stability of VHH-Fc fusion protein

[0742] Hinge regions, especially those of the IgG3 type, are considered readily susceptible to 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 cause the VHH (antigen-specific binding portion) to separate from the Fc portion (which is responsible for extended half-life and effector function). Furthermore, since the hinge region represents the site of toxin conjugation, cleavage in the hinge region can adversely affect DAR, as demonstrated in later examples. Therefore, any cleavage occurring in an antibody, particularly within the hinge region, can have a detrimental effect on the efficacy of the ADC.

[0743] To investigate the stability of antibodies containing IgG1-derived and IgG3-derived hinges, antibodies exhibiting D1 or D3 hinge designs (A1B3-D1-IgG1Fc and A1B3-D3-IgG1Fc) were incubated in human serum at 37°C for up to two weeks. The human serum contained all proteins found in the serum of living humans, including proteases, and was therefore a suitable source of proteases that the antibodies would encounter in vivo. As a control, the same antibodies were incubated in PBS at 4°C for the same time range.

[0744] To test the integrity of the antibodies, cells expressing antigen 1 were stained with the antibodies incubated in serum, and binding was tested using anti-human antibodies. VHH binding to the antigen could only be tested by detecting the human Fc moiety when the hinge region remained uncut and intact. No loss of binding was observed on target-positive CHO cells during incubation in serum at 37°C, very similar to the binding profile observed in PBS. Although data using tumor cells expressing antigen 1 showed more background noise, samples from day 1 and day 14 showed comparable MFI values, highlighting the stability of IgG1-hinge and IgG3-hinge antibodies in human serum over extended time periods. Figure 3 ).

[0745] Surprisingly, and despite literature indicating a proteolytic tendency in IgG3-derived hinges, no cleavage was observed during the experiment. These unexpected findings allow for further investigation of heterogeneous hinge technology for ADC development.

[0746] Example 8 – Generation of ADC using an antibody targeting antigen 1

[0747] Because the VHH-Fc fusion protein tested in the previous embodiments exhibited highly favorable properties, it was subsequently tested in conjugation experiments to generate an ADC. Here, a topoisomerase I (TopoI) inhibitor payload, along with the thiol-reactive moiety, was tested as a linker-payload. Following partial reduction of the antibody (which leads to the opening of interchain disulfide bridges), free cysteine ​​residues within the hinge sequence were used as conjugation sites. Therefore, the inventors hypothesized that the predetermined number of cysteine ​​residues within the hinge, designed by the antibody and hinge, predetermined the DAR that the ADC would achieve. The resulting ADC was analyzed by mass spectrometry to experimentally determine the drug-antibody ratio (Table 7).

[0748] Table 7 – Describes the expected and achieved DAR of the tested VHH-Fc-based ADC targeting antigen 1, as determined by mass spectrometry.

[0749]

[0750] In all cases, the expected DAR was achieved with very high homogeneity. Here, the inventors of this application demonstrate for the first time that by employing a heterogeneous hinge region, the DAR of a VHH-Fc-based ADC can be shifted to a higher value while simultaneously achieving a highly homogeneous product.

[0751] Example 9 – Characterization of ADC targeting antigen 1

[0752] The ADC exhibiting the D3 design generated in previous experiments represents the first such ADC where DAR is controlled by utilizing heterogeneous hinge regions. Since the hinge regions are surface-exposed, there is a possibility that conjugation with the payload could affect aggregation behavior. Furthermore, the specific orientation of the binding portions relative to each other changes after opening the tightly packed disulfide bridges, and the introduction of linker-payload molecules may mediate specific steric effects. These changes may affect the binding properties of the ADC. To verify the full functionality of the generated ADC of the present invention, the biophysical characterization of the ADC against unconjugated parental antibodies was repeated (Table 8).

[0753] Table 8 - Biophysical characterization of VHH-Fc ADCs targeting antigen 1. SEC purity and thermal denaturation values ​​as determined by NanoDSF are described for the VHH-Fc fusions.

[0754]

[0755] As expected, the reduction in the correlation between conjugated and stable inter-chain disulfide bonds decreases the overall stability of the ADC to a small extent. Surprisingly, however, no hinge- or DAR-dependent loss of stability was observed. This suggests that the constructs of this invention can be further developed for use as ADCs, and that the heterologous IgG3-derived hinge presented by the inventors is not inferior to prior art hinges of the IgG1 type. Furthermore, analytical SEC revealed high purity for all tested items, even for the higher DAR 8 ADC. Next, the antibody binding properties were evaluated in a manner similar to previous experiments (Table 9).

[0756] Table 9 - Affinity of VHH-Fc-based ADCs targeting antigen 1. BLI was used to determine affinity for the primary antigen VHH-Fc and for CD64.

[0757]

[0758] Furthermore, for all ADCs, high-affinity binding was detected, with no noteworthy differences dependent on DAR. In some cases, the dissociation rate was so slow that it exceeded the instrument's limits. Further, a cell binding assay was performed to examine whether the retained binding to the recombinant target protein translated into the retained binding to target-positive cells (Table 10). In this experiment, two EC50 values / subjects were determined using either the antibody portion of the ADC (using an anti-human detection antibody) or the intact ADC (using an antitoxin detection antibody).

[0759] Table 10 - Cell binding of VHH-Fc ADC on antigen 1 positive cells. Target-positive tumor cells were stained with different concentrations of VHH-Fc, and EC50 values ​​were measured.

[0760]

[0761] Typically, slightly weaker ADC binding was observed compared to unconjugated antibodies. However, again, no DAR- or hinge-dependent differences in EC50 values ​​were observed.

[0762] In summary, by selecting appropriate hinge sequences, well-behaved ADCs containing predetermined 8 DARs can be generated in a highly homogeneous manner. This is achieved without compromising the stability, binding affinity, or monomer content of the ADC product.

[0763] Example 10 – ADC serum stability

[0764] In Example 7, the inventors tested the stability of the antibodies in human serum and observed that these molecules were surprisingly difficult to cleave by proteolytic hydrolysis. However, in these antibodies, the interchain disulfides of the hinges were closed. This may have stabilized the hinges, and therefore hinges that are not structurally restricted by disulfides (as found in ADCs) may be more susceptible to proteolytic hydrolysis. Therefore, the inventors sought to test the serum stability of the conjugated ADCs. For this experiment, a three-week incubation period was chosen to better reflect the expected half-life that these ADCs should have in an in vivo setting.

[0765] After incubation in PBS (4°C) or human serum (37°C) for the given time, samples were analyzed by Gyros ELISA. Intact protein was detected by capture with biotinylated antigen 1 followed by detection with anti-human AF647 antibody, or by performing the same capture reaction but detecting with antitoxin AF647 antibody. Figure 4 ).

[0766] For antibody or toxin detection measurements, the inventors observed no decrease in concentration. This underscores that the ADC remains fully functional and intact during incubation in serum: the hinge sequence was neither cleaved nor was the toxin released from the ADC. These findings are relevant to prior art IgG1-derived hinges (DAR 4; Figure 4 A) and the heterologous IgG3-derived hinge exhibiting 8 DAR ( Figure 4 B) is true. Therefore, no DAR- or hinge-dependent effects were observed, highlighting the suitability of the D3 hinge design for therapeutic development.

[0767] Example 11 – Cytotoxicity assessment of ADC

[0768] One of the most important characteristics of ADCs is their target-specific and potent killing effect on target-positive cells. Therefore, the inventors investigated cytotoxicity using antigen-1-positive HEK cells. For an ADC to exhibit its effect, it needs to bind to the antigen, be internalized, and allow for toxin release, which ultimately kills the target cells. All these parameters were tested simultaneously by studying cytotoxicity. Furthermore, the specificity of the ADC was investigated by also performing this experiment on target-negative cells, as only non-specific binding leads to decreased cell viability in target-negative cells. Further, free ethanotecan (a topoisomerase I inhibitor) was tested alongside the ADC and another non-specific DAR8 ADC for antigen-1.

[0769] All ADCs mediated potent, target-specific, dose-dependent, and DAR-dependent killing. While esaxetidine mediated cell killing in both target-positive and target-negative cells, only the tested item showed specificity for target-positive cells. Target-negative cells were killed only at very high concentrations, a characteristic also observed with the non-binding isotype control (DAR8). VHH-Fc-based ADCs showed significant dose dependence, with DAR8 ADCs killing cells in all cases with lower IC50 values ​​compared to their DAR4 counterparts (Table 11). Figure 5 ).

[0770] Table 11 - IC50 values ​​of the tested items for target-positive cells.

[0771]

[0772] In summary, the inventors have demonstrated that ADCs with increased DAR compared to prior art IgG1-derived hinges can be achieved using IgG3-derived heterogeneous hinges, thereby mediating more potent cytotoxicity against target-positive cells.

[0773] Example 12 – ADCC and CDC Activity

[0774] In addition to cell killing mediated by antibody-conjugated cytotoxic payloads, ADCs can further mediate killing in a toxin-independent manner. Among these are antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). To investigate these properties, ADCC and CDC assays were performed using unconjugated antibodies and ADCs exhibiting D1- and D3-design hinges. In either case, ADCC and CDC activities were confirmed, highlighting that conjugation to the payload did not deactivate these antibody-dependent effector functions for IgG1- or IgG3-derived hinges.

[0775] Example 13 – Antibody PK and ADC Stability in Vivo

[0776] The inventors sought to investigate whether hinge modifications led to altered pharmacokinetic behavior. Therefore, A1B3-D1-IgG1Fc and A1B3-D3-IgG1Fc were tested in a PK study, with serum samples collected at various time points. To ensure the accuracy of the results, LLOD (lower limit of detection) and LLOQ (lower limit of quantification) settings from a Gyros ELISA setup were also included for quantifying the levels of the two antibodies. Figure 6The concentrations of the two antibodies, A1B3-D1-IgG1Fc and A1B3-D3-IgG1Fc, in serum over time exhibited almost identical characteristic profiles. This indicates that modifications to the hinge region do not affect the PK characteristic profiles of the antibodies in any way.

[0777] Because the detection setup in this experiment was based on VHH antigen 1 binding and used Fc detection, it was further demonstrated that no hinge region cleavage occurred in vivo. In the next study, the inventors utilized the ADC forms of the two antibodies (which exhibit 4 or 8 DAR) for in vivo experiments. Figure 7 ).

[0778] The objective of this study was to analyze the stability of antibody-drug conjugates (ADCs) A1B3-D1-IgG1Fc-DAR4 and A1B3-D3-IgG1Fc-DAR8. To this end, the concentrations of total antibody (detected using anti-human antibody) and ADC (detected using antitoxin antibody) were measured. The results demonstrated a significant correlation between the proportion of ADC and total antibody in serum. The latter was present at higher concentrations between 0.1 and 8 hours. However, at later time points, the concentrations of ADC in serum decreased slightly in both constructs. This highlights that the antibody containing the D3-hinge did not deconjugate in vivo and formed a stable ADC comparable to the prior art D1 construct.

[0779] Example 14 – Characterization of VHH-Fc fusion protein against a second antigen

[0780] The promising results of antigen 1 binding antibody and ADC encouraged the inventors to test their protein modification method on another set of VHHs bound to a second, different antigen (antigen 2). Similarly, a standard IgG1-derived hinge sequence (D1) was used as a control, and an IgG3-derived hinge (D3), which allows for higher DAR, was used in combination with five different VHHs. Proteins were successfully produced in high yields and subjected to biophysical analysis after purification (Table 12).

[0781] Table 12 - Biophysical characterization of the VHH-Fc fusion protein targeting antigen 2. SEC purity and thermal denaturation values ​​as determined by NanoDSF are described for the VHH-Fc fusions.

[0782]

[0783] Once again, the differences in SEC purity and thermal stability were mediated solely by the binding moiety, with no notable differences between the hinge designs. The same was true for antigen affinity with recombinant proteins (Table 13) and with antigen 2-positive cells (Table 14).

[0784] Table 13 - Affinity of VHH-Fc fusions targeting antigen 2. BLI was used to determine the affinity for the primary antigen of VHH-Fc.

[0785]

[0786] Table 14 - Cell binding of VHH-Fc ADC targeting antigen 2. Target-positive tumor cells were stained with different concentrations of VHH-Fc, and EC50 values ​​were measured.

[0787]

[0788] In summary, using a second set of binders (targeting a different antigen than previously evaluated), the inventors observed that hinge designs with different DARs exhibited identical performance at the protein level when conjugated, and that the variable sequence, rather than the selected hinge region, determined the antibody's properties. This further supports the inventive concept of designing hinges based on the intended DAR rather than other parameters, thus allowing for high flexibility during drug development.

[0789] Example 15 – Characterization of VHH-Fc fusion protein against a third antigen

[0790] The promising results of antigen 1 and 2 binding antibodies and ADCs encouraged the inventors to test their observations using a third group of VHHs targeting a third, different antigen (antigen 3). In this paper, nine different VHHs, as tested in previous experiments, were tested in VHH-Fc fusion patterns exhibiting either a conventional IgG1-derived hinge (D1) or a modified IgG3-derived hinge (D3). Following successful production of these antibodies, biophysical characterization was performed (Table 15).

[0791] Table 15 - Biophysical characterization of VHH-Fc fusions targeting antigen 3. SEC purity and thermal denaturation values ​​as determined by NanoDSF are described for the VHH-Fc fusions.

[0792]

[0793] In the presented conjugate groups, the TM values ​​again showed no significant difference between the two hinge designs. While some exhibited lower purity, no obvious trend was observed between the two hinge designs. Although the IgG1-derived hinge produced higher purity compared to the IgG3-derived hinge when using the A3B1 VHH, the opposite effect was observed for the A3B7 conjugate. Therefore, the biochemical properties of the VHH itself are responsible for the behavior of the antibody in any given pattern, rather than the hinge sequence employed.

[0794] For antigen 3, affinity measurements were performed either in an affinity setting where the bivalent antibody was immobilized and the monomeric antigen was used as the analyte, or in an affinity setting where the antigen was immobilized and VHH-Fc was used as the analyte (Table 16). Due to the longer sequence of the IgG3-derived hinge, altered affinity can be expected, particularly in the affinity setting. These variations 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).

[0795] Table 16 - Affinity and affinity measurements of VHH-Fc fusions bound to antigen 3. BLI was used to determine the affinity and affinity for the primary antigen of VHH-Fc.

[0796]

[0797] While affinity assays typically yielded high affinity values, as expected, no differences were observed based on the hinge design. Particularly in the affinity settings, the observed dissociation rates exceeded instrumental limits. These findings further underscore the modularity of the heterogeneous hinge approach. This is further emphasized by the determination of EC50 values ​​for antigen 3-positive tumor cells (Table 17).

[0798] Table 17 - Cell binding of VHH-Fc fusion on antigen 3 positive cells. Target-positive tumor cells were stained with different concentrations of VHH-Fc, and EC50 values ​​were measured.

[0799]

[0800] Similarly, in this group of binders, the inventors attempted to verify successful closure of the interchain disulfide using mass spectrometry (Table 18). As with antigen 1 above, intact mass was confirmed, and no unpaired cysteine ​​residues were detected in the hinge region. This further underscores the suitability of these designs for the development of therapeutic antibodies and ADCs.

[0801] Table 18 - Complete quality data for VHH-Fc fusion protein targeting antigen 3. Complete quality of deglycosylated VHH-Fc fusion protein containing IgG1-derived hinge and IgG3-derived hinge regions.

[0802]

[0803] Example 16 – Generation of ADC targeting antigen 3

[0804] As with antigen 1, the antibody group exhibiting the IgG3-derived hinge (D3) targeting antigen 3 was tested in a conjugation assay via thiol reaction coupling with a topoisomerase I inhibitor. The DAR of the resulting ADCs was determined using mass spectrometry. In all cases, the expected DAR of 8 was achieved with very high homogeneity (Table 19).

[0805] Table 19 – describes the expected and achieved DAR of the tested VHH-Fc fusions targeting antigen 3, as determined by mass spectrometry.

[0806]

[0807] In all cases, a DAR of 8 was achieved with high homogeneity, similar to that observed for the ADC targeting antigen 1 previously. Since prior art IgG1-derived VHH-Fc fusions are only capable of having a DAR of 4, the inventors have now demonstrated with a wide variety of binders that, when utilizing the hetero-hinge method, the desired DAR of 8 can be reliably achieved with up to 100% high homogeneity in the final ADC.

[0808] Example 17 – ADC characterization of antigen 3

[0809] The generated ADC against antigen 3 was characterized in terms of its biophysical properties. The fluorescence of the toxin interfered with the precise determination of the unfolding temperature because it overlapped with the intrinsic fluorescence of the protein measured during unfolding; therefore, approximations based on visual observation of the raw data were used for certain data points (labeled in Table 20). ).

[0810] Table 20 - Biophysical characterization of VHH-Fc ADCs targeting antigen 3. SEC purity and thermal denaturation values ​​as determined by NanoDSF are described for the VHH-Fc fusions.

[0811]

[0812] While the ADC exhibited remarkable thermal stability, a favorable aggregation profile was also observed. Furthermore, binding affinity was determined in both affinity-based and affinity-based settings, as previously done with unconjugated antibodies (Table 21).

[0813] Table 21 - Affinity of VHH-Fc ADC targeting antigen 3. BLI was used to determine affinity and cohesion to the primary antigen of the VHH-Fc ADC.

[0814]

[0815] To examine binding to antigen 3-positive tumor cells, a FACS assay was performed. The bound ADC was detected using either an anti-human antibody (which detects the antibody portion of the ADC) or an antitoxin antibody (which detects the intact ADC when bound to the target antigen). For both methods, EC50 values ​​were measured (Table 22). Comparable EC50 values ​​were observed between the two methods.

[0816] Table 22 - Cell binding of VHH-Fc ADC targeting antigen 3. Target-positive tumor cells were stained with different concentrations of VHH-Fc, and EC50 values ​​were measured.

[0817]

[0818] In summary, the inventors demonstrated, using different sets of binding agents, that the ADC with a DAR of 8 generated by utilizing a heterologous IgG3-hinge exhibits advantages in conjugation, stability, purity, and binding assays. These results further emphasize the modularity of the method and highlight that the hinge region of the ADC of the present invention can be selected solely based on the desired DAR without compromising on other aspects.

[0819] Example 18 – Cytotoxicity assessment of ADCs targeting antigen 3

[0820] To verify the full functionality of the ADC targeting antigen 3, the inventors conducted a cytotoxicity experiment using antigen 3-positive cells. As controls, a DAR 0 (unconjugated antibody) control and a non-binding DAR 8 ADC control were included. The experiment was performed independently twice, and... Figure 8 Table 23 describes the mean and standard deviation of the two independent experiments for target-positive cells.

[0821] Table 23 - IC50 values ​​of ADCs targeting antigen 3. The IC50 values ​​indicate the concentration of the ADC at which viability reaches 50%.

[0822]

[0823] For all ADCs, sub-nM IC50 values ​​were achieved, highlighting the potency of these candidates. Furthermore, specificity was investigated by testing cytotoxicity using antigen 3-negative cells. Figure 9 No nonspecific killing events were observed at the relevant concentrations.

[0824] In summary, effective and target-specific killing was observed for all ADCs. While target-negative cells showed the expected decrease in viability only at extremely high ADC concentrations, target-positive cells were killed in a dose-dependent manner with sub-nM IC50 values. This highlights that the inventive method of using heterologous hinge regions to generate ADCs that mediate effective killing is not limited to a specific target antigen (e.g., antigen 1) but represents a platform solution transferable to other antigens.

[0825] Example 19 – Bispecific Fab:VHH-Fc fusion (containing heterogeneous hinge region)

[0826] Above, the inventors of this application demonstrate the suitability of heterogeneous hinge-Fc pairing and IgG3-derived hinges for generating VHH-Fc fusion ADCs (which have a higher performance than conventionally designed DARs containing IgG1-derived hinge regions).

[0827] To further evaluate the platform and broaden the potential applications of the method, the inventors aimed to generate bispecific antibodies exhibiting the modified hinge region according to the invention. Therefore, bispecific antibodies were constructed, one binding moiety being a Fab fragment derived from an IgG1 antibody, and the second binding moiety being VHH. Both binding moieties were fused to the N-terminus of the hinge region, which itself was fused to the Fc of the IgG1 antibody as in previous constructs. Heterodimation of the Fc moiety was enhanced using a "mortar and pestle" technique. Because Fab exhibits heavy-chain-light-chain disulfide bonds, in these constructs, the potential DAR is not only mediated by the heavy-chain disulfide in the hinge region. An overview of the potential DAR mediated by the hinge region and the overall possible DAR is described in Table 24, which is the hinge DAR + 2 (caused by the Fab fragment).

[0828] Table 24 - An overview of the designs used in the test projects and the expected DARs that can be achieved with these bispecific antibodies.

[0829]

[0830] Bispecific antibodies were successfully generated in mammalian cells and subjected to biophysical analysis after purification (Table 25).

[0831] Table 25 - Biophysical characterization of the Fab:VHH-Fc bispecific antibody. Thermal denaturation values, measured by NanoDSF, are described for the bispecific antibody.

[0832]

[0833] As expected, no hinge-dependent differences were revealed in these bispecific antibodies, and very good thermal stability was observed. Furthermore, the binding properties of the bispecific antibodies to their respective antigens were elucidated (Table 26).

[0834] Table 26 - Affinity of Fab:VHH-Fc bispecific antibodies. Affinity to both antigens was determined using BLI.

[0835]

[0836] As observed above, the binding affinity for the antigen is independent of the hinge region. Furthermore, binding to the Fc receptor was elucidated, showing that neither CD64 nor FcRn binding was impaired by the use of the IgG3-derived hinge (Table 27).

[0837] Table 27 - Affinity of Fab:VHH-Fc bispecific antibody to Fc receptor. Affinity to CD64 and FcRn was determined using BLI.

[0838]

[0839] One of the most important characteristics that bispecific antibodies must satisfy is the ability to simultaneously bind two antigens. For this purpose, the distance between the binding regions is crucial. To test whether it is possible to simultaneously bind two antigens using engineered hinges, the inventors performed a sandwich-like BLI assay, in which one antigen was immobilized, followed by association of the bispecific antibody and then the second antigen. Through this assay, the inventors clearly demonstrated that the engineered hinge region mediates sufficient flexibility to allow for the simultaneous binding of two antigens (data not shown).

[0840] In this paper, the inventors demonstrate that even when the two distinct binding regions (Fab and VHH) are asymmetrically fused into the engineered hinge in more complex bispecific antibodies, no negative impact on the overall stability of the antibody or impairment of binding is observed. Simultaneous binding of the bispecific antibody to the antigen is also confirmed. This further underscores the modularity of the modified and heterologous hinge regions presented herein and further opens up the possibility of using this technique in constructing bispecific ADCs with personalized DARs.

[0841] Example 20 – Bispecific VHH:VHH-Fc antibody (containing heterologous hinge region)

[0842] Following the encouraging results of the Fab:VHH-based bispecific antibodies, the inventors sought to further test the heterojunction platform using two different VHHs in the bispecific approach. Therefore, VHHs targeting antigen 1, antigen 3, or antigen 6 were fused to IgG1 Fc exhibiting the well-known "mortar and pestle" mutation via IgG1-derived or IgG3-derived hinges (D3 or D11) to generate bispecific antibodies. These molecules were well expressed, and their biophysical properties were analyzed (Table 28).

[0843] Table 28 - Biophysical characterization of VHH:VHH-Fc bispecific antibodies. Thermal denaturation values, measured by NanoDSF, are described for each bispecific antibody.

[0844]

[0845] While differences in purity and stability were revealed in these antibodies, no clear trend toward IgG1-hinge or IgG3-hinge was observed. However, most antibodies exhibited high thermal stability and sufficient purity (after single-step purification). Therefore, these molecules were further investigated regarding their binding affinity to their respective antigens (Table 29).

[0846] Table 29 - Affinity of VHH:VHH-Fc bispecific antibodies. Affinity to both antigens was determined using BLI.

[0847]

[0848] Based on the hinge design utilized, no differences in binding were observed. Further binding profiles for the Fc receptor were determined, revealing no hinge-dependent differences in these bispecific antibodies (Table 30).

[0849] Table 30 - Affinity of VHH:VHH-Fc bispecific antibody to Fc receptor. Affinity to CD64 and FcRn was determined using BLI.

[0850]

[0851] To test the ability of these antibodies to mediate simultaneous binding to two antigens, a sandwich-like BLI assay was performed. After fixation of the first antigen and subsequent association of the bispecific antibody, a second antibody was applied. Association of the second antibody depended on the simultaneous binding of the bispecific antibody to both antigens. Simultaneous binding of the bispecific antibody to both antigens was confirmed (data not shown). The inventors demonstrated that heterojunctions can mediate such a binding mode. This unexpected finding further supports the heterojunction platform for the development of bispecific ADCs presented herein, as the hinge region is responsible for the spatial orientation of the binding portion.

[0852] Example 21 – ADC Generation Using Bispecific VHH:VHH-Fc Fusion (Including Heterogeneous Hinge Region)

[0853] Using a topoisomerase I inhibitor payload along with the thiol-reactive linker, bispecific antibodies exhibiting the D3 hinge region and displaying DAR 8 were tested in conjugation experiments. In all cases, the antibodies were successfully conjugated to the toxin, achieving the desired DAR value (Table 31).

[0854] Table 31 describes the expected and achieved DAR of the tested bispecific VHH-Fc fusions, as determined by mass spectrometry.

[0855]

[0856] This further highlights the suitability of the heterogeneous hinge method for designing dual-specific ADCs with personalized DARs.

[0857] Example 22 – IgG2 and IgG4 Fc combined with IgG1 or IgG3 hinges

[0858] To this end, the inventors tested IgG3-derived hinges, using an IgG1-derived counterpart as a reference, combined with IgG1 Fc. To broaden the applications of the heterologous hinge region, the inventors then analyzed IgG3-derived hinge design 3 (D3) in the context of IgG2 and IgG4 Fc regions. To allow for comparison with previous data, the A1B3 binder was used again.

[0859] Antibodies were successfully generated and subsequently tested for a wide range of biophysical parameters (Table 32).

[0860] Table 32 - Biophysical characterization of IgG3 hinge-IgG2 / 4 Fc paired constructs. Thermal denaturation values ​​were determined by NanoDSF.

[0861]

[0862] High purity was achieved after single-step purification of protein A. The measured melting temperatures were highly similar to those previously tested for IgG1 Fc fused to IgG1-derived or IgG3-derived hinges. Similarly, binding experiments revealed no change in binding behavior after switching Fc isotypes (Table 33).

[0863] Table 33 – Showing the affinity of IgG2 or IgG4 Fc for VHH-Fc. Affinities for the primary antigen and FcRn of VHH-Fc, and binding to antigen-positive cancer cells, as determined using BLI.

[0864]

[0865] Furthermore, cell binding assays confirmed the single-digit nM EC50 value, which was comparable to previous data.

[0866] In the field of antibodies, Fc isotypes are often selected based on the intended mode of action. For example, IgG2 and IgG4 Fc are naturally silenced and elicit less ADCC and CDC activity compared to their IgG1 counterparts. This embodiment emphasizes that, with the constructs of the present invention, Fc isotypes can be selected solely based on the intended effector function and can be combined with hinge regions to achieve the DAR envisioned for the final ADC. This modularity does not negatively impact purity or antigen binding.

[0867] Example 23 – Characterization of VHH-Fc with a wide range of DAR (2-12) using IgG1-derived, IgG2-derived, IgG3-derived, and IgG4-derived hinge regions.

[0868] The inventors have demonstrated that the IgG3-derived hinges (D3, D9, D10, D11) tested to date can be utilized in a modular manner. These hinges are truncated to exhibit a predetermined number of interchain disulfides to allow for the desired DAR. For example, for D3, by utilizing four heavy interchain disulfides, the hinge exhibits a DAR of 8. In the following steps, the inventors endeavor to demonstrate the modularity of the concept by utilizing hinge regions from modified hinge sequences derived from IgG1-derived, IgG2-derived, IgG3-derived, and IgG4-derived hinges to drive a wide range of DARs (from as low as 2 to as high as 12).

[0869] To achieve this very unique technique, the inventors applied their hinge shortening and PTM modification method to multiple hinges.

[0870] For the anticipated 2 DAR, the inventors used the IgG1 hinge (which exhibits an inter-heavy chain disulfide) as a starting point and exchanged each individual cysteine ​​for serine (or alanine) (D4, D5).

[0871] The DAR4 form of VHH-Fc ADC is prior art and utilizes the IgG1 hinge region. However, the inventors investigated complementary methods using modified IgG2 hinges (D6), modified IgG3 hinges (D7), or modified IgG4 hinges (D8). In D6, the two most N-terminal Cys residues are substituted because those involve different disulfide isoforms described for IgG2 antibodies. In D7, the IgG3 hinge is truncated to include the intended two interchain disulfides and an additional mutation to avoid post-translational modifications such as O-glycosylation. D8 carries the well-known S228P mutation, which avoids Fab-arm exchange in IgG4 antibodies.

[0872] When utilizing the human hinge region, higher DAR can be achieved by utilizing the modified IgG3 hinge. By using a variety of truncated and PTM-optimized forms, the inventors constructed D9 (capable of having DAR 6), D10 (capable of having DAR 10), and D11 (capable of having DAR 12).

[0873] Antibodies exhibiting these hinge designs in combination with the A1B3 binder were generated and purified. Surprisingly, as with the D3 design above, no hinge-dependent effects were observed for any antibody in terms of overall stability or monomer content after purification (Table 34).

[0874] Table 34 – Biophysical characterization of VHH-Fc antibodies exhibiting a wide variety of modified and heterologous hinge regions. Thermal denaturation values ​​as determined by NanoDSF are described.

[0875]

[0876] Furthermore, the inventors unexpectedly observed no difference in binding affinity for antigen 1 or even CD64 (Table 35).

[0877] Table 35 – Showing the affinity of VHH-Fc for a wide variety of modified and heterologous hinge regions. BLI was used to determine the primary antigen affinity for VHH-Fc and the affinity for CD64.

[0878]

[0879] Cell binding assays using antigen 1-positive tumor cells revealed an EC50 value of approximately 1 nM. Although slight differences were observed, no clear trend could be derived in terms of hinge sequence length or allotype dependence (Table 36).

[0880] Table 36 – Showing cell binding of VHH-Fc with various modified and heterologous hinge regions. Target-positive tumor cells were stained with different concentrations of VHH-Fc, and EC50 values ​​were measured.

[0881]

[0882] In the next step, full mass spectrometry measurements were performed on the non-deglycosylated antibodies. As expected for antibodies containing IgG-Fc, distinct glycosylation patterns were observed. The Δmass (Da) of these antibodies relative to their calculated mass is described in Table 37.

[0883] Table 37 - The mass error of the intact molecule relative to the calculated mass of VHH-Fc, which exhibits a wide variety of modified and heterologous hinge regions.

[0884]

[0885] The antibody formed the expected dimer, and the low Δ mass suggests that no modification occurred.

[0886] In this paper, the inventors tested forms D4 and D5 (SEQ ID NO: 8 and 9), where X = S. These hinges are IgG1-derived and therefore not heterogeneous hinges in the presented context. In both designs, additional O-glycosylation was observed within the hinge region (data not shown). This modification was avoided by replacing X with A in D5 (SEQ ID NO: 9) (data not shown). No modification was observed in the hinges comprising designs D6–D11.

[0887] In summary, these experiments surprisingly demonstrate that the hinge region of an antibody can be swapped with a truncated or otherwise modified sequence that is heterologous to the Fc, and a fully functional fusion protein can still be produced. These hinge modifications do not affect cell or protein binding, nor do they affect the overall stability or purity of the antibody.

[0888] Example 24 – ADC generation using VHH-Fcs with DAR 2-12 via IgG1-derived, IgG2-derived, IgG3-derived, and IgG4-derived hinge regions.

[0889] Because the inventors were able to generate antibodies exhibiting a wide variety of heterogeneous hinge regions, they sought to investigate whether the resulting variants would be suitable for ADC generation. Therefore, as described above, a topoisomerase I inhibitor was conjugated to a partially reduced antibody via thiol reaction chemistry. The DAR of the resulting ADC was examined by mass spectrometry (Table 38).

[0890] Table 38 describes the expected and realized DAR of the tested VHH-Fc fusions exhibiting a wide variety of modified and heterogeneous hinge regions, as determined by mass spectrometry.

[0891]

[0892] With high homogeneity, the inventors were able to generate ADCs with a wide variety of DARs. From low DARs, such as 2, to very high DARs, such as 12, the inventors showed that the heterogeneous hinge method provides a reliable sizing characteristic spectrum. This demonstrates that the desired DAR can be easily achieved in this invention by means of existing sizing methods.

[0893] Example 25 – Characterization of ADCs exhibiting IgG1-derived, IgG2-derived, IgG3-derived, and IgG4-derived hinge regions.

[0894] Next, the inventors characterized the previously generated ADC based on its biophysical properties. The fluorescence of the toxin complicates the precise determination of the melting temperature due to interference from the intrinsic fluorescence of the protein measured during heat-induced unfolding. Therefore, approximations were used for certain data points (labeled in Table 39). ).

[0895] Table 39 – Biophysical characterization of VHH-Fc ADCs for a wide variety of DARs. Thermal denaturation values ​​determined by NanoDSF, and purity assessments performed by SEC.

[0896]

[0897] Interestingly, the higher DAR ADCs generated using the techniques presented herein maintained their thermal stability and purity compared to the prior art IgG1-hinge tested in Example 9. Furthermore, these results indicate that the higher DAR (e.g., DAR10, DAR12) ADCs did not exhibit significantly increased aggregation.

[0898] Affinity measurements of ADC for antigen 1, CD64, and FcRn did not show a DAR-dependent effect (Table 40).

[0899] Table 40 shows the affinity of various DAR VHH-Fc ADCs for antigen 1 and Fc receptors. Affinity for antigen 1, CD64, and FcRn was determined using BLI.

[0900]

[0901] Similarly, the cell binding profiles of the tested ADCs were very similar, with EC50 values ​​of approximately 1–2 nM (Table 41). This further emphasizes that the hinge and DAR tested in this paper do not affect the binding properties of the ADCs.

[0902] Table 41 – Showing the cell binding of various DAR-VHH-Fc ADCs. Target-positive tumor cells were stained with different concentrations of VHH-Fc ADC, and EC50 values ​​were measured.

[0903]

[0904] In summary, an ADC with a wide range of DARs was generated by utilizing heterogeneous hinge regions. The expected DAR for these ADCs is predetermined by the sequence of the selected hinge. The inventors not only demonstrated the homogeneous achievement of these DARs, but they further proved that the antibodies exhibit very comparable performance in terms of biophysical properties crucial for the development of therapeutics. Among those parameters are monomer content and binding capacity.

[0905] Example 26 – Demonstrates cytotoxicity assays for ADCs with IgG1-derived, IgG2-derived, IgG3-derived, and IgG4-derived hinge regions and a wide variety of DARs.

[0906] Finally, the inventors aimed to verify the cytotoxicity of the ADCs generated in the foregoing examples. Again, they tested HEK cells expressing antigen 1 and target-negative HEK cells for dose-dependent cytotoxicity at a series of ADC concentrations. For comparison, the A1B3-D3-IgG1-DAR8 ADC from Example 11 was tested again. Further, as a negative control, unconjugated VHH-Fc, i.e., A1B3-D3-IgG1, was tested. Non-binding DAR8 ADC was further included as a non-specific control. Figure 10 ).

[0907] The inventors observed significant cytotoxicity against target-positive cells. Furthermore, it was evident that high DAR mediated lower IC50 values ​​and lower baseline values. The latter was defined as the minimum observable residual viability (…). Figure 11 ).

[0908] These results clearly show that higher DAR leads to lower IC50 values ​​and also lower baseline values. To test whether this effect is mediated not only by using a higher DAR ADC, but also by higher DAR combined with high specificity, the experiment was repeated for target-negative cells. Figure 12 In this experiment, without considering DAR, no killing effect was observed at the relevant concentrations, and only a very weak effect was observed at extremely high concentrations.

[0909] Using these results, the inventors demonstrated for the first time a clear difference between ADCs, namely that higher DAR mediated higher potency in cytotoxicity assays. However, based on the targeted antigen and intended indication, one may want to strive for lower DAR to avoid on-target off-tumor toxicity. In other cases, where the antigen is highly tumor-selective, higher DAR may lead to more effective drugs. In any case, it is necessary to select the optimal DAR for each ADC. In this paper, the inventors demonstrate a simple, straightforward, and novel modification method in which antibodies can be adaptively tuned using heterologous hinge / Fc region pairings to allow any desired DAR to be achieved. Since all other properties of the antibody remain substantially unaffected by the method of this invention, including stability, aggregation behavior, and binding affinity, the determination of DAR can be made solely within the biological context, and is not limited to prior art hinges and the associated DAR mediated by those hinges, while also achieving highly homogeneous ADC products.

[0910] discuss

[0911] The inventors of this application recognize the limitations in the art regarding the provision of ADCs in which DAR can be selected, and still provide ADCs with acceptable in vivo properties (e.g., stability) and available in a substantially homogeneous population. In response, the inventors of this application propose a simple and straightforward invention that allows for personalized DAR values ​​without compromising any other properties of the antibody or ADC.

[0912] The inventors of this application have generated antibody patterns that enable control (or predetermined) of the DAR of an ADC by designing engineered IgG hinge regions containing a predetermined number of cysteine ​​residues. This novel class of antibodies containing engineered IgG hinge regions provides functional antibody patterns that are generated as efficiently as conventional antibody patterns. The inventors' method also addresses the issues of immunogenicity (by utilizing human sequences) and association with free thiol groups (by using paired cysteine ​​residues).

[0913] Further evidence shows that modifications to avoid O-glycosylation within the engineered IgG hinge are well tolerated. VHH-Fc constructs incorporating such modifications are functional and are expected to provide homogeneous ADC products suitable for large-scale production for clinical use.

[0914] FACS experiments showed that all tested VHH-Fc cells containing engineered IgG hinge regions were able to bind to target-positive cells. This highlights the full functionality of VHH-Fc.

[0915] Therefore, this work provides novel antibodies that can be engineered to have the desired DAR, provide homogeneous ADC products, and retain or enhance ADC properties compared to conventional antibodies, including stability, cell binding, and internalization.

[0916] Furthermore, the inventors demonstrated that antibodies exhibiting engineered IgG3 hinges are stable in human serum (Example 7). This was unexpected, as antibody hinge regions have been considered readily susceptible to proteolytic cleavage since the 1960s. In the serum stability studies presented herein, it was surprisingly demonstrated that antibodies containing IgG3-derived hinges were stable in serum for up to two weeks without observed proteolytic cleavage. This is a prerequisite for development into therapeutic molecules and is an unexpected finding that contradicts expectations based on the literature.

[0917] By utilizing a standard method—partial reduction of the antibody with TCEP followed by conjugation of the linker payload molecule via a thiol-reactive moiety—the inventors demonstrated successful ADC generation (Example 8). Surprisingly, the desired DAR was achieved precisely in a highly homogeneous manner. Thus, the inventors not only demonstrated for the first time that VHH-Fc ADCs with more than 4 DARs (mediated by hinges) are possible, but also presented a general proof of concept for the heterojunction technique. Subsequent examples (Examples 9-13) showed that the DAR 8 molecule behaved very similarly to prior art DAR 4 ADCs, while mediating more efficient cell killing. Subsequently, the inventors showed that this was not antigen-specific, and repeated the most critical analyses with a large number of VHHs targeting antigens 2 and 3 (Examples 15-18), clearly demonstrating the modularity of the method. This modularity is further emphasized because the inventors have demonstrated that the heterojunction technology is compatible with Fab:VHH and VHH:VHH bispecific antibodies (Examples 19-21), and also with Fc regions from other isotypes (Example 22). In particular, given the results achieved with Fab:VHH bispecific antibodies, it can be assumed that bispecific Fab:Fab molecules and monospecific IgG are also compatible with the technology presented herein.

[0918] After determining that the hinge region of the antibody could be homogeneously exchanged for heterologous sequences allowing for precise DAR values, the inventors went further and used a large number of hinge sequences from all four human IgG isotypes to generate ADCs exhibiting DARs ranging from as low as 2 to as high as 12. The antibodies performed identically across all measured parameters, except in cytotoxicity assays, where higher DARs mediated more effective cell killing (Examples 23-26). It should be noted that design 11 (D11), which mediates a DAR of 12, is also a truncated and PTM-modified form of the IgG3 hinge. Furthermore, using the techniques described in this application, ADCs with DARs of 14, 16, 18, etc., can also be readily achieved by correspondingly modifying the hinge sequences.

[0919] 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 been previously reported that different hinges and antibody isotypes can affect antibody binding properties (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). However, this was not observed in any cell-free or cell-based binding assays, whether examining the ADC or its unconjugated parent molecule.

[0920] Furthermore, quite surprisingly, no differences in stability were observed when VHH was combined with several different hinge regions. Chiu et al., in their 2019 review article, argued that human IgG hinges vary significantly in terms of the number of residues and disulfide bridges, and that the selection of these parameters “contributes to the overall stability of the antibody” (ibid.). However, unexpectedly, the inventors observed that thermal stability was mediated almost solely by the variable region, as antibodies exhibiting IgG4-derived or IgG3-derived hinges had melting temperatures very similar to their IgG2-derived or IgG1-derived counterparts (Table 34). While conjugation of the antibody to toxin decreased thermal stability for all antibodies, surprisingly, the inventors did not observe a hinge-mediated effect, thus allowing them to select hinge regions solely based on the expected DAR without compromising thermal stability.

[0921] By utilizing heterohinged technology, the inventors avoided the damaging half-life effect of IgG3 Fc demonstrated in Example 13. Furthermore, no proteolytic cleavage was observed in any of the antibodies or ADCs described herein. Skilled scientists in the art would expect that the problems described in the literature regarding significant antibody hinge modifications would hinder the heterohinged approach from providing ADCs. However, the inventors surprisingly found that the antibodies described herein performed very favorably. Mass spectrometric analysis of many antibodies containing heterohinged regions (Examples 5, 15, 23) revealed no cysteylation, trisulfide formation, glutathioneization, or any other anticipated but undesirable modifications. Affinity for CD64 (Fcγ receptor 1) remained unchanged across a wide range of molecules tested, and no cleavage effect was observed in vitro (Example 10) and in vitro (Example 13). Furthermore, in PK experiments, the behavior of antibodies with heterohinged regions was indistinguishable from that of antibodies exhibiting heterohinged regions (Example 13). In conclusion, it is quite surprising and unexpected that the hinge area does indeed perform so favorably in heterogeneous situations.

[0922] In summary, the literature clearly suggests that hinge sequences are associated with numerous problems, and their transfer leads to heterogeneous mixtures of disulfide isoforms. Surprisingly, the inventors discovered (truncated) hinge sequences and methods for incorporating them into antibodies to circumvent these known obstacles.

[0923] All publications mentioned in the foregoing specification are incorporated herein by reference. Various modifications and variations to the methods and systems of the invention described herein 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 conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications to the described modes for carrying out the invention will be apparent to those skilled in molecular biology or related fields and are intended to be within the scope of the following claims.

Claims

1. A polypeptide comprising an immunoglobulin fragment crystallizable (Fc) region, at least one variable domain at the N-terminus 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 at the N-terminus 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 through cysteine-based site-specific conjugation, wherein the antibody comprises a first polypeptide comprising an Fc region, at least one variable domain at the N-terminus 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 of claim 2 or 3, wherein the antibody further comprises a second polypeptide comprising an Fc region and an engineered IgG hinge region at the N-terminus 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 of claim 4, wherein the second polypeptide further comprises at least one variable domain at the N-terminus of the engineered IgG hinge region.

6. An antibody-drug conjugate (ADC), wherein the antibody is conjugated to the drug through 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 crystallizable (Fc) region, at least one variable domain at the N-terminus of the Fc region that is a VHH, and an engineered IgG hinge region between the Fc region and the at least one VHH, 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 of any of the preceding claims, wherein the engineered IgG hinge region of each polypeptide is heterologous to the Fc region of the polypeptide. ​ ​ ​ 8. The polypeptide, antibody or ADC of 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 of any one of the preceding claims, wherein the engineered IgG hinge region of the first polypeptide additionally comprises at least one 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 additionally comprises at least one cysteine residue compared to the native hinge region of the Fc region of the second polypeptide.

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

11. The polypeptide, antibody or ADC of any one of the preceding claims, wherein the engineered IgG hinge region is an IgG1 hinge region, an IgG2 hinge region, an IgG3 hinge region or an IgG4 hinge region.

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

5.

13. The polypeptide, antibody or ADC of any one of the preceding claims, wherein the engineered IgG hinge region has an amino acid sequence set forth in any one of SEQ ID NOs: 8-118, preferably the amino acid sequence 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 of any one of the preceding claims, wherein the engineered IgG hinge region of the first and / or second polypeptide comprises at least one amino acid modification to avoid O-glycosylation as compared to the native IgG hinge region of the Fc region of the first and / or second polypeptide.

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

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

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

18. The ADC of 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 to a 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 crystallizable (Fc) region and at least one variable domain at the N-terminus 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 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 to a drug.

21. A method for generating an antibody-drug conjugate (ADC) having a predetermined drug-antibody ratio (DAR), comprising the following steps: (i) (a) Provides 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 crystallizable fragment (Fc) region and at least one of the N-terminus of the Fc region as a variable domain of VHH. (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 in 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 mutated to provide a predetermined number of cysteine ​​residues; (c) Introducing the at least one polynucleotide sequence into the cell; (d) The cells are cultured under conditions suitable for expressing the at least one polynucleotide sequence; (e) Isolate the antibodies expressed by the cells; (ii) Reduce the antibody with a reducing agent; and (iii) The reduced antibody is conjugated 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 interchain disulfide 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 containing a thiol reactive group; and / or (b) Step (iii) is performed using a thiol reaction coupling strategy.

24. The method according to any one of claims 19 to 23, wherein the method provides a homogeneous ADC.

25. The method of 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 at the N-terminus of the Fc region of the second polypeptide, wherein the engineered IgG hinge region is heterologous and / or mutated for the Fc region of the second polypeptide 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) Prior to step (ii), all cysteine ​​residues within the hinge region of the engineered IgG form stable interchain disulfide bonds; (b) In step (iii), the antibody is conjugated to the drug by cysteine-based site-specific conjugation, through all cysteine ​​residues in the hinge region of the engineered IgG; (c) The ADC has a drug-to-antibody ratio (DAR) of about 2 to about 12, preferably about 4 to about 8; and / or (d) The ADC has a drug-antibody ratio (DAR) of approximately 2, 4, 6, 8, 10 or 12, preferably approximately 8.

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

28. A pharmaceutical composition comprising the ADC of 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 a 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 diseases or conditions selected from the group consisting of: cancer, autoimmune diseases, infections, infectious diseases, cardiovascular diseases, and liver metabolic disorders.

31. One or more nucleic acid sequences capable of expressing a polypeptide or antibody according to any one of claims 1 to 18 or 27.

32. Nucleic acid particles comprising the nucleic acid sequence according to claim 31.

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

34. A method for preparing an ADC, comprising: i) Expressing the nucleic acid sequence or nucleic acid particles according to claim 31 or 32 to generate antibodies; ii) Reduce the antibody with a reducing agent; and iii) Conjugate the reduced antibody with the drug.

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