Anti-PROTAC antibodies and conjugates

Abstract

The present invention relates to monospecific or bispecific antibodies or antibody fragments or fusion proteins thereof capable of binding to the degradative portion (degron) of the VHL ligand VH032 (or derivatives thereof) of a targeted proteolytic chimera (PROTAC), and optionally to a target protein. The present invention also relates to conjugates of such antibodies or antibody fragments or fusion proteins with PROTACs (PAXs), as well as methods for their production, and their respective medical and non-medical uses.

Description

[Technical Field]

[0001] 1.Technical Field The present invention relates to monospecific or bispecific antibodies or antibody fragments or fusion proteins thereof capable of binding via their VHH moiety to the degron (degron) of the VHL ligand VH032 (or a derivative thereof) of a targeted proteolytic chimera (PROTAC) and, optionally, to a target protein. The present invention also relates to conjugates of such antibodies or antibody fragments or fusion proteins with PROTACs (PAXs), as well as methods for their production and their respective medical and non-medical uses. [Background technology]

[0002] 2.Background 2.1 Degradation of Unwanted Proteins - PROTAC Controlled degradation of cellular proteins is necessary for cell maintenance and normal function. For example, degradation of regulatory proteins triggers events during the cell cycle, such as DNA replication and chromosome segregation. Therefore, such degradation of proteins affects cell proliferation, differentiation, and death. While protein inhibitors can block or reduce protein activity in cells, proteolysis is another possibility for reducing or completely eliminating the activity of target proteins. Therefore, utilizing cellular proteolytic pathways can provide a means to reduce or eliminate protein activity. One of the major cellular degradation pathways is known as the ubiquitin-proteasome system. In this system, proteins are marked for proteasomal degradation by E3 ubiquitin ligases, which bind to the protein and transfer ubiquitin molecules to it. E3 ubiquitin ligases are part of a pathway that includes E1 and E2 ubiquitin ligases, which make ubiquitin available for transfer to proteins catalyzed by the E3 ubiquitin ligase. PROTACs have been developed to harness this degradation pathway for desired proteins. PROTACs can bring E3 ubiquitin ligases into close proximity with desired proteins, resulting in ubiquitination and marking them for degradation. PROTACs are heterobifunctional molecules that contain a structural motif that binds to E3 ubiquitin ligases and another motif that binds to the protein desired for degradation. These groups are usually connected using a linker.

[0003] Only a small percentage of the approximately 600 E3 ligases, namely MDM2, inhibitors of apoptosis proteins (IAPs), HECT and RBR family members, RNF4, DCAF16, EAP1-Nrf2, von Hippel-Lindau (VHL), and cereblon (CRBN), have been successfully applied to targeted protein degradation, with the last two playing the largest role. VHL is a well-established E3 ligase substrate receptor that tightly binds hydroxylated HIF-1α. Based on peptide structures surrounding the hydroxyl-prolyl binding site of HIF-1α, more drug-like small molecule ligands have been derived and successfully applied to create chimeric proteolytic agents (Shanique, A. and Crews, C., J. Biol. Chem 296 (2021) 100647).

[0004] A widely applied ligand is VH032 (Figure 1A), a VHL ligand that binds to VHL with strong affinity (Galdeano, C. et al. J. Med. Chem. 57 (2014) 8657-8663). VH032 is particularly tolerant of acetyl group substitution (Ciulli, A and Ishida, T. SLAS 7Discovery 26 (2021) 484-502), paving the way for the development of additional VHL ligands such as VH298 (Soares, P. et al. J. Med. Chem. 61 (2018) 599-618).

[0005] The elucidation of the mode of action of thalidomide as a CRBN ligand has opened up the possibility of CRBN being used for targeted protein degradation, where various target proteins have already been degraded through CRBN association (Shanique, A. and Crews, C., J. Biol. Chem. 296 (2021) 100647).

[0006] Targeted proteolysis has already been achieved for a large number of proteins by association of these E3 ligases with chimeric degraders. Examples include CRBN, VHL, Tau, DHODH, FKBP12, AR, ERα, RAR, CRABP-II, ALK, CK2, CDK8 and CDK9, BTK, PI3K, TBK1, FLT3, BTK, RTKs such as EGFR, HER2 and cMET, ERK1 and ERK2, BCR-ABL, RIPK2, BCL6, PCAF / GCN5, BRD4 and HDAC6, TRIM24, SIRT2, BRD9 (Scheepstra, M., Comput. Struct. Biotec. 17 (2019) 160-176; U.S. Patent Application Publication Nos. 2018 / 0125821; 2015 / 0291562; 2017 / 0065719).

[0007] There are numerous synthetic strategies for assembling heterobifunctional degraders. In one example (US Patent Application Publication No. 2017 / 0065719), degraders were primarily synthesized via condensation reactions of activated carboxyl functional groups with amides. Thus, the VHL ligand VH032 and its derivatives were reacted with activated carboxylic acid-containing linker structures. The linker structures possessed terminal amines, which, after deprotection, reacted with the activated carboxylic acid functional groups of protein binders. However, synthetic strategies are highly dependent on the chemical nature of the ligand that must be modified. In another example, the hydroxyl group of 7-hydroxy-thalidomide was modified using an alkylation reaction with propargyl bromide or propargyl tosylate. The resulting compounds retained click chemistry handles, which were then used to obtain the complete degraders via copper(I)-catalyzed azide-alkyne cyclization (Wurz, RP et al. J. Med. Chem. 61 (2018) 453-461).

[0008] The androgen receptor degrading ARV-110 and the estrogen receptor degrading ARV-471 (Arvinas, Inc.) are the two most advanced PROTACs in clinical development, having recently reached Phase II. However, several heterobifunctional degraders have already reached Phase I clinical development for various targets, such as the BCL-XL PROTAC DT2216 degrader (Dialectic, Inc.) and the IRAK4 degrader KT474 (Kymera / Sanofi SA).

[0009] Many reported PROTACs are highly efficient degraders, but they are generally non-tissue specific because they utilize E3 ligases with broad expression profiles. Tissue-specific degradation may enable optimization of the therapeutic window and minimize side effects, while increasing the potential of broad-spectrum PROTACs as drugs or chemical tools. However, no PROTACs utilizing E3 ligases with restricted tissue distribution have been reported to date, and the development of novel E3 ligase ligands remains a significant challenge (Maneiro, M. et al. ACS Chemical Biology 5 (2020) 1306-1312). Another challenge in PROTAC development is their short circulatory half-life in the range of several hours in mice (Pillow, TH et al., ChemMedChem 15 (2020) 17-25; Burslem, GM et al., J. Am. Chem. Soc. 140 (2018) 16428-16432).

[0010] Furthermore, the effectiveness of PROTACs is often hindered by their poor permeability (Klein, VG et al., ACS Med. Chem. Lett. 11 (2020) 1732-1738), which limits their ability to enter cells and induce proteolysis.

[0011] Therefore, there is currently a need in the art for enhanced, targeted delivery of PROTACs to cells containing the protein target to be degraded.

[0012] To address this need, attempts have been made to enhance the delivery of PROTACs to specific cells by using covalent antibody-PROTAC conjugates similar to antibody-drug conjugates (ADCs). Such constructs take advantage of the cell target-selective binding and enhanced pharmacokinetics conferred by antibodies.

[0013] 2.2 Targeted Drug Delivery – Antibody-Drug Conjugates (ADCs) The basic concept of ADCs is fairly simple. All that is required is an antigen that allows for molecular differentiation between cancer and healthy cells. This could be, for example, a specific cell surface receptor that is highly upregulated in tumor cells. Antibodies against such antigens can serve as highly potent targeting vehicles for cytotoxic drugs, or "payloads." To form an ADC, the cytotoxic drug must be covalently attached to the antibody via a linker that is stable in circulation to avoid premature release of the payload. After administration, the ADC distributes throughout the patient's body and binds to its antigen on the surface of tumor cells. The antibody-antigen complex is then internalized by the cell and directed to lysosomes via endogenous intracellular transport pathways. After reaching the lysosomes, the ADC is degraded, thereby releasing its toxic cargo. The free toxin can then bind to its intracellular target and thus induce apoptosis and cancer cell death. In some cases, the toxin can leave the cancer cell and act similarly on neighboring, ideally cancerous, cells. This process is called the bystander effect, the extent of which varies depending on the linker and drug applied. On the other hand, healthy cells are largely spared, since the antibody should only bind and deliver the toxin to cancer cells that express the antigen.

[0014] ADCs approved for the treatment of cancer include the HER2-targeting DM1 conjugates Kadcyla and Adcetris, an anti-CD30 ADC bearing the tubulin inhibitor MMAE, and the CD33-targeting calicheamicin ADC Mylotarg.

[0015] The design of ADCs is a multidisciplinary effort because they are composed of biotechnologically generated biomolecules and chemically synthesized, highly potent small molecule drugs. Both entities are generated separately and then combined into highly complex hybrid molecules. Therefore, the entire process of ADC development, from the design of the individual components to the final production of the conjugate, poses significant technical challenges. According to the term "antibody-drug conjugate," the main components of an ADC are the drug and the antibody. However, coupling these entities requires a linker connecting the mAb to the drug. Careful selection of this linker, taking into account both the mAb and the payload, is crucial for the efficacy and safety of the final ADC. In the bloodstream, the linker should be as stable as possible to prevent premature payload release, which could otherwise cause systemic off-target toxicity. However, once the ADC reaches the target cells, the payload must be active without being hindered by the attached linker. Furthermore, the length and chemical nature of the linker can have a strong impact on the pharmacokinetics and dynamics of the ADC. Linkers used in ADCs are mainly classified into non-cleavable and cleavable. Non-cleavable linkers are stable both in circulation and in cells, while cleavable linkers are designed to be degraded by specific intracellular mechanisms in target cells. From the above, it becomes clear that engineering an appropriate linker for a given ADC is a challenge in itself.

[0016] While all three parts of an ADC—antibody, linker, and cytotoxic payload—determine important properties of the final conjugate, an equally important parameter is how these components are assembled. Linkers and payloads are generated by chemical synthesis as combined linker-payload structures that are directly conjugated to the mAb, or as individual components that are sequentially assembled during ADC construction. In both cases, small molecules must be conjugated to mAbs without compromising their favorable properties, which poses a significant technical challenge. Key parameters that must be controlled during ADC construction are the number of linker-drugs conjugated to each antibody, referred to as the drug-antibody ratio (DAR), and the location on the antibody surface where the structures are attached (conjugation site). Both parameters can critically affect several properties of the ADC, including its stability and pharmacokinetic behavior, and ultimately its toxicity and efficacy profile. On the other hand, the warheads used in ADCs are largely hydrophobic, and increasing the DAR can significantly alter the overall hydrophobicity and significantly disrupt the protein stability of the final conjugate. On the other hand, to achieve a fully active ADC, a certain amount of drug is required depending on its potency. However, not only the DAR but also the conjugation site and chemistry have a significant impact on these parameters. For example, several studies have shown that certain sites exhibit superior tolerance to difficult payloads and result in more stable conjugates than others by providing a favorable microenvironment and steric shielding on the antibody surface. Therefore, finding an optimal combination of the individual components (linker, drug, and mAb) as well as a suitable DAR, conjugation strategy, and conjugation site is key to developing efficient and safe therapeutic drugs (Dickgiesser, S. et al., Introduction to Antibody Engineering, Springer (2021) 189-214).

[0017] 2.3 ADCs with PROTACs as payloads A special form of ADC is degrader-ADC, in which the drug is represented by a proteolytic agent. In this case, a linker needs to be attached to the degrader to facilitate conjugation to an antibody. In addition to selecting the correct linker, it is also important to identify a suitable attachment site on the degrader (either the warhead, degron, or linker moiety). Several publications have demonstrated the feasibility of this concept.

[0018] One example is an estrogen receptor alpha (ERα) degrader covalently attached to a HER2-targeting antibody via conjugation to an engineered cysteine. Therefore, the degrader had to be chemically modified with a protease-cleavable linker either on the ERα-targeting moiety or on the XIAP-binding moiety. In the case of a degrader-ADC in which the linker was attached via a warhead, ERα degradation was achieved in HER2-overexpressing MCF7 cells, but significantly less degradation was observed in parental MCF7 cells. Additional linker options were tested. The hydroxyl group of the hydroxyprolyl residue of a VHL ligand was modified with a carbonate linker, which was then conjugated to a HER2 antibody via an activated disulfide. Furthermore, a diphosphate-containing linker was attached to the hydroxyprolyl residue of the VHL ligand. In both cases, the conjugates lacked selectivity (Dragovich, PS et al., Bioorg. Med. Chem. Lett. 30 (2020) 126907).

[0019] In addition to ERα as an intracellular PROTAC target for degrader-ADCs, BRD4 has also been intensively studied as a target protein. One example demonstrated selective delivery of a BRD4 degrader to HER2-positive cells, leading to BRD4 degradation via a HER2-targeting antibody. The degrader was conjugated to a hydroxyprolyl residue of a VHL ligand via a combination of cysteine ​​conjugation and click chemistry using an acid-cleavable ester bond (Maneiro, M. et al. ACS Chemical Biology 5 (2020) 1306-1312). In another example, the BRD4 degrader GNE987 was conjugated to an engineered cysteine ​​of a CLL1-targeting antibody, achieving a DAR of 6. Therefore, PROTACs were modified with an acid-cleavable carbonate linker containing an activated disulfide for conjugation. The conjugate was well tolerated and significantly improved the pharmacokinetic profile and in vivo efficacy of the PROTAC in a mouse xenograft model (Pillow, TH et al., ChemMedChem 15 (2020) 17-25).

[0020] BRD4 degrader conjugates have been thoroughly investigated by this group in two further publications (Dragovich, PS et al., J. Med. Chem. 64 (2021) 2534-2575; Dragovich, PS et al., J. Med. Chem. 64 (2021) 2576-2607). Multiple conjugates of BRD4 degraders based on STEAP1 and HER2 antibodies have been prepared. The focus of the research was on the ideal linker connecting the ADC and the degrader, as well as the ideal attachment point for this linker in either the target protein ligand, the E3 ligase ligand, or the linker between the target protein ligand and the E3 ligase ligand. Therefore, several target protein ligands were evaluated, including JQ1 derivatives incorporating suitable chemical handles for linker attachment. In the case of the linker between the target protein and the E3 ligase ligand, multiple variants were tested, including versions incorporating PEG and lipid chains as well as chemical handles for linker attachment. Furthermore, derivatives of VHL ligands chemically modified to allow for linker attachment have been evaluated. Although the conjugates were able to induce receptor-selective proteolysis, only a few exhibited selective cytotoxicity. These publications highlight the complexity of conjugating chimeric degraders to antibodies. Two patent applications have been filed for the described degrader conjugates (WO 2020 / 086858, WO 2017 / 201449).

[0021] In addition, BRD4 degrader conjugates have also been found in patent literature targeting HER2 (WO 2019 / 140003), and dual degraders of BRD4 and PLK1 have been investigated as payloads for CD33-targeting antibodies (WO 2020 / 073930). Furthermore, TGFβR2 degraders have been conjugated to HER2 and TROP2 antibodies for targeted delivery (WO 2018 / 227018, WO 2018 / 227023).

[0022] 2.4 Noncovalent approaches to drug delivery to target cells Several approaches have been described for non-covalent drug delivery, where the drug must always be chemically linked to a ligand or hapten that binds to or can be subjected to antibody binding.

[0023] For example, gemcitabine was chemically modified with 4-mercaptoethylpyridine, an affinity ligand that binds to several sites on antibodies. By combining an antibody with the affinity-ligand-modified gemcitabine, selective toxicity against target-positive cancer cells was induced, resulting in an ADC with a pharmacokinetic profile comparable to that of the unmodified antibody. Tumor regression of the gemcitabine ADC was observed in a mouse xenograft model (Gupta, N. et al., Nat. Biomed. Eng. 3 (2019) 917-929).

[0024] Furthermore, to facilitate drug delivery into cells, several approaches have used modification of the anticancer drug doxorubicin or small molecules such as fluorophores Cy5, siRNA, proteins like GFP, and saporin with the hapten digoxigenin (Metz, S. et al., Proc. Natl. Acad. Sci. 108 (2011) 8194-8199; Schneider, B. et al., Mol. Ther. - Nucleic Acids 1 (2012) e46; Mayer, K. et al., Int. J. Mol. Sci. 16, (2015) 27497-27507).

[0025] Furthermore, the cytotoxic drug duocarmycin DM could be delivered to EGFR-positive cells using a bispecific antibody conjugated to EGFR and simultaneously to cotinine. To deliver duocarmycin DM to target cells, a peptide bearing cotinine at the C- and N-termini was synthesized, and four duocarmycin DM molecules were attached to the peptide via a cleavable valine-citrulline linker. The construct was tested in a mouse EGFR-expressing A549 xenograft model and demonstrated superior antitumor efficacy to an isotype control construct (Jin, J. et al., Exp. Mol. Med. 50 (2018), 67). A similar construct was used to deliver duocarmycin DM to mPDGFRβ-positive cells (Kim, S. et al., Methods 154 (2019) 125-135).

[0026] Various other publications have detailed the concept of conjugation using hapten-modified compounds and anti-hapten antibodies (Yu, B. et al., Angew. Chemie - Int. Ed. 58 (2019) 2005-2010; Kim, H. et al., Mol. Pharm. 16 (2019) 165-172; Kilian, T. et al., Nucleic Acids Res., 47 (2019) e55).

[0027] A comparable approach uses covalent conjugation of tubulysin A to Fc-binding proteins such as protein A or G to assemble complexes with antibodies for targeted drug delivery (Maso, K. et al., Eur J Pharm Biopharm 142 (2019) 49-60).

[0028] Although there are numerous examples of noncovalent drug delivery using haptenized compounds together with anti-hapten antibodies or affinity ligands / proteins that bind to antibodies, examples of noncovalent drug delivery using unmodified drugs are rare.

[0029] Despite all these attempts, there remains a need for a well-defined, efficient, and specific delivery platform for PROTACs that can be widely applied and has effective release of the payload at the target. Summary of the Invention

[0030] 3. Summary of the invention The present invention relates to monospecific or bispecific antibodies, or antibody fragments or fusion proteins thereof, capable of binding via their VHH moieties to the degron (degron) of the VHL ligand VH032 (or a derivative thereof) of a targeted proteolytic chimera (PROTAC), and, in the case of bispecific antibodies, to a target protein. The present invention also relates to conjugates of such antibodies, or antibody fragments or fusion proteins thereof, with PROTACs, methods for their production, and their respective medical and non-medical uses. Such PROTAC-antibody conjugates are hereinafter referred to as "PAX."

[0031] In one embodiment, the target protein is a cell surface antigen on the target cell to which the PROTAC is delivered. Upon delivery, the PROTAC is released into the cytosol of the target cell, where it binds to the target protein for degradation, thereby initiating its degradation via the cellular proteasome.

[0032] An advantage of PAX compared to covalently linked antibody-drug conjugates (ADCs) is that no specific manufacturing steps are required to link the PROTAC to the antibody. Another advantage is that once PAX releases its PROTAC payload, it is ready for a new cycle of PROTAC binding and targeted delivery of the PROTAC molecule, for example, away from the target cell to which it was previously delivered.

[0033] Another advantage is that the conjugation of the PROTAC to PAX is predicted to improve the pharmacokinetic profile, extending the half-life of the PROTAC in the patient's body. Because of the complexation of the PROTAC with the anti-PROTAC antibody, the complex stability determines the clearance of the PROTAC. As long as the PROTAC is conjugated to the antibody, it cannot be eliminated nephrolytically due to the antibody's high molecular weight.

[0034] In one embodiment, a bispecific antibody comprises a) a monospecific bivalent antibody consisting of two full-length antibody heavy chains and two full-length antibody light chains, each chain comprising only one variable domain, b) two heavy chain single domains (VHH), each consisting of one antibody variable domain, and optionally c) two or more further copies of VHH(b) fused to said VHH, and optionally d) a peptide linker connecting a), b) and / or c), which peptide linker may be protease-cleavable or non-cleavable.

[0035] Those skilled in the art will understand that the presence or length of the peptide linker does not affect the performance of the present invention, however, in one embodiment, the peptide linker consists of 1 to 50 amino acids, preferably 1 to 35 amino acids, more preferably 3 to 20 amino acids, even more preferably 12 to 18 amino acids, for example, 15 amino acids.

[0036] In one embodiment, a peptide linker connects the C-terminus of the antibody heavy and / or light chain to the N-terminus of the VHH.

[0037] In one embodiment, the VHH is fused to the C-terminus of the heavy chain of the antibody.

[0038] In certain embodiments, each of the two heavy and light chains of antibody (a) carries up to three heavy chain single domain (VHH) antibodies (b) fused to the C-terminus of said antibody chain (a). Optionally, a peptide linker connects the C-terminus of (a) to the N-terminus of (b).

[0039] In one embodiment, the antibody does not comprise an additional copy of the VHH.

[0040] In one embodiment, the variable regions of the monospecific bivalent antibody bind to the target protein and the VHH binds to the PROTAC.

[0041] In one embodiment, the bispecific antibody is characterized in that the target protein is a cell surface antigen, for example, a tumor antigen. In a preferred embodiment, the target protein is HER2, CD33, CLL1, EGFR, CD19, CD20, CD22, B7H3 (CD276), CD30, CD37, CEACAM5, cMET, MUC1, ROR1, CLDN18.2, TROP2, BCMA, CD25, CD70, CD74, CD79b, TROP2, cMET, STEAP1, NaPi2b, PSMA, integrin alpha-V, FRα, MUC16, Mesothelin, CEACAM5, CanAg-MUC1 glycoform, EpCAM, HER3, or TNC. In a more preferred embodiment, the target protein is HER2, CD33, CLL1, or EGFR.

[0042] However, one of skill in the art will understand that the present invention will work with any targeting protein that establishes a subset of cells for targeted PROTAC delivery compared to any cells present in a patient's body.

[0043] Another aspect of the invention is a method for treating a disease in which degradation of a particular target protein is suspected, wherein PAX is administered to a patient in need thereof.

[0044] It is contemplated that the PAX disclosed herein can be used to treat various diseases or disorders.Exemplary hyperproliferative disorders include benign or malignant solid tumors and blood disorders, such as leukemia and lymphatic tumors.Others include neuronal, glial, astrocytic, hypothalamic, glandular, macrophage, epithelial, interstitial, blastocoelic, inflammatory, angiogenic and immunological disorders, including autoimmune.

[0045] Another aspect of the invention is a pharmaceutical composition comprising PAX according to the invention. In yet another aspect, said pharmaceutical composition is used in targeted cancer therapy.

[0046] In still other embodiments, the antibodies of the present invention serve to detect and / or quantify PROTACs or to purify a PROTAC of interest from impurities / by-products of the manufacturing process.

[0047] As shown herein below by protein crystallography as well as binding data from single amino acid mutants, the VHH according to the invention uses only its complementarity determining region (CDR) 3 for binding to the VHL ligand VH032 (or a derivative thereof), without involving CDRs 1 and 2. 4 List of Figures [Brief explanation of the drawings]

[0048] [Figure 1] Figure 1 shows the chemical structures of the VHL ligand VH032 and derivatives. (A) Structure of VH032. (B) Markush structure of VH032-based VHL-ligands. (C) Representation showing different exit vectors (R1, R2, R3) for the linker connecting the VH032-based degron to different warheads, exemplarily shown for the MZ1, AT1, and ACBl1 warheads. The MIC2 antibody accepts the exit vectors R1 and R2, resulting in the binding of the PROTACs MZ1 and AT1. [Figure 2-1]Figure 2 shows the amino acid sequences of bispecific fusion proteins against cell surface antigens and PROTACs. Bold: sequence of anti-PROTAC antibody MIC2, CDR sequences are underlined; italics: linker sequences; underlined: antibody fragment sequences (anti-EGFR VHH sequences or anti-HER2 scFv. [Figure 2-2] Same as above. [Figure 3] FIG. 3 is a diagrammatic depiction of the range of BsAb variants possible according to the present invention. [Figure 4] FIG. 4 shows the chemical structures of VH032-based haptens. [Figure 5] FIG. 5 shows the hapten-to-carrier protein ratios of cBSA and huFc and the corresponding individual haptens derived from MALDI-MS measurements. [Figure 6] FIG. 6 shows the research plan for hybridoma screening to identify anti-VH032 antibodies. [Figure 7] Figure 7 illustrates the assay principle for affinity determination. A) MIC2 is immobilized on an SPR chip. The analyte flows past the antibody and is captured. After the PROTAC is captured, the buffer is exchanged and the PROTAC is allowed to dissociate again. B) Association of the PROTAC with the antibody is observed as an increase in signal, while dissociation leads to a decrease in signal. This is exemplarily shown for the binding of MIC2 to the PROTAC, MZ1. [Figure 8-1] Figure 8(a) shows VH032-based PROTACs tested for binding in an SPR assay. [Figure 8-2] Figure 8(b) shows VH032-based PROTACs tested for binding in an SPR assay. [Figure 9] Figure 9 shows the binding evaluation of bispecific antibodies aEGFRxMIC2 and aHER2xMIC2 to several PROTACs compared to the parent antibody MIC2. Affinity parameters were categorized by on- and off-rates and affinity. [Figure 10] Figure 10 shows loading-dependent complex formation analyzed by SE-HPLC. The peak distribution shifts with increasing theoretical loading from a peak of unconjugated antibody (0% loading) (left) to peaks of half-loaded (50% loading; antibody:PROTAC molar ratio = 1:1) antibody to fully loaded (100% loading; antibody:PROTAC molar ratio = 1:2) antibody. [Figure 11] Figure 11 shows the SE-HPLC profiles of crude and purified aEGFRxMIC2+GNE987 complexes. Blue-violet: crude sample; blue: desalted sample. [Figure 12] FIG. 12 shows the peak distribution of unpurified and purified aEGFRxMIC2+GNE987 complexes. [Figure 13] FIG. 13 shows the peak distribution of aEGFRxMIC2+GNE987 complex over time. [Figure 14] FIG. 14 shows the chemical structure of GNE987 modified with a linker. [Figure 15] Figure 15 shows exemplary fluorescence images of BRD4 levels. High green fluorescence correlates with high BRD4 abundance. Untreated cells had the strongest fluorescence, but fluorescence decreased for cells treated with 4 nM GNE987 and EGFR-targeted C225-L328C-GNE987 and aEGFRxMIC2 loaded with GNE987. Fluorescence increased with 4 nM GNE987-loaded non-binding aHER2xMIC2 compared to the EGFR-targeted complex. Green fluorescence is represented by gray shading. [Figure 16]Figure 16 shows BRD4 level quantification. A) GNE987, C225-L328C-GNE987, and aEGFRxMIC2+GNE987 had comparable effects on BRD4 levels across the concentration range investigated, while aHER2xMIC2+GNE987 degraded BRD4 to a lesser extent. B) BRD4 degradation was induced at a concentration of 4 nM for all analytes. [Figure 17] Figure 17 shows dose-response curve plots for aEGFRxMIC2+GNE987 and controls. Serial dilutions of test compounds were added to MDAMB468 cells, and after 3 days of incubation, the effect of each individual compound on cell viability was assessed. 50% (1:1) loading of the EGFR-targeted aEGFRxMIC2+GNE987 and benchmarks C225-L328C-GNE987 and GNE987 had comparable potency, while the non-binding control MIC2+GNE987 and 50% (1:1) loading of aHER2xMIC2+GNE987 had reduced potency. [Figure 18] Figure 18 shows dose-response curve plots for aEGFRxMIC2+GNE987 and controls. The PROTAC GNE987 had the highest efficacy, followed by aEGFRxMIC2+GNE987 at 25% loading. The non-binding control MIC2+GNE987 and aHER2xMIC2+GNE987 at 50% (1:1) loading had a reduced effect on cell viability. [Figure 19] FIG. 19 shows the IC50 value plots of the investigated molecules in N=3 biological replicates. [Figure 20] Figure 20 shows dose-response curves for HEPG2 cells treated with PROTAC-ADC and PROTAC shuttle. [Figure 21] FIG. 21 shows the molecular structures of BRD4-degrading GNE987 and its analog GNE987P, which bear a PEG linker. [Figure 22]Figure 22 shows that compared to GNE987P alone, complexed aEGFRxMIC2+GNE987P exhibits increased cytotoxicity against EGFR-expressing MDAMB468 cells at concentrations ranging from 0.1 to 10 nM, demonstrating targeted delivery. Complexation with non-targeted MIC2+GNE987P completely reduces the cytotoxicity of GNE987P. [Figure 23] FIG. 23 shows mouse plasma stability of GNE987 alone or in complex with aEGFRxMIC2 over 72 hours. [Figure 24] FIG. 24 shows mouse plasma stability of bispecific antibody aEGFRxMIC2 complexed with GNE987 over 96 hours. [Figure 25] Figure 25 shows the stability of the 50% loaded aEGFRxMIC2+GNE987 complex in mouse plasma over 96 hours. The aEGFRxMIC2+GNE987 complex was captured on beads, and the supernatant was collected for LC-MS analysis of unbound GNE987. The bead-bound aEGFRxMIC2+GNE987 complex was then eluted from the beads and subjected to GNE987 quantification using LC-MS. [Figure 26] FIG. 26 shows an immunization schedule for New World camelid immunization to generate anti-hapten antibodies. [Figure 27] FIG. 27 shows biotinylated VH032 for antibody discovery by phage display. [Figure 28] FIG. 28 shows the expression rate of VHH fusion proteins versus the unmodified parent antibody. [Figure 29] Figure 29 shows the sequential numbering of the indicated VHHs for MIC5, MIC7 and MIC10 wild type. Numbering starts at the VHH N-terminus. [Figure 30-1] FIG. 30 shows the co-crystal structures of MIC5 (top), MIC7 (middle) and MIC10 (bottom) in complex with the VH032 ligand. [Figure 30-2] Same as above. [Figure 31]FIG. 31 shows a superposition of the co-crystal structures of MIC5, MIC7 and MIC10 in complex with GNE987P. [Figure 32] FIG. 32 shows exemplary cell viability data for humanized MIC7 variants compared to the parental MIC7. [Figure 33] FIG. 33 is a diagram showing the molecular structure of CD33xMIC7[N-2H]. [Figure 34] Figure 34 shows a schematic diagram of SEED constructs with bivalent binding to EGFR (top) or monovalent EGFR binding (bottom) combined with the capabilities of two PROTACs (left) or one PROTAC (right). The gray ovals represent VL and VH. The white ovals represent the heavy and light chain constant regions. The black oval represents VHH MIC7. Figure 35 is intentionally omitted. [Figure 36] Figure 36 shows a commercially available IgG-based antibody being site-specifically conjugated to a VHH using microbial transglutaminase (MTG). The conjugated PROxAb shuttle as a bispecific protein can specifically bind to a PROTAC due to its MIC7 binding specificity for the ligand-binding subunit of VHL. [Figure 37] Figure 37 shows the measured DAR of CD33xMIC7[2H] shuttle complexed with GNE987 PROTAC over time by native SEC / MS. [Figure 38] FIG. 38 shows flow cytometry analysis of cell binding of CD33xMIC5 or EGFRxMIC5 to MV411 and MDAMB468, respectively, compared to the parental antibody lacking VHH MIC5. [Figure 39] Figure 39 shows a comparison of cell binding of the CD33-binding CD33xMIC7, loaded and unloaded with the PROTAC GNE987, to CD33-expressing cell lines. [Figure 40] FIG. 40 shows the structure of the pH-responsive VH032-pHAb dye. [Figure 41]FIG. 41 shows flow cytometry analysis of internalization of CD33xMIC7 into CD33-positive cells MOLM13, MV411 and U937 and CD33-negative RAMOS cells over a 6-hour period. [Figure 42] Figure 42 shows a Western blot of CD33xMIC7+GNE987 (1:1) and DIGxMIC7+GNE987 (1:1) on CD33-positive MV411 cells. Concentrations above the plot are shown in mol / L. Marker sizes (right) are shown in kDa. [Figure 43] FIG. 43 shows Western blot analysis of the degradation patterns of CD33xMIC7+GNE987 (1:1) and DIGxMIC7+GNE987 (1:1) in MV411 cells. [Figure 44] Figure 44 shows a comparison of cell viability data depending on CD33 receptor expression levels. 50% loading of CD33xMIC5+GNE987 induced cytotoxicity against CD33-positive MV411 and MOLM13 cells, but had little effect on RAMOS cells lacking CD33. [Figure 45] Figure 45 shows the cytotoxicity of 25, 50, and 75% of CD33xMIC5 loaded with the PROTAC GNE987 compared to the cytotoxicity of GNE987 against CD33-positive MV411 cells. [Figure 46] Figure 46 shows cell viability data for CD33xMIC5 antibodies loaded with various amounts of PROTAC GNE987P per antibody compared to PROTAC GNE987P alone. [Figure 47] Figure 47 shows cell viability data for CD33xMIC5 antibodies loaded with the PROTAC FLT3d1 per antibody compared to the PROTAC GNE987P alone. Cell viability was analyzed after 6 days of treatment. [Figure 48]Figure 48 shows cell viability assays of pre-complexed or non-pre-complexed CD33xMIC5+GNE987 at 75% loading (1:1.5) on CD33-positive MV411 and CD33-negative RAMOS cells. For non-pre-complexed samples, the antibody (CD33xMIC5) and PROTAC (GNE987) were added separately to the cell suspension as treatment. The PROTAC, GNE987, was tested on the cells for reference. [Figure 49] Figure 49 shows cell viability assays of CLL1xMIC7+GNE987P and DIGxMIC7+GNE987P at 75% loading (1:1.5) on CLL1-positive MOLM13 and U937, and on CLL1-negative K562 cells. The PROTAC, GNE987 alone, was tested on the cells for reference. [Figure 50] Figure 50 shows cell viability assays of CLL1xMIC7+GNE987, CLL1xMIC7+GNE987P, and CLL1xMIC7+SIM1 at 75% loading (1:1.5) on CLL1-positive MV411 and U937 cells, and on CLL1-negative RAMOS and K562 cells. PROTACs GNE987, GNE987P, and SIM1 were tested on cells for reference. [Figure 51] Figure 51 shows cell viability assays of B7H3xMIC7+GNE987P or B7H3xMIC7+SIM1 at 75% loading (1:1.5) on B7H3-positive MV411 and U937 cells, and on B7H3-negative RAMOS cells. PROTACs alone (GNE987P and SIM1) were tested on the cells for reference. [Figure 52] Figure 52 shows cell viability assays of B7H3xMIC7+GNE987 and DIGxMIC7+GNE987 at 75% loading (1:1.5) on B7H3-positive MV411 and U937 cells, and on B7H3-negative RAMOS cells. The PROTAC, GNE987, was tested on the cells for reference. [Figure 53] FIG. 53 shows the dose-response curves of cell viability assays of B7H3xMIC7[6H]+GNE987P (DAR4.5) and control in A2780 cells. [Figure 54-1] Figure 54 shows cell viability assays of NAPI2BxMIC7 and DIGxMIC7 loaded with GNE987, GNE987P, or SIM1 at 50% loading (1:1) on NAPI2B-positive OVCAR3 and NAPI2B-negative SKOV3 cells. PROTACs GNE987, GNE987P, and SIM1 were tested on the cells as references. [Figure 54-2] Same as above. [Figure 55] Figure 55 shows dose-response curves for cell viability assays on OVCAR3 and NCIH1437 cells. PAX binding to NAPI2B-expressing OVCAR3 and NCIH1437 cells was studied. As a negative control, a non-binding control isotype antibody (binding to digoxigenin) was tested. As a positive control, PROTAC alone was studied. The top panel shows the dose-response curves for cells treated with either PAX from PROTAC GNE987 or the PROTAC control, and the bottom panel shows the curves for either PAX from GNE987P or the PROTAC control. [Figure 56] Figure 56 shows dose-response curves for a cell viability assay of TROP2-binding PAX in TROP2-expressing A431 (left panel) and SNU840 cells (right panel) loaded with BRD4 PROTACs GNE987 (top panel) and GNE987P (bottom panel). As positive controls, PROTACs GNE987 and GNE987P alone were tested, and an isotype control binding to digoxigenin was used as a negative control to assess nonspecific effects. [Figure 57] Figure 57 shows dose-response curves for PAX-targeted PSMA (PSMAxMIC7[6H]+GNE987P, DAR4.5) and control on PSMA-expressing LNCAP cells. [Figure 58]Figure 58 shows a dose-response curve for the cell viability assay BRD4 PAX. The cMET-binding cMETxMIC7[2H] antibody was loaded with GNE987 or GNE987P at DARs of 1.0 and 1.5. The non-binding control antibody DIGxMIC7[2H] was similarly loaded. PROTAC alone was tested as a positive control. [Figure 59] Figure 59 shows dose response curves for a cell viability assay of CLDN18.2 targeting PAX (CLDN18.2xMIC7[6H]) loaded with GNE987 (DAR1.5 and DAR3), GNE987P (DAR4.5), or SIM1 (DAR3 and DAR4.5) and a negative control targeting digoxigenin (DIGxMIC7[6H]) loaded with the same PROTAC at the same ratio, compared to PROTAC and antibody alone. [Figure 60] Figure 60 shows the dose response curves of the CD33-targeting PROTAC-antibody conjugate CD33xMIC7[N-2H]+GNE987P DAR1 with an N-terminal fusion of VHH MIC7 to the heavy chain and a control on MV411 cells. [Figure 61] Figure 61 shows dose-response curves for SEED antibody complexed with PROTAC GNE987 (top) and SEED antibody alone (bottom) studied in EGFR-expressing A431 cells. Figure 62 is intentionally omitted. Figure 63 is intentionally omitted. [Figure 64] Figure 64 shows dose-response curves for TROP2-positive SNU840 and TROP2-negative SW620 cells treated with cathepsin B-cleaving PAX constructs targeting TROP2 (TROP2xMIC7[2H_CL]) or non-binding (DIGxMIC7[2H_CL]) loaded with GNE987, GNE987P, or SIM1 PROTAC (PROTAC to antibody ratio 1.5:1). Unloaded PAX and PROTAC alone were studied as controls. [Figure 65]FIG. 65 shows dose-response curves of EGFR-targeting cPAX (EGFRxMIC7[C2]+GNE987P, DAR1) and control in EGFR-positive A431 and MDAMB468 cells. [Figure 66] Figure 66 shows a comparison of the cytotoxicity of 50% PROTAC-loaded EGFRxMIC5+GNE987 and a cetuximab-based EGFR-binding PROTAC-ADC (DAR=1.62) against EGFR-negative HEPG2 and EGFR-positive MDAMB468 cells. [Figure 67] Figure 67 shows a PK study of CD33xMIC5+GNE987 and CD33xMIC7+GNE987 PROTAC-antibody conjugates with 100% theoretical loading in C57BL / 6N mice after IV administration of 30 mg / kg. Detected concentrations of GNE987 are shown. [Figure 68] Figure 68 shows the clearance of compared unmodified antibody CD33 Ab, antibody-VHH fusion proteins CD33xMIC5 and CD33xMIC7, and CD33xMIC5 and CD33xMIC7 loaded with GNE987. [Figure 69] Figure 69 shows an MV411 xenograft efficacy study of CD33xMIC7+GNE987 in female CB17 SCID mice. CD33xMIC7+GNE987 at 30 mg / kg was given once or twice compared to GNE987 at 0.38 mg / kg given once or twice (days 1 and 8). Additionally, the efficacy of CD33xMIC5+GNE987 at 30 mg / kg given once (day 1) was evaluated, as was the effect of antibody alone (30 mg / kg CD33xMIC7) as a control. [Figure 70] FIG. 70 shows a tumor growth chart of the MV411 sc model in CB17-scid mice treated with a single dose of the indicated molecule and dose on day 0. [Figure 71] FIG. 71 shows the weight changes of treated mice. [Figure 72]Figure 72 shows PD biomarker studies in female CB17-scid mice with MV411 sc grafts (top, A and B) and A431 sc grafts (bottom, C and D). BRD4 levels were quantified in tumors of mice receiving vehicle control (white bars) or BRD4-depleting therapy (black bars) after the indicated time points. Treatments were 30 mg / kg CD33xMIC7+GNE987(DAR2) (corresponding to 0.38 mg / kg GNE987) (A) or 0.38 mg / kg GNE987 (B and D) or 30 mg / kg EGFRxMIC7+GNE987(DAR2) (corresponding to 0.38 mg / kg GNE987) (C). [Figure 73-1] Figure 73 (a-e) shows antibody VHH sequences obtained from immunisation and phage display screening of New World camelids. [Figure 73-2] Same as above. [Figure 73-3] Same as above. [Figure 73-4] Same as above. [Figure 73-5] Same as above. [Figure 73-6] Same as above. [Figure 73-7] Same as above. [Figure 73-8] Same as above. [Figure 73-9] Same as above. [Figure 73-10] Same as above. [Figure 73-11] Same as above. [Figure 73-12] Same as above. [Figure 73-13] Same as above. [Figure 73-14] Same as above. [Figure 73-15] Same as above. [Figure 74-1] Figure 74 shows the antibody sequence. [Figure 74-2] Same as above. [Figure 74-3] Same as above. [Figure 74-4] Same as above. [Figure 74-5] Same as above. [Figure 74-6] Same as above. [Figure 74-7] Same as above. [Figure 74-8] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0049] 5 List of tables Table 1: Binding epitopes of the antibodies of the present invention.

[0050] Table 2: Affinity parameters K for MIC2 and PROTAC combinations (structures, see Figure 8) obtained using a 1:1 kinetic binding model of MIC2 and KD for PROTAC binding to MIC1 derived from a steady-state model. D , meeting speed k on , dissociation rate k off Summary. NM = Not Measured; NB = Not Binding. 41

[0051] Table 3: Summary of required final PROTAC concentrations to achieve desired loading.

[0052] Table 4: IC of EGFR-binding aEGFRxMIC2 and non-binding control MIC2 complexed with GNE987 at 25 and 50% loading. 50 Value: 45

[0053] Table 5: IC of EGFR binding aEGFRxMIC2+GNE987 complex and control for MDAMB468 50 Values. Potencies and standard deviations were derived from three independent experiments. 46

[0054] Table 6: Storage stability evaluation of antibody-PROTAC conjugates in PBS pH 6.8, 5% DMSO final.

[0055] Table 7: Library characteristics of antibody hit discovery campaigns using phage display. 49

[0056] Table 8: Affinity (K) of VHH clones to PROTACs determined using SPR D). VHHs were studied as antibody fusion proteins by C-terminal addition to the heavy chain of either an anti-CD33 or anti-CLL1 antibody. N / D - not detected (the full PROTAC structure can be found in Figure 8). 52

[0057] Table 9. Data collection and refinement statistics. 54

[0058] Table 10: Interaction between MIC5 and E3 ligase binding domain VH032. 55

[0059] Table 11: Interaction between MIC7 and E3 ligase binding domain VH032. 56

[0060] Table 12: Interaction between MIC10 and E3 ligase binding domain VH032 for Monomer A. 56

[0061] Table 13. Interaction between MIC10 and E3 ligase binding domain VH032 for monomer B. 57

[0062] Table 14: Parental MIC7 and CDR1 and CDR2 single point mutant melting temperatures (T M ), parent reference MIC7(ΔT M ), the melting temperature change (ΔT M(リガンド) ) and the deviation of this change with respect to the parent (ΔΔT M(リガンド) ). 58

[0063] Table 15. Parental MIC7 and exemplary CDR2 single point mutant ITC data using VH032. 59

[0064] Table 16: Single point mutant melting temperature (T M ), parent reference MIC7(ΔT M ), the melting temperature change (ΔT M(リガンド) ) and the deviation of this change with respect to the parent (ΔΔT M(リガンド) ). 59

[0065] Table 17. Additional point mutations in the CDR3-loop based on rational design. 60

[0066] Table 18. Affinity data of humanized CD33xMIC7 variants compared to parental CD33xMIC7 for the VHO32 E3-ligase binding moiety of PROTACs. 61

[0067] Table 19: Cell profiling of different humanized CD33xMIC7 variants compared to parental CD33xMIC7 in combination with PROTACs GNE987, GNE987P and ARV711 (1:1 ratio) on CD33-positive MV411 and RAMOS cells and CD33-negative RAMOS cells. IC50 values ​​are expressed in M. 62

[0068] Table 20: IgG-type antibody scaffolds for fusion with VHH antibody fragments. 63

[0069] Table 21: Nomenclature of PAX-targeted CD33. 65

[0070] Table 22: Intentionally omitted

[0071] Table 23: Cellular profiling of CD33-binding gemtuzumab (G)- and EGFR-binding cetuximab (c)-based VHH fusion proteins in combination with the PROTAC GNE987 in EGFR-positive MDAMB468 cells and MDAMB468-negative HEPG2 cells. IC50 values ​​were used to calculate the selectivity index. 69

[0072] Table 24: Primary antibodies used for Western blot analysis. 71

[0073] Table 25: Cell profiling of various CD33xMIC7 in combination with PROTACs GNE987 and GNE987P or PROTACs alone on CD33-positive MV411 cells and CD33-negative RAMOS cells. IC50 values ​​are expressed in M. 76

[0074] Table 26. Cellular profiling of different EGFRxMIC7 variants in combination with the PROTAC GNE987P, or the PROTAC alone, in EGFR-positive MDAMB468 and A431 cells and EGFR-negative HEPG2 cells. IC50 values ​​are expressed in M. 77

[0075] Table 27. Cell profiling of different CD33xMIC7 variants in combination with PROTAC GNE987P or PROTAC alone in CD33-positive MV411 and MOLM13 cells and CD33-negative RAMOS cells. IC50 values ​​are expressed in M. 78

[0076] Table 28: Cellular profiling of PROTACs ARV771, GNE987, GNE987P and EGFR-positive and EGFR-negative HEPG2 cells. IC50 values ​​are expressed in M. 81

[0077] Table 29: Cellular profiling of EGFRxMIC7 in combination with PROTACs GNE987, GNE987P, and SIM1 at 50% loading in EGFR-positive and EGFR-negative HEPG2 cells. Digoxigenin-binding DIGxMIC7 fusion protein was utilized as a non-internalization control. IC50 values ​​are expressed in M. 82

[0078] Table 30: Cellular profiling of EGFRxMIC7 in combination with PROTACs ARV771, GNE987, GNE987P, and SIM1 at 75% loading in EGFR-positive and EGFR-negative HEPG2 and EGFR-low MCF7 cells. Digoxigenin-binding DIGxMIC7 fusion protein was utilized as a non-internalization control. 82

[0079] Table 31: Cellular profiling of HER2xMIC7 in combination with PROTACs GNE987, GNE987P and SIM1 at 75% loading in HER2-positive and HER2-negative MDAMB468 cells. 84

[0080] Table 32: Cellular profiling of TROP2xMIC7 in combination with the PROTAC GNE987 at 75% loading in TROP2-positive and TROP2-negative SW620 cells. 85

[0081] Table 33: Efficacy of TROP2-binding PAX and controls in SNU840 cells. 86

[0082] Table 34: IC50 values ​​of PSMA-targeted PAX loaded with GNE987 or GNE987P in PSMA-expressing LNCAP cells. Negative control binding digoxigenin (DIGxMIC7 [2H] ) was studied as a PROTAC-antibody conjugate. The PROTAC alone was also tested. 87

[0083] Table 35: Potency of conjugated cPAX and respective controls. 91

[0084] Table 36: Efficacy of STEAP1-binding PAX and controls on STEAP1-PC3 and PC3 cells.

[0085] Table 37: Summary of pharmacokinetic parameters of CD33-based VHH fusions with MIC5 and MIC7 and parental antibody CD33 Ab, loaded and unloaded with PROTAC GNE987. Analytes were administered at 30 mg / kg, and PK parameters of quantified total antibody (tAntibody) and PROTAC GNE987 are presented. Abbreviations: t: half-life; C: maximum serum concentration; AUC: area under the curve to infinity; Cl: clearance; V: steady-state volume of distribution. SD: standard deviation. 96

[0086] Table 38: PAX CD33xMIC7 complexed with PROTAC GNE987 [4H] , CD33xMIC7 [4L] and CD33xMIC7 [2H2L]Summary of pharmacokinetic parameters of tAntibody. Analyte was administered at 30 mg / kg and PK parameters for quantification of total antibody (tAntibody) and PROTAC GNE987 are presented. Abbreviations: t 1 / 2 : Half-life; Cmax: Maximum serum concentration; AUC0-inf: Area under the curve to infinity; Cl: Clearance; Vss: Steady-state volume of distribution. SD: Standard deviation. 98

[0087] Table 39: Overview of the scope of this study. The investigated combinations are presented in tabular form. 103

[0088] 6 Detailed Description of the Invention 6.1 Definition "PROTACs" (targeted proteolytic chimeras) are heterobifunctional small molecules composed of two active domains and a linker that can remove specific, unwanted proteins. PROTACs work by inducing selective protein degradation rather than acting as traditional enzyme inhibitors. PROTACs consist of two covalently linked protein-binding molecules: one that is (in most cases) capable of associating, and the other that binds to the target protein, directing it for degradation. Recruitment of an E3 ligase to the target protein results in ubiquitination and subsequent degradation of the target protein by the proteasome. This concept was first described by Deshaies and coworkers in 2001 (Skamoto, KM et al., Proc. Natl. Acad. Sci. USA 98 (2001) 8554-8559).

[0089] The term "antibody" includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with poly-epitopic specificity, multispecific antibodies, in particular bispecific antibodies, diabodies and single chain molecules (e.g., scFv), single domain antibodies (nanobodies, e.g., VHHs derived from New World camelid species, e.g., llamas), and antibody fragments (e.g., Fab, F(ab')2 and Fv).

[0090] The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units plus an additional polypeptide called a J chain and contain 10 antigen-binding sites, whereas IgA antibodies contain two to five basic four-chain units, which can polymerize and combine with the J chain to form multivalent aggregates. In the case of IgG, the four-chain unit is generally approximately 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the alpha and gamma heavy chain isotypes and four CH domains for the mu and epsilon heavy chain isotypes. Each L chain has a variable domain (VL) at the N-terminus, followed by a constant domain at the other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain of the heavy chain (CH1). Specific amino acid residues are thought to form an interface between the light and heavy chain variable domains. The pairing of the VH and VL together forms a single antigen-binding site. For the structure and properties of various classes of antibodies, see, for example, Schroeder, H., Cavacini, L., J. Allergy Clin. Immunol. 125 (2010), S41-S52. L chains from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Immunoglobulins can be assigned to different classes or isotypes depending on the amino acid sequence of the constant domain (CH) of their heavy chains. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains designated alpha, delta, epsilon, gamma, and mu, respectively.The gamma and alpha classes are further divided into subclasses based on relatively minor differences in CH sequence and function, eg, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

[0091] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domains of the heavy and light chains are sometimes referred to as "VH" and "VL," respectively. These domains are generally the most variable parts of an antibody (relative to other antibodies of the same class) and contain the antigen-binding site.

[0092] The term "variable" refers to the fact that certain segments of variable domains vary widely in sequence among antibodies. V domains mediate antigen binding and define the specificity of an antibody for its antigen. However, variability is not uniformly distributed throughout the span of the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Native heavy-chain and light-chain variable domains each contain four FR regions that largely adopt a beta-sheet configuration and are connected by three HVRs, which form loops that connect the beta-sheet structure and, in some cases, form part of the beta-sheet. The HVRs in each chain are held together in close proximity by the HVRs and FR regions from the other chain and contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institutes of Health, Bethesda, MD (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0093] The term "CDR" as used herein refers to the complementarity-determining regions of antibody variable domains that are hypervariable in sequence and / or form structurally defined loops. Antibodies generally contain six CDRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In natural antibodies, H3 and L3 are the most diverse of the six CDRs, and H3 in particular is thought to play a unique role in conferring fine specificity to antibodies. See, for example, Xu et al., Immunity 13 (2000) 37-45; Johnson and Wu, Methods Mol. Biol. 248 (2003) 1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains. See, for example, Hamers-Casterman et al., Nature 363 (1993) 446-448; Sheriff et al., Nature Struct. Biol. 3 (1996) 733-736. Several CDR delineations are used. The ImMunGeneTics (IMGT) specific Lefranc numbering (IMGT numbering) (Lefranc, M.-P. et al., Dev. Comp. Immunol. 27 (2003) 55-77) takes into account sequence conservation, structural data from X-ray diffraction studies, and characterization of hypervariable loops to define FRs and HVRs. Kabat CDRs are based on sequence variability and are also commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196 (1987) 901-917). The CDR delineations used herein follow the IMGT numbering.

[0094] "Framework" or "FR" residues are those variable domain residues other than the CDR residues as herein defined.

[0095] The terms "full length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, a whole antibody includes one having a heavy chain and a light chain, including an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, an intact antibody may have one or more effector functions.

[0096] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding and / or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments and a remaining "Fc" fragment, a name reflecting their ability to readily crystallize. The Fab fragment consists of an entire L chain, the variable region domain (VH) of the H chain, and the first constant domain (CH1) of one heavy chain. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody produces a single large F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0097] An "scFv" (single-chain Fv) is a covalently linked VH::VL heterodimer that is usually expressed from a gene fusion containing genes encoding VH and VL linked by a peptide-encoding linker. Human scFv fragments of the invention comprise CDRs held in the appropriate conformation, for example, by using genetic engineering techniques. Bivalent and multivalent antibody fragments can form spontaneously by association of monovalent scFvs or can result from coupling monovalent scFvs via a peptide linker, e.g., bivalent sc(Fv)2. A "dsFv" is a VH::VL heterodimer stabilized by a disulfide bond. "(dsFv)2" refers to two dsFvs coupled by a peptide linker.

[0098] The term "bispecific antibody" or "BsAb" refers to an antibody that contains two different antigen-binding sites. Thus, BsAbs can simultaneously bind to two different antigens. Genetic engineering has been used with increasing frequency to design, modify, and generate antibodies or antibody derivatives with desired sets of binding properties and effector functions, for example, as described in EP 2050764.

[0099] The term "multispecific antibody" refers to an antibody that contains two or more different antigen-binding sites.

[0100] The term "hybridoma" refers to cells obtained by subjecting B cells, prepared by immunizing a non-human mammal with an antigen, which produce the desired monoclonal antibody having antigen specificity to cell fusion with myeloma cells derived from a mouse, for example.

[0101] The term "diabody" refers to small antibody fragments prepared by constructing scFv fragments (see previous paragraph) using a short linker (approximately 5-10 residues) between the VH and VL domains such that interchain, rather than intrachain, pairing of the V domains is achieved, thereby resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" scFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described in more detail, for example, in EP 0 404 097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448.

[0102] As used herein, monoclonal antibodies specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies so long as they exhibit the desired biological activity.

[0103] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a recipient HVR (defined herein below) are replaced by residues from an HVR of a non-human species (donor antibody), e.g., mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and / or capacity. In some cases, framework ("FR") residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody. These modifications may be made to further refine antibody performance, e.g., binding affinity. Generally, a humanized antibody will comprise substantially all of at least one, and usually two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence and all or substantially all of the FR regions are those of a human immunoglobulin sequence, but the FR regions may include one or more individual FR residue substitutions which improve antibody performance, e.g., binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FR will typically not exceed six in the H chain and not exceed three in the L chain. The humanized antibody also optionally will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321 (1986) 522-525; Riechmann et al., Nature 332 (1988) 323-329; and Presta, Curr. Op. Struct. Biol. 2 (1992) 593-596.See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol. 1 (1998) 105-115; Harris, Biochem. Soc. Transactions 23 (1995) 1035-1038; Hurle and Gross, Curr. Op. Biotech. 5 (1994) 428-433; and U.S. Pat. Nos. 6,982,321 and 7,087,409.

[0104] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or produced using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol. 227 (1991) 381; Marks et al., J. Mol. Biol., 222 (1991) 581. Also available for preparing human monoclonal antibodies is the method described in Dijk and van de Winkel, Curr. Opin. Pharmacol. 5 (2001) 368-74. Human antibodies can be prepared by administering antigen to transgenic animals that have been genetically modified to produce partially or fully human antibodies in response to antigen challenge, but whose endogenous gene loci have been disabled, such as the OmniAb therapeutic antibody platform (Ligand Pharmaceuticals), immunized xenomouse (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 for xenomouse technology), etc. See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA 103 (2006) 3557-3562 for human antibodies produced by human B cell hybridoma technology.

[0105] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies; i.e., the individual antibodies comprising the population are identical except for potential naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Each monoclonal antibody is directed against a single determinant on the antigen, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes). In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by a hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used according to the present invention can be produced by, for example, hybridoma methods (e.g., Kohler and Milstein, Nature 256 (1975) 495-497; Hongo et al., Hybridoma 14 (1995) 253-260; Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, for example, U.S. Pat. No. 4,816,567), phage display technology (e.g., Sidhu et al., J. Mol. Biol. 338 (2004) 299-310; Lee et al., J. Mol. Biol. 340 (2004) 1073-1093; Fellouse, Proc. Natl. Acad. Sci. USA 101 (2004) 12467-12472; and Lee et al., J. Immunol.Methods 284 (2004) 119-132), and techniques for producing human or human-like antibodies in animals that have part or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551; Jakobovits et al., Nature 362 (1993) 255-258; Bruggemann et al., Year in Immunol. 7 (1993) 33; Fishwild et al., Nature Biotechnol. 14: (1996) 845-851; Neuberger, Nature Biotechnol. 14 (1996) 826; and Lonberg and Huszar, Intern. Rev. Immunol. 13 (1995) They can be made by a variety of techniques, including ion exchange (see, e.g., pp. 65-93).

[0106] An "affinity matured" antibody is one that contains one or more alterations in one or more of its HVRs that result in improved affinity of the antibody for antigen compared to a parent antibody lacking those alteration(s). In one embodiment, the affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are produced by procedures known in the art. For example, Marks et al., Biotechnology 10 (1992) 779-783, describes affinity maturation by VH- and VL-domain shuffling. Random mutagenesis of HVR and / or framework residues has been described, for example, by Barbas et al. Proc Nat. Acad. Sci. USA 91 (1994) 3809-3813; Schier et al. Gene 169 (1995) 147-155; Yelton et al. J. Immunol. 155 (1995) 1994-2004; Jackson et al, J. Immunol. 154 (1995) 3310-9; and Hawkins et al, J. Mol. Biol. 226 (1992) 889-896.

[0107] As used herein, the terms "specifically binds to" or "specific for" refer to a measurable, reproducible interaction, e.g., binding between a target and an antibody, that is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds to a target (which may be an epitope) is one that binds to this target with greater affinity, avidity, more readily, and / or for a longer period of time than it binds to other targets.

[0108] "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity," "binds to," "binds to," or "binding to" refers to the intrinsic binding affinity that reflects a one-to-one interaction between members of a binding pair (e.g., an antibody Fab fragment and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigens slowly and tend to dissociate easily, whereas high-affinity antibodies generally bind antigens faster and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative and exemplary embodiments for measuring binding affinity, i.e., avidity, are described below.

[0109] "K" according to the present invention D " or "K D The "value" can be measured by a radiolabeled antigen binding assay (RIA) performed using the Fab version of the antibody and the antigen molecule, or by using a surface plasmon resonance assay using a BIACORE instrument (BIAcore, Inc., Piscataway, NJ), or by using a biolayer interferometry assay using an Octet instrument (Forte bio, Fremont, CA).

[0110] As used herein, the term "conjugate" refers to a chemical (non-biological) therapeutic agent covalently linked to an antibody, in contrast to "conjugate," which refers to a chemical (non-biological) therapeutic agent non-covalently attached to the variable regions (CDRs) of an antibody.

[0111] "Purified" or "isolated," when referring to a polypeptide (e.g., an antibody) or nucleotide sequence, means that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. As used herein, the term "purified" refers to the presence of at least 75%, 85%, 95%, 96%, 97%, or 98% by weight of biological macromolecules of the same type. An "isolated" nucleic acid molecule encoding a particular polypeptide refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the polypeptide of interest, although the molecule may contain some additional bases or moieties that do not adversely affect the essential characteristics of the composition.

[0112] The term "degron," as used herein, refers to the degradable portion of a PROTAC that is a von Hippel-Lindau (VHL) ligand.

[0113] The term "warhead," as used herein, refers to the portion of a PROTAC that binds to a protein to be degraded (e.g., an inhibitor or such target protein). The warhead portion is also referred to hereinafter as a "target protein binder" or "protein binder" or "PB."

[0114] 6.2 Antibodies and Antibody-PROTAC Conjugates (PAX) of the Invention The inventors have succeeded in generating and selecting specific anti-PROTAC antibodies, in particular anti-VHL-ligand antibodies, in which the antibodies specifically bind to the VHL ligand degron of the PROTAC.

[0115] In one aspect, the present invention relates to an antibody that binds to the VHL ligand VH032 or a derivative thereof.

[0116] The anti-PROTAC antibodies generated by the present inventors can bind to the VHL ligand VH032 while tolerating various modifications, as outlined in Figure 1 and Table 1 for MIC1- and MIC2-derived antibodies. The antibodies tolerate all investigated substitutions at positions R1 and R2, which contain several distinct linker structures connecting VH032 and the target protein binder. Hydrogen and hydroxyl substitutions are tolerated at R3, but antibody binding was inhibited when the target protein binding moiety was connected to R3 via a linker. R4 can be a hydrogen atom of methyl. Both R5 and R6 can contain a hydroxyl group, assuming hydrogen at each of the other positions. No intolerant substitutions were identified at positions R1, R2, R5, and R6.

[0117] Structural analysis based on a literature review revealed that 49.2% of VHL-associated PROTACs are based on the VHL ligand VH032. 29.5% of VHL-based PROTACs utilize a close derivative of VH032 that carries an additional methyl group (R4 = Me, Figure 1). The linker for warhead attachment is now attached at position R1 (Figure 1). The remaining VHL-based PROTACs use a different buildup in which the connection to the warhead is performed by linker attachment to R3 or carries other modifications, such as hydroxymethyl, in R4. In summary, the anti-PROTAC antibodies disclosed in this invention can bind to at least 79% of currently publicly known VHL-based PROTACs.

[0118] [Table 1]

[0119] Thus, in one embodiment, a VH032 derivative can be described by formula I:

[0120] [ka] During the ceremony, One of R1 or R2 is a linker connected to the warhead (target protein binder, PB), provided that: When R2 is a warhead-linker, R1 is acetyl, and When R1 is a warhead-linker, R2 is methyl; R3 is H, OH, cyano, F, Cl, amino or methyl; R4 is H or methyl; R5, R6 are H or OH, provided that: When R6 is H, R5 is OH, and When R5 is H, R6 is OH.

[0121] In a more particular embodiment, R1 is PB-Q-(CH2-CH2-O) n -(CH2-CH2-CH2-O) m -(CH2) p -(C=O)-, During the ceremony, PB is a protein-binding warhead, Q is NH, C=O or absent; n and m are independently 0, 1, 2, 3, or 4; p is 0 to 10; R2 is methyl; R3, R4, R5 and R6 are as defined above.

[0122] In an even more particular embodiment, R1 is PB-Q-(CH2-CH2-O) n -(CH2-CH2-CH2-O) m -(CH2) p -(C=O)-, During the ceremony, PB is a protein-binding warhead, Q is NH, C=O or absent; (i) n, m, and p are 1, or (ii) n is 3 or 4, m is 0, and p is 1; or (iii) n is 1, m is 0, and p is 2, or (iv) n is 2, m is 0, and p is 2, or (v) n and m are 0, and p is 6, 7, 8, 9, or 10; R2 is methyl; R3, R4, R5 and R6 are as defined above.

[0123] In a very particular embodiment, R1 is PB-NH-(CH2-CH2-O) n -(CH2-CH2-CH2-O) m -(CH2) p -(C=O)-, During the ceremony, PB is a protein-binding warhead, (vi) Q is NH and n, m, and p are 1; or (vii) Q is NH, n is 3 or 4, m is 0, and p is 1; or (viii) Q is absent, n is 1, m is 0, and p is 2; or (ix) Q is absent, n is 2, m is 0, and p is 2; or (x) Q is NH or C=O, n and m are 0, and p is 6, 7, 8, 9, or 10; R2 is methyl; R3, R4, R5 and R6 are as defined above.

[0124] In another more particular embodiment, R1 is acetyl; R2 is PB-NH-(CH2) p -S-, where PB is a protein-binding warhead and p is 1, 2, 3, 4, 5, or 6; R3, R4, R5 and R6 are as defined above.

[0125] In one embodiment, the antibody is a VHH comprising a CDR3 involved in PROTAC binding and has the following sequence: CDR3: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 (SEQ ID NO: 1) It has.

[0126] As demonstrated herein below, crystallographic data (Section 7.8.7, Figures 30, 31) show that single amino acid mutant PROTAC binding data reveals that residues X4, X5, X7, X 10 , X 12 , X 14 , X 15 and X 20 It has been determined that is solvent exposed, i.e., does not contribute to PROTAC binding, and therefore may remain unclear (Sections 7.8.8, 7.8.9).

[0127] So in this array: X1 is A, S or T, X2 is A, V or I, X3 is S, A, I or D, X4 is any amino acid, X5 is any amino acid, X6 is V, S, L or T, X7 is any amino acid, X8 is S, A, C or P, X9 is T, A, K, S or N, and X 10 is any amino acid, and X 11 is absent, V or A, and X 12 is any amino acid or is absent, and X 13 is V, G or P, and X 14 is any amino acid, and X 15 is any amino acid, and X 16 is T, V, K or R, and X 17 is R, I or Y, and X 18 is Y, Q, F or A, and X 19 is V or L, and X20 is any amino acid, and X 21 is V, Y or A, Or more particularly, X1 is A, S or T, X2 is A, V or I, X3 is S, A, I or D, X4 is K, T, Y, R or A, X5 is E, R, Y or G, X6 is V, S, L or T, X7 is K, L, G, S or C, X8 is S, A, C or P, X9 is T, A, K, S or N, and X 10 is K, P, I, V or D, and X 11 is absent, V or A, and X 12 is E, D, S, R or absent, and X 13 is V, G or P, and X 14 is K, D, T, G or R, and X 15 is K, Q, I, T or R, and X 16 is T, V, K or R, and X 17 is R, I or Y, and X 18 is Y, Q, F or A, and X 19 is V or L, and X 20 is K, E, P or D, and X 21 is V, Y or A, Or even more particularly, X1 is A or S, X2 is V or A, X3 is A or I, X4 is K, T or Y, X5 is E, G or R, X6 is L or S, X7 is K, C or S, X8 is P or C, X9 is A, K or S, and X 10 is K, V or D, and X 11 is absent or V, and X 12 is E, R or absent, and X 13 is G or P, and X 14 is K, T or G, and X 15 is K, Q or I, and X 16 is T, K or R, and X 17 is R, I or Y, and X 18is F or A, and X 19 is L and X 20 is K, E or D, and X 21 is V or Y.

[0128] In a preferred embodiment, the CDR3 sequences are the CDR3 sequences of the VHH antibodies shown in Figures 73(a) to (n).

[0129] In a more preferred embodiment, the CDR3 sequence is the CDR3 sequence of a VHH antibody YU734-F06 (MIC7) shown in Figure 73(a) CDR3: SAIYRLSCSVVRPTIRYALDY; (SEQ ID NO: 2) MIC7_2.12(R112K) shown in Figure 73(f) CDR3: SAIYRLSCSVVRPTIKYALDY; (SEQ ID NO: 3) MIC7_2.13(R112T) shown in Figure 73(f) CDR3: SAIYRLSCSVVRPTITYALDY; (SEQ ID NO: 4) MIC7_2.21 (S105K) shown in Figure 73(f) CDR3: SAIYRLSCKVVRPTIRYALDY; (SEQ ID NO: 5) MIC7_2.39(Y100K) shown in Figure 73(f) CDR3: SAIKRLSCSVVRPTIRYALDY; (SEQ ID NO: 6) MIC7_2.40(R101E) shown in Figure 73(f) CDR3: SAIYELSCSVVRPTIRYALDY; (SEQ ID NO: 7) MIC7_2.41(S103K) shown in Figure 73(f) CDR3: SAIYRLKCSVVRPTIRYALDY; (SEQ ID NO: 8) MIC7_2.42(V106K) shown in Figure 73(f) CDR3; SAIYRLSCSKVRPTIRYALDY; (SEQ ID NO: 9) MIC7_2.43(R108E) shown in Figure 73(g) CDR3: SAIYRLSCSVVEPTIRYALDY; (SEQ ID NO: 10) MIC7_2.44(T110K) shown in Figure 73(g) CDR3: SAIYRLSCSVVRPKIRYALDY; (SEQ ID NO: 11) MIC7_2.45 (I111K) shown in Figure 73(g) CDR3: SAIYRLSCSVVRPTKRYALDY; (SEQ ID NO: 12) MIC7_2.46(D116K) shown in Figure 73(g) CDR3: SAIYRLSCSVVRPTIRYALKY; (SEQ ID NO: 13) is.

[0130] In one embodiment, the above described sequence is part of a BsAb, and the N-terminus of said sequence is fused, optionally via a peptide linker, to the C-terminus of a full-length antibody capable of binding to a target protein.

[0131] In a preferred embodiment, the BsAb comprises a peptide linker. In a more preferred embodiment, the peptide linkers each consist of one, two, or three repeats of GSGGGSGGSGGGGSG (SEQ ID NO: 14). In an even more preferred embodiment, the peptide linkers each consist of one repeat of GSGGGSGGSGGGGSG (SEQ ID NO: 14).

[0132] In the case of full-length antibodies, the antibodies are preferably of the IgG1 or IgG4 type, which allows FcRn receptor binding.

[0133] In one embodiment, the antibody is monospecific and binds only to the PROTAC. The antibody may also be a bispecific antibody (BsAb), in which the second specificity is for the target protein.

[0134] In the case of BsAbs, PROTAC binding is achieved by VHH antibodies fused to either the C-terminus or N-terminus of either the heavy or light chain, or both chains, of the full-length antibody, while target protein binding is achieved by the six CDRs of the variable region of the full-length antibody.

[0135] Examples of BsAb variants according to the invention are shown in FIG.

[0136] In a preferred embodiment, the target protein of a BsAb according to the invention is a cell surface protein, for example, a tumor antigen, such as HER2 or EGFR.

[0137] 6.3 Nucleic Acids, Vectors and Host Cells Another aspect of the present invention relates to an isolated nucleic acid comprising or consisting of a nucleic acid sequence encoding an antibody of the present invention as defined above.

[0138] Typically, the nucleic acid is a DNA or RNA molecule and may be comprised in any suitable vector, for example a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

[0139] The terms "vector," "cloning vector," and "expression vector" refer to a vehicle by which DNA or RNA sequences (e.g., foreign genes) can be introduced into a host cell in order to transform the host and promote expression (e.g., transcription and translation) of the introduced sequences. Accordingly, a further aspect of the present invention relates to a vector comprising a nucleic acid of the present invention as defined above. Such a vector may contain regulatory elements, e.g., promoters, enhancers, terminators, etc., to cause or direct expression of the polypeptide upon administration to a subject.

[0140] A further aspect of the present invention relates to host cells which have been transfected, infected or transformed with nucleic acids and / or vectors according to the invention.

[0141] The term "transformation" refers to the introduction of a "foreign" (i.e., exogenous) gene, DNA, or RNA sequence into a host cell, such that the host cell expresses the introduced gene or sequence to produce a desired substance, usually a protein or enzyme, encoded by the introduced gene or sequence. A host cell that receives and expresses the introduced DNA or RNA has been "transformed."

[0142] The nucleic acids of the invention can be used to produce the antibodies of the invention in a suitable expression system. The term "expression system" refers to a host cell and a compatible vector under suitable conditions for the expression of a protein encoded by, for example, foreign DNA carried by the vector and introduced into the host cell.

[0143] Common expression systems include E. coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, yeast of the genus Kluyveromyces or Saccharomyces, and mammalian cell lines (e.g., Vero cells, CHO cells, HEK cells, 3T3 cells, COS cells, etc.).

[0144] 6.4 Methods of Producing Antibodies of the Invention The antibodies of the present invention may be produced by any technique known in the art, including, but not limited to, any chemical, biological, genetic or enzymatic technique, alone or in combination.

[0145] Knowing the amino acid sequence of a desired antibody, one of skill in the art can readily produce said antibody or immunoglobulin chains using standard techniques for producing polypeptides. For example, they can be synthesized using a commercially available peptide synthesizer (e.g., one manufactured by Applied Biosystems, Foster City, California) according to the manufacturer's instructions using the well-known solid-phase method. Alternatively, the antibodies and immunoglobulin chains of the present invention can be produced by recombinant DNA techniques as are well known in the art. For example, these polypeptides (e.g., antibodies) can be obtained as DNA expression products after incorporating a DNA sequence encoding the desired polypeptide into an expression vector and introducing such a vector expressing the desired polypeptide into a suitable eukaryotic or prokaryotic host, from which they can then be isolated using well-known techniques.

[0146] In a further aspect, the invention relates to a method of producing an antibody of the invention, the method comprising the steps of: (i) culturing a transformed host cell according to the invention; (ii) expressing the antibody; and (iii) recovering the expressed antibody.

[0147] Antibodies of the invention can be conveniently separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0148] The Fab of the present invention can be obtained by treating an antibody (e.g., IgG) of the present invention with a protease, such as papain. Alternatively, the Fab can be produced by inserting DNA sequences encoding both chains of the Fab of the antibody into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic cell (as appropriate) to express the Fab.

[0149] The F(ab')2 of the present invention can be obtained by treating the antibody (e.g., IgG) of the present invention with the protease pepsin. Alternatively, the F(ab')2 can be produced by linking the Fab' described below via a thioether bond or a disulfide bond.

[0150] The Fab' of the present invention can be obtained by treating the F(ab')2 of the present invention with a reducing agent, such as dithiothreitol. Alternatively, the Fab' can be produced by inserting a DNA sequence encoding the Fab' chain of the antibody into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic cell (as appropriate) to effect expression.

[0151] 6.5 Solubilizing and stabilizing PROTACs PROTACs are often hydrophobic, which limits their in vivo applicability, whereas antibodies are generally sufficiently soluble. Thus, binding of the anti-PROTAC antibody of the present invention to the degron portion of a PROTAC partially masks the PROTAC from the surrounding solvent. The net result is a solubilizing effect of antibody binding, i.e., improved solubility, which is advantageous for in vivo administration and xenograft studies.

[0152] Furthermore, PROTACs have several metabolic soft spots in the warhead, linker, and degron moieties (Goracci, L. et al., J. Med. Chem. 63 (2020) 11615-11638), which limit their metabolic stability. Conjugation of PROTACs with the antibodies of the present invention limits the steric accessibility of the PROTACs to metabolic enzymes, leading to improved metabolic stability.

[0153] 6.6 Pharmaceutical Compositions The PAX of the present invention can be combined with pharmaceutically acceptable carriers, diluents and / or excipients, and optionally sustained release matrices, including but not limited to classes of biodegradable polymers, non-biodegradable polymers, lipids or sugars, to form pharmaceutical compositions.

[0154] Therefore, another aspect of the present invention relates to a pharmaceutical composition comprising the PAX of the present invention and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0155] "Pharmaceutical product" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other unwanted reactions when administered to a mammal, particularly a human, as appropriate. A pharmaceutically acceptable carrier, diluent or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating agent or formulation aid of any type.

[0156] As used herein, "pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, etc. Examples of suitable carriers, diluents, and / or excipients include, but are not limited to, one or more of water, amino acids, saline, phosphate buffered saline, buffered phosphate, acetate, citrate, succinate; amino acids and derivatives such as histidine, arginine, glycine, proline, glycylglycine; inorganic salts such as sodium or calcium chloride; sugars or polyhydric alcohols such as dextrose, glycerol, ethanol, sucrose, trehalose, mannitol; surfactants such as polysorbate 80, polysorbate 20, poloxamer 188, etc., and combinations thereof. It is often useful to include isotonic agents, for example, sugars, polyalcohols or sodium chloride in the pharmaceutical composition; the formulation may also contain antioxidants, for example, tryptamine and / or stabilizing agents, for example, Tween 20.

[0157] The form of the pharmaceutical composition, the route of administration, the dosage and the regimen will naturally vary depending on the condition to be treated, the severity of the disease, the age, weight and sex of the patient, etc.

[0158] The pharmaceutical compositions of the present invention can be formulated for parenteral, intravenous, intramuscular, or subcutaneous administration, and the like.

[0159] In one embodiment, the pharmaceutical composition contains a pharmaceutically acceptable medium for the preparation of an injection, which may be an isotonic, sterile saline solution (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc. or a mixture of such salts) or a dried, especially lyophilized, composition that can be made up into an injection solution upon addition of sterile water or saline, as the case may be.

[0160] The pharmaceutical composition can be administered by a drug combination device.

[0161] The dose used for administration can be adapted as a function of various parameters, for example as a function of the mode of administration used, of the pathology involved or of the desired duration of treatment.

[0162] To prepare a pharmaceutical composition, an effective amount of the PAX of the present invention can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0163] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions; in all such cases the form must be sterile, injectable, with an appropriate device or system for delivery without degradation, stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0164] Sterile injectable solution can be prepared by incorporating the required amount of active compound into a suitable solvent with any of the other ingredients listed above as needed, followed by sterile filtration.Generally, dispersion can be prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the other ingredients required from those listed above.In the case of sterile powder for preparing sterile injectable solution, preparation methods include vacuum drying and freeze-drying technology, which can obtain powder of active ingredient and any additional desired ingredients from its previously sterile-filtered solution.

[0165] For parenteral administration in aqueous solution, for example, the solution can be suitably buffered, if necessary, and the liquid diluent can first be rendered isotonic with sufficient saline or glucose. These aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous vehicles that can be used will be known to those skilled in the art in light of the present disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion therapy solution or injected at the proposed infusion site (see, e.g., "Remington's Pharmaceutical Sciences," 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dose for the individual subject.

[0166] The PAX of the present invention may be formulated into a therapeutic mixture containing, for example, about 0.01 to 100 milligrams per dose.

[0167] In certain embodiments, the first pharmaceutical composition comprises PAX and the second pharmaceutical composition comprises only the PROTAC portion of said PAX.

[0168] In another specific embodiment, the first pharmaceutical composition comprises only the antibody portion of PAX and the second pharmaceutical composition comprises only the PROTAC portion of said PAX.

[0169] 6.7 Treatment Methods and Uses As described above and below, the inventors have found that the PAX of the present invention can effectively deliver a given PROTAC to a target cell. Furthermore, they have shown that the PAX releases the PROTAC payload into the cytosol of the target cell, where the PROTAC mediates degradation of the target protein.

[0170] Thus, in one embodiment, the present invention provides PAX or a pharmaceutical composition thereof for use as a medicament.

[0171] In another aspect, the present invention provides a method for treating a disease that benefits from degradation of a target protein of a PROTAC, such as cancer, comprising administering PAX or a pharmaceutical composition of the present invention to a subject in need thereof.

[0172] In certain embodiments, PAX or a pharmaceutical composition comprising said PAX is administered first, followed by subsequent administration of the PROTAC moiety of PAX alone or a pharmaceutical composition comprising said PROTAC, allowing the antibody to release its PROTAC payload and bind to additional PROTAC moieties and deliver them to target cells.

[0173] In another specific embodiment, the antibody portion of PAX or a pharmaceutical composition comprising said antibody is administered first, followed by the PROTAC portion of PAX or a pharmaceutical composition comprising said PROTAC, allowing the antibody to bind to the PROTAC "antigens" and deliver them to target cells in vivo.

[0174] In further embodiments, the antibody portion of PAX is used (i) to increase the in vivo half-life of the PROTAC (i.e., to slow down degradation), (ii) as a sustained-release formulation of the PROTAC (i.e., allowing it to be effective for a longer period of time), or (iii) as an antidote to counter the toxic effects of the PROTAC by temporarily neutralizing them, thereby lowering the toxicity threshold.

[0175] In one embodiment, the antibody portion of PAX is an antibody fragment, eg, an Fc fragment.

[0176] 6.8 Non-therapeutic use The anti-PROTAC antibodies of the present invention, preferably monospecific antibodies, may also be used in non-therapeutic applications, for example, to detect, quantify or purify PROTACs.

[0177] In one embodiment, for such uses, the antibody is immobilized on a chromatography column or some other solid support.

[0178] 6.9 Kits Finally, the present invention also provides kits comprising at least one antibody or PAX of the present invention.

[0179] The kits containing PAX of the present invention may be used for therapeutic purposes, which may be monotherapy or combination therapy, and in this case, they contain one or more additional pharmaceutical compositions containing additional pharmaceutical ingredients. The therapeutic kits may also contain a package insert with administration instructions.

[0180] Kits containing the antibody of the present invention can also be used for diagnostic or detection purposes.In such kits, the antibody is usually coupled to a solid support, such as a tissue culture plate or beads (for example, Sepharose beads), and is used to detect and / or quantify PROTAC in vitro, for example, in ELISA or Western blot.Such antibodies useful for detection may be provided with a label, for example, a fluorescent or radioactive label. [Example]

[0181] 7 Working Example 7.1 Anti-PROTAC antibodies MIC1 and MIC2 7.1.1 Hapten conjugation For immunogen preparation and compound screening, VH032-based haptens (VHL-1, VHL-6, VHL-7, VHL-c (Figure 4)) were dissolved separately in conjugation buffer (0.1 M MES, 0.9 M NaCl, 0.02% sodium azide; pH 4.7) to a final concentration of 4 mg / mL and mixed with either 10 mg / mL bovine serum albumin (BSA) or a solution of 10 mg / mL keyhole limpet hemocyanin (KLH), 10 mg / mL cationic BSA (cBSA), and 7 mg / mL human Fc (huFc) (final protein:hapten molar ratio of 1:100).

[0182] To this mixture, a 10 mg / mL aqueous solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was added (final protein-EDC molar ratio 1:1750), and the reaction was incubated overnight. The reaction mixture was purified on a Zeba Spin Desalting column pre-equilibrated in phosphate-buffered saline (PBS, 0.137 M NaCl, 0.0027 M KCl, 0.01 M NaHPO, 0.0018 M KHPO, pH 7.4) (7K MWCO, Thermo Scientific). Protein concentration was determined using Bradford reagent with unconjugated KLH or BSA as a standard. Conjugation efficiency was tested for BSA and huFc conjugates by MALDI-MS. An approximate hapten-to-carrier protein ratio could be derived for each individual conjugation (Figure 5).

[0183] 7.1.2 Immunization and Hybridoma Screening BALB / c and CD-1 mice and SD rats were immunized with an equimolar mixture of haptens VHL-1, VHL-6, and VHL-7 conjugated to keyhole limpet hemocyanin (KLH) (Figure 4) as the immunogen carrier protein. After immunogen injection, sera from all animals were immediately examined for antibody responses using BSA-hapten conjugates using ELISA. The animals demonstrated an immune response, leading to immune repertoire screening for VHL-ligand binding antibodies using hybridoma screening. Figure 6 shows the detailed work plan.

[0184] 7.2 Production of MIC antibodies Monospecific antibodies were expressed by transient transfection of heavy and light chains in Expi293F cells using the corresponding transfection kits and media from Life Technologies according to the manufacturer's instructions. 50 μg of plasmid DNA for the heavy chain and 100 μg of plasmid DNA for the light chain were diluted in 10 mL of OptiMEM medium. 536 μL of ExpiFectamine was added to 10 mL of OptiMEM, followed by incubation at room temperature for 5 minutes. The plasmid dilutions were then added. After incubation at room temperature for 20 minutes, 2.9 × 10 plasmids per mL were obtained. 6 The mixture was added to 180 mL of Expi293 cells at a cell density of 100 viable cells. The cell suspension was incubated at 37°C, 5% CO2, and 80 rpm in a humidified atmosphere. After 18–22 hours, 1 mL of Enhancer 1 and 10 mL of Enhancer 2 were added. After a further incubation of 4 days in a humidified atmosphere at 37°C with 5% CO2 and shaking, the antibody was collected by centrifugation (3000 rpm for 30 minutes) and sterile filtered using a 0.22 μm bottle-top filter.

[0185] The supernatant was purified by Protein A affinity chromatography using the AktaXpress system, followed by preparative SEC (HiLoad 16 / 60 Superdex 200 prep grade) to remove aggregates. The antibody was concentrated and sterile filtered using ultracentrifugal filter units (30k MWCO, Amicon), and the protein concentration was determined by UV-VIS spectroscopy at 280 nm. The antibody was characterized for identity by analytical SEC, SDS-PAGE, and LC-MS.

[0186] 7.3 Versatility of Antibodies Binding to PROTACs The binding affinities of the hit antibodies MIC1 and MIC2 to a diverse set of VH032-based PROTACs (Figure 8) were determined by surface plasmon resonance (SPR) on a Biacore T200 instrument. The running buffer consisted of PBS, 0.05% Tween-20, and 2% DMSO, with the temperature and flow rate set at 30°C and 30 μL / min, respectively. The assay setup is exemplarily depicted in Figure 7. A CM5 sensor chip was coated with antibody (=ligand; 2500 RU) using standard EDC / NHS chemistry. The PROTAC (=analyte) was serially diluted in running buffer (1 μM to 1.9 nM) and injected into the instrument in successive runs. It was captured by the antibody, resulting in a corresponding increase in SPR response. After the association step (300 s), the running buffer was injected for 600 s, and PROTAC dissociation from the antibody led to a signal decrease. After each run, the remaining bound PROTAC was removed from the immobilized antibody by 2 × 30-second injections of 10 mM glycine / HCl, pH 1.5, regenerating the antibody for the next association and dissociation cycle. To compensate for matrix effects, the measured SPR response signal was subtracted by the analyte response to a deactivated (EDC / NHS, ethanolamine) reference surface omitting the ligand. Additionally, a DMSO solvent correction was performed, and the analyte response was subtracted by the running buffer signal. The corrected response was fitted by a 1:1 kinetic binding model to determine the binding rate (k) of the PROTAC. on ) and dissociation rate (koff ) and its dissociation constant (K D ) was obtained.

[0187] For MIC2 in combination with 16 distinct PROTACs, affinity parameters were calculated using the corresponding K D , meeting speed k on and dissociation rate k off The results are summarized in tabular form (Table 2) using the . Thirteen of the 16 (81.3%) PROTACs had a nanomolar K D Table 2 also contains affinity data for MIC1, which was able to bind to one PROTAC that was not bound by MIC2.

[0188] [Table 2]

[0189] 7.4 Production of PAX using MIC2 and PROTAC The antibody MIC2 was engineered into a bispecific format by genetically fusing a glycine-serine linker sequence followed by an anti-EGFR VHH antibody sequence or an anti-HER2 scFv sequence to the C-terminus of the heavy chain of MIC2. Generation was performed as previously described for monospecific antibodies. EGFR and HER2 were chosen because they are tumor-associated antigens expressed on the cell surface of cells and therefore accessible for antibody binding. Furthermore, they have already been applied in the development of antibody-drug conjugates, confirming their utility as targets for tumor model availability, efficient expression, and internalization.

[0190] Complexation was performed as follows: 10 μM (final) aEGFRxMIC2 or aHER2xMIC2 was mixed with GNE987 in DMSO in PBS pH 7.4 to achieve the desired loading (Table 3). 5% Tween-20 was added to a final concentration of 0.3% in the PBS pH 7.4 solution. Samples were incubated at 25°C for 3 hours in a ThermoMixer with shaking at 650 rpm. Aqueous Tween-20 was not added for complexation studies using size exclusion chromatography.

[0191] [Table 3]

[0192] Affinity against a set of PROTACs was reassessed to confirm PROTAC binding. Overall, the affinity of the bispecific antibody was comparable to that of MIC2 (Figure 9).

[0193] 7.4.1 Loading-dependent complex formation aEGFRxMIC2 was loaded with 0, 10, 25, 50, 75, and 100% GNE987 PROTAC and immediately injected into the SEC system. Antibody aEGFRxMIC2 eluted at 3.15 minutes. A second peak appeared at 3.48 minutes with increased loading, corresponding to antibody aEGFRxMIC2 loaded with one PROTAC molecule. Further increases in loading led to the appearance of a third peak at 4.04 minutes (two PROTACs per antibody). Overall, the peak distribution shifted to later elution times as loading increased (Figure 10).

[0194] 7.4.2 Purification of Fully Loaded Conjugates and Complex Stability The aEGFRxMIC2 antibody was loaded with 200% GNE987. The sample was split in half and one portion was desalted in PBS pH 7.4 using a Zeba Spin Desalting Column, 40K MWCO, 75 μL, according to the manufacturer's instructions. The chromatogram shows the removal of DMSO and the PROTAC eluting at approximately 5.7 minutes (FIG. 11).

[0195] The peaks from Figure 11 were integrated and the peak distribution plotted for the unpurified and purified antibody-PROTAC conjugates (Figure 12). The peak distribution remained unaffected by the desalting process, indicating that PAX can be purified from excess PROTAC or other small molecules without any effect on PROTAC loading.

[0196] The aEGFRxMIC2+GNE987 complex was studied over the course of 70 hours at room temperature at a protein concentration of 10 μM in PBS pH 7.4 to assess complex stability over time. Peak distribution was unaffected by increasing incubation time (FIG. 13), demonstrating complex stability over 70 hours.

[0197] 7.5 Functionality of the PAX Approach 7.5.1 Covalent PROTAC-ADC A control PROTAC-ADC was prepared according to WO2020086858 by conjugation of 1 (Figure 14) to the anti-EGFR antibody cetuximab (C225), which harbored the L328C mutation. The final conjugate had a drug-to-antibody ratio of 1.62 by mass spectrometry, while having a monomer content of 97.0%.

[0198] 7.5.2 BRD4 Decomposition Due to the strong pharmacological effect (cell death) induced upon BRD4 degradation, BRD4 was selected as a model protein for the disclosed invention. To evaluate targeted BRD4 degradation, MDA-MB-468 cells were seeded (10,000 cells / well) into black 96-well clear-bottom plates and then incubated overnight in a humidified chamber (37°C, 5% CO2). Test compounds were added using a D300e digital dispenser (Tecan) and incubated for 43 hours (37°C, 5% CO2, humidified chamber). Cells were washed three times with PBS, fixed in 2% (v / v) formaldehyde for 15 minutes at room temperature, and washed three times. To permeabilize cells, 0.2% (v / v) Triton-X-100 was added, left at room temperature for 10 minutes, and then removed by washing with PBS three times. Wells were blocked with 3% (w / v) BSA in PBS for 60 minutes at room temperature, washed immediately, and incubated overnight at 4°C with 2.3 μg / mL rabbit anti-BRD4 antibody (Abcam) diluted in 3% BSA / PBS. After three PBS washes, cells were incubated with secondary conjugated AF488 goat anti-rabbit antibody (5 μg / mL in 3% BSA / PBS) in the dark for 120 minutes at room temperature, washed immediately, and nuclei were stained with Hoechst 33342 (5 μg / mL in 3% BSA / PBS) in the dark for 90 minutes at room temperature. After a final wash step, cells were stored in 0.1% (w / v) sodium azide and transferred to a Cytation 5 cell imaging reader (Biotek). Images were captured using DAPI (nuclei) and green fluorescent protein (BRD4) filter cubes and processed using BioTek gen5 data analysis software. For quantitative analysis of nuclear BRD4 levels, only green fluorescence (AF488) colocalizing with DAPI staining was counted. Green fluorescence was normalized to cell number and expressed relative to untreated cells.

[0199] Figure 15 shows an example image of BRD4 levels. It is important to note that higher fluorescence indicates higher availability of BRD4 in MDAMB468 cells. Untreated cells exhibited the strongest green fluorescence, which could be suppressed by BRD4 degradation mediated by GNE987, an anti-EGFR PROTAC-antibody-drug conjugate, C225-L328C-GNE987 based on the EGFR antibody cetuximab (short C225), and aEGFRxMIC2 loaded with 50% GNE987. These molecules have comparable effects on BRD4 degradation. The induction of degradation by GNE987 could be reduced by complex formation with aHER2xMIC2, which does not bind to MDAMB468 cells.

[0200] Fluorescence in the nucleus can be used to quantify the degradation effect of the analyte (Figure 16). The trend already observed in the fluorescence images becomes visible again across the entire concentration range (Figure 16A). To facilitate easier comparison, a zoomed-in display of the BRD4 values ​​at a treatment concentration of 4 nM was created (Figure 16B). GNE987 had the strongest degradation effect, reaching 39.0% of remaining BRD4. GNE987-loaded aEGFRxMIC2 and C225-L328C-GNE987 degraded BRD4 in an almost identical manner (44.0% and 44.2%, respectively). Degradation induced by GNE987-loaded aHER2xMIC2 amounted to 57.3%, which was 13.3% lower than the corresponding aEGFRxMIC2 + GNE987 complex.

[0201] conclusion aEGFRxMIC2 complexed with GNE987 induced BRD4 degradation in EGFR-expressing MDAMB468 cells, similar to the PROTAC GNE987 and the PROTAC-antibody-drug conjugate C225-L328C-GNE987. Because the aHER2xMIC2+GNE987 complex cannot enter MDAMB468 cells due to the lack of HER2 expression, reduced BRD4 degradation was observed for aHER2xMIC2 complexed with GNE987.

[0202] 7.5.3 Selective cell killing by antibody-mediated delivery of BRD4-degrading PROTACs BRD4 degradation has been shown to induce potent cell death in several cell lines, with potency in the nanomolar to subnanomolar range (Pillow, TH et al., ChemMedChem 15 (2020) 17-25).

[0203] 2000 cells per well were seeded in white-opaque 384-well plates followed by overnight incubation in a humidified chamber at 37°C and 5% CO. Complex formation was performed as described in section 7.4.

[0204] Using a Tecan D300e dispenser, solutions were added to the cells based on antibody concentration, and all wells were normalized to the same volume using 0.3% TW20 and DMSO in PBS pH 7.4. The assay was performed after 3 days unless otherwise noted using the CellTiter-Glo Luminescent Cell Viability Assay as described in the manufacturer's protocol. Briefly, plates were equilibrated to room temperature for 30 minutes. 100 mL of CellTiter-Glo buffer was added to the CellTiter-Glo substrate flask and mixed thoroughly. 30 μL of reagent was transferred to each well. After 3 minutes of incubation at room temperature with shaking at 550 rpm, the plate was incubated for an additional 10 minutes at room temperature. Luminescence was read on an Envision reader. Evaluation was performed using GraphPad Prism 8 by normalizing sample-treated cells to untreated cells. Data were fitted with a four-point logistic curve to determine IC. 50 value was determined.

[0205] In EGFR-highly expressing MDAMB468 cells, the BRD4-degrading PROTAC GNE987, the EGFR-binding PROTAC-antibody conjugate C225-L328C-GNE987 (DAR=1.62), and the EGFR-targeting bispecific antibody aEGFRxMIC2 (1:1) complexed with 50% GNE987 had comparable potency. The cytotoxic effect of GNE987 was suppressed by incubation with the VH032-binding antibody MIC2 and the non-binding aHER2xMIC2+GNE987 (1:1) (Figure 17). The bispecific antibody without the PROTAC had no effect on cell viability over the range of concentrations tested.

[0206] Reducing the loading of the bispecific antibody and anti-VH032 antibody MIC2 to 25% reduced the potency of the EGFR-targeting conjugate aEGFRxMIC2+GNE987 (1:0.5). Concurrently, the effect of the non-binding controls aHER2xMIC2 and MIC2 on cell viability was also reduced at lower loadings (Figure 18).

[0207] The potency of the non-binding control conjugate MIC2+GNE987 and the EGFR-targeted aEGFRxMIC2+GNE987 varies depending on the loading of the conjugate (Table 4).

[0208] [Table 4]

[0209] The selectivity index can be calculated by dividing the potency of the non-binding control conjugate by the EGFR-targeted aEGFRxMIC2+GNE987 conjugate for each respective loading, resulting in a selectivity index of 12.9 for 50% (1:1) loading and 29.4 for 25% (1:0.5) loading.

[0210] Experiments were performed in biological triplicates. The potencies of the molecules are summarized in Table 5 and represented graphically in Figure 19.

[0211] [Table 5]

[0212] Complexation of GNE987 with EGFR-binding aEGFRxMIC2 leads to 42-fold higher potency compared to complexing non-binding aHER2xMIC2 with GNE987, and 21-fold higher potency compared to complexing non-binding MIC2 antibody with GNE987.

[0213] Furthermore, aEGFRxMIC2 and aHER2xMIC2 complexed with 50% GNE987 were investigated in the EGFR-negative cell line HEPG2 alongside several benchmarks, including the PROTAC GNE987 alone, a non-targeting MIC2+GNE987 complex, and an EGFR-targeting PROTAC-ADC (C225_L328C-GNE987) (Figure 20). The PROTAC itself had a strong antiproliferative effect on HEPG2 cells, with an IC of 3.1 nM. 50 values, but all antibody-based constructs had IC values ​​>100 nM 50 aEGFRxMIC2 and MIC2 complexed with GNE987 most potently suppressed GNE987 toxicity, inducing only initial toxicity (approximately 10%) at 100 nM.

[0214] conclusion Cell viability assay data corroborate the BRD4 degradation data. A) The aEGFRxMIC2+GNE987 (50%) conjugate exhibits similar cytotoxic effects against EGFR-expressing MDAMB468 cells compared with the PROTAC-ADC C225-L328C-GNE987 and the PROTAC GNE987. B) Antibody conjugates that cannot enter cells because they completely lack a targeting moiety (MIC2+GNE987 (50%)) or because the antibody's target receptor is not expressed on MDAMB468 (aHER2xMIC2+GNE987 (50%)) have reduced antiproliferative effects. Furthermore, in HEPG2 cells, which do not express EGFR, the cytotoxicity of all antibody-based conjugates and conjugates was reduced compared with the PROTAC alone, which lacks a targeting moiety.

[0215] 7.5.4 Targeted Delivery of Additional PROTACs To demonstrate targeted delivery of another PROTAC, a GNE987 analog with a hydrophilic PEG linker, GNE987P (Figure 21), was complexed with MIC2 or aEGFRxMIC2 and incubated with EGFR-expressing MDAMB468 cells (Figure 22). aEGFRxMIC2+GNE987P mediated increased cytotoxicity compared to GNE987P alone in the concentration range of 0.1–10 nM, demonstrating targeted intracellular delivery of GNE987P by aEGFRxMIC2, whereas the nonbinding control construct, MIC2+GNE987P, was significantly less toxic.

[0216] 7.6 Mouse serum stability The antibody aEGFRxMIC2 was mixed with GNE987 to a final concentration of 40 μM each. The mixture was incubated at room temperature for 2 hours with shaking at 650 rpm. 15% (vol / vol) of 2 M HEPES buffer, pH 7.55, was added to Biowest mouse serum (lot number S18169S2160), followed by sterile filtration.

[0217] aEGFRxMIC2+GNE987 and GNE987 were diluted to 5 μM using a mouse serum-HEPES mixture and incubated at 37°C and 5% CO for 0, 2, 4, 6, 24, 48, 72, and 96 hours. Incubation was stopped by freezing at -20°C.

[0218] The concentration of the PROTAC GNE987 was quantified using LC-MS. GNE987 and GNE987 in complex with aEGFRxMIC2 were stable over 72 hours (Figure 23).

[0219] Furthermore, the concentration of intact aEGFRxMIC2 antibody was quantified in samples incubated in mouse serum for 96 hours. Quantification was performed using a total antibody ELISA (Figure 24). The concentration of intact antibody was unaffected, demonstrating the high plasma stability of the bispecific antibody.

[0220] Furthermore, aEGFRxMIC2 in complex with GNE987 was incubated in mouse serum for 0 and 96 hours, and the samples were then subjected to affinity capture assays. Therefore, the beads were vortexed and transferred into a 1.5 mL LoBind tube, followed by washing three times with 500 μL of HBS-E buffer. 0.2 μg / μL of biotin-SP (long spacer) AffiniPure goat anti-human IgG (Fcγ fragment specific) was added to the beads, which were then incubated on a rotating plate for 2 hours. The beads were washed three times with 500 μL of HBS-E buffer. 20 μL of a 0.5 μg / μL sample was diluted to 0.1 μg / μL with HBS-E buffer, added to the beads, and incubated on a rotating plate for 2 hours. The supernatant was collected. 100 μL of acetonitrile was added to the beads, followed by incubation at 1000 rpm for 30 minutes, and the eluate was collected. GNE987 was quantified from the supernatant and eluate using LC-MS / MS.

[0221] Although GNE987 was not detected in the serum supernatant, the eluate still contained 96.7% intact GNE987 bound to the antibody. Surprisingly, antibody-bound GNE987 was still detectable in the eluate after 96 hours, demonstrating high stability (Figure 25).

[0222] 7.7 Storage stability The storage stability of antibody-PROTAC conjugates was assessed after conjugation (6 mg / mL, 650 rpm, 3 hours, room temperature) by incubation in a 4°C refrigerator for 96 hours and by flash-freezing the conjugates and storing them at -80°C for 24 hours or at -20°C for 96 hours. Samples were then examined for visible changes, polydispersity was assessed using DLS measurements, and loading was measured by SE-HPLC (Table 6). Samples with polydispersity up to 15% are considered monodisperse.

[0223] [Table 6]

[0224] The data not only indicated high storage stability of the complex, as polydispersity and loading remained largely unchanged, but also observed a shielding effect on PROTAC hydrophobicity, as hydrophobic GNE987 did not precipitate in the presence of MIC2.

[0225] 7.8 VHH-based antibodies and PAX 7.8.1 Immunization New World camelids (NWC) were immunized with alternating KLH-based and cBSA-based immunogens (Schedule Figure 26). Serum ELISA assays were performed using huFc-hapten conjugates to monitor VHL ligand-specific immune responses. All animals demonstrated an immune response after immunization.

[0226] 7.8.2 Antibody gene library An immune library was generated from the immune repertoire of three immunized NWC individuals for the selection of VHL ligand-specific antibodies. Peripheral blood mononuclear cells (PBMCs) were isolated from the animals' blood. RNA was extracted, purified, and used for cDNA synthesis. The cDNA pool was then used to amplify VHH gene sequences by PCR, cloned into a VHH antibody-phage display vector, and used to transform Escherichia coli (E. coli). The size of the antibody-gene library was determined by serial dilution and colony counting. Furthermore, the insertion rate was determined by cPCR, and the number of clones with functional ORFs was determined by DNA sequence analysis. The transformed bacteria were then grown and used for packaging antibody-phage particles. After purification of the antibody-phage particles, the presence of antibody-pIII fusion proteins was examined by SDS-PAGE, Western blotting, and anti-pIII immunoblot staining of the antibody-phage particles. A summary of the library characteristics is shown below (Table 7):

[0227] [Table 7]

[0228] 7.8.3 Antibody Discovery First, the library was cleared from nonspecific or cross-reactive antibody-phages. To this end, the library was incubated in the presence of immobilized streptavidin, magnetic streptavidin beads, and BSA. Antibody-phages that bound to negative antigens were removed from further selection.

[0229] The cleared library was then selected for target antigen-specific antibodies. To this end, a conjugate of VH032 and a PEGylated crosslinker bearing a pendant amine was obtained (Sigma-Aldrich; Figure 27) and biotinylated by biotin-NHS-ester coupling. Biotinylated VH032 was purified and analyzed by HPLC. First, biotinylated VH032 was added to the cleared library preparation. Antibody-phage bound to the biotinylated target antigen were captured and recovered from the solution using magnetic streptavidin beads. The beads were washed multiple times with BSA solution (containing 0.05% Tween 20) and PBS to remove nonspecific or weakly bound antibody-phage particles. Antigen-specific antibody phage were eluted from the beads by trypsin treatment and rescued by E. coli infection. After a short period of growth, the bacteria were co-infected with M13K07 helper phage to induce antibody-phage amplification. Amplified phage from the immune library were used for two further selection cycles as described above.

[0230] 7.8.4 Antibody Screening After antibody-phage selection, the binding characteristics of monoclonal antibody clones were analyzed by ELISA. For the immune library, the selection output after selection cycles 2 and 3 was used. 384 single clones were picked for VHH antibody expression in bacteria. Streptavidin + biotinylated VH032 Streptavidin Human Fc-VHL-1 Human Fc The products were tested for their binding specificity by ELISA on .

[0231] An antibody clone was identified as antigen-specific if: ELISA binding signal for positive antigen was ≥ 0.1 ELISA binding signal for negative antigen was ≤0.1 The signal-to-noise (S / N) ratio between positive and negative antigens was ≧10.

[0232] All 562 hits were used for DNA-sequence analysis to identify antibodies with unique antibody sequences (≥1 amino acid difference in the CDRs). 113 unique clones were identified from the NWC library.

[0233] To identify clones with the best binding affinity, we performed BLI dissociation rate measurements. First, we generated VHH-containing culture supernatants of unique clones. We then measured the association and dissociation of antibody fragments to biotinylated VHL immobilized on a BLI streptavidin sensor. Binding curves were fitted with a 1:1 binding model to calculate dissociation rates.

[0234] Based on dissociation rates and antibody sequence information, 10 lead clones (designated MIC5–MIC14) were selected for conversion into the final format.

[0235] 7.8.5 Antibody conversion The VHH genes were amplified from phagemid DNA by PCR and cloned into two different IgG expression vectors. One expression vector encoded the EGFR-targeting cetuximab IgG antibody. The other expression vector encoded the CD33-targeting gemtuzumab IgG antibody. To insert the VHH antibody fragment into the expression vector, it was genetically fused to the C-terminus of the heavy chain of an IgG antibody. The antibody fragment was separated from the IgG heavy chain by a short GS linker (GSGGGSGGSGGGGSG) (SEQ ID NO: 14), resulting in the following format: HC-linker-VHH.

[0236] After sequence verification and preparation of transfection-grade DNA, the expression vector of one VHH clone was transiently transfected into HEK cells. The antibody was produced by HEK cells for 7 days and secreted into the culture medium. After clearance of the culture supernatant from the cells by centrifugation, the IgG antibody was purified by Protein A affinity chromatography. After adjusting the buffer to PBS, the antibody protein concentration was determined by UV / VIS spectroscopy. The antibody integrity and purity were assessed by SDS-PAGE under reducing conditions. The functional binding activity of the antibody to the target antigen was measured by ELISA.

[0237] Furthermore, parental antibodies were produced using the same procedure so that expression rates of the parental antibodies versus the fusion proteins could be compared. The data are illustratively presented in Figure 28 for VHH fusions to cetuximab and gemtuzumab, demonstrating no significant impact on the productivity of the VHH fusions.

[0238] 7.8.6 Versatility of VHH conjugation to PROTACs The kinetic and affinity parameters of protein-PROTAC interactions were evaluated by SPR. Anti-PROTAC VHH clones MIC5–MIC14 were immobilized as CD33 or CLL1 antibody fusions on a CM5 (Series S) sensor chip at 25°C using standard amine coupling procedures (the preparation of the fusion proteins and the linkers used are described in Section 7.9). Prior to immobilization, the carboxymethylated surface of the chip was activated with 400 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 100 mM N-hydroxysuccinimide for 7 minutes. To reach 3,000–6,000 response units (RU), hit anti-PROTAC VHHs as CD33 and CLL1 antibody fusions were diluted to 10 μg / mL in 10 mM acetate, pH 4.5, and immobilized on the activated surface chip for 7 minutes. Remaining activated carboxymethylated groups were blocked with a 10-minute injection of 1 M ethanolamine, pH 8. HBS-N, consisting of 10 mM HEPES pH 7.4 and 150 mM NaCl, was used as background buffer during immobilization.

[0239] PROTACs were pre-diluted in DMSO and diluted 1:50 in running buffer (12 mM phosphate pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, 2% DMSO) and injected at 10 different concentrations, ranging from 1 µM to 0.002 µM, using a two-fold dilution series. DMSO solvent correction (1%–3%) was performed to account for bulk signal variability and achieve high-quality data. The interaction analysis cycle consisted of a 300-second sample injection (30 µL / min; association phase), followed by a 900-second buffer flow (dissociation phase).

[0240] All sensorgrams were processed by first subtracting the binding response recorded from the control surface (reference flow-channel) and then subtracting a buffer blank injection from the active flow-channel (immobilized target protein). All data sets were fitted to a simple 1:1 Langmuir interaction model to determine kinetic rate constants. Experiments were performed on a Biacore 8k+ (Cytiva, Uppsala, Sweden) at 25°C, and interactions were evaluated using the provided Biacore Insight evaluation software.

[0241] The results are summarized in Table 8. The PROTACs in this study were selected based on their chemical structure in order to test as diverse a set of molecules as possible. In general, all antibodies bound to the various PROTACs with sub-nanomolar to triple-digit nanomolar affinities.

[0242] Of all the variants tested, the MIC7 anti-PROTAC VHH had the most favorable binding profile, binding to most PROTACs with single-digit nanomolar to even subnanomolar affinities. The MIC7 clone tolerates all PROTACs in which the linker to the protein-binding moiety is present in R1 and R2, but not in R3. No negative effect on MIC7-PROTAC binding was observed for any type of linker chemistry. Furthermore, MIC7 tolerates the common methyl group in R4. Finally, SPR PROTAC binding measurements using a CLL1-binding antibody MIC7 VHH fusion (CLL1xMIC7) instead of a CD33-binding antibody fusion showed that PROTAC binding was not affected by fusion to other antibodies, as binding affinity was unaffected (Table 8).

[0243] [Table 8]

[0244] 7.8.7 Co-crystallization of MIC5, MIC7, and MIC10 with GNE987P To further elucidate the binding properties of the VHH moieties of our bispecific fusion proteins, the MIC5, MIC7, and MIC10 VHHs were each crystallized as a single VHH in complex with the GNE987P PROTAC. MIC5 was concentrated to 60.9 mg / mL in a 20 mM HEPES, 150 mM NaCl, pH 7.0 buffer solution. The protein was incubated with 2 mM GNE987P and 2% DMSO overnight at 277 K to prepare the complex for crystallization. Crystals of the complex were obtained using the sitting-drop vapor diffusion method by equilibrating the protein solution against 100 nL of reservoir solution (50% PEG 400, 0.1 M Na acetate, pH 4.5, 0.2 M Li sulfate) at 293 K. MIC7 was concentrated to 38.65 mg / mL in a 50 mM histidine, 150 mM NaCl, pH 5.5 buffer solution. The protein was incubated with 2 mM GNE987P and 2% DMSO at 277 K overnight to prepare the complex for crystallization. Crystals of the complex were obtained using the sitting-drop vapor diffusion method by equilibrating the protein solution against 100 nL of reservoir solution (30% PEG8000, 0.2 M ammonium sulfate, 0.1 M Tris, pH 8.5) at 293 K. The MIC was concentrated to 43.31 mg / mL in a 20 mM HEPES, 150 mM NaCl, pH 7.0 buffer solution. The protein was incubated with 2 mM GNE987P and 2% DMSO at 277 K overnight to prepare the complex for crystallization.

[0245] Crystals of the complex were obtained using the sitting-drop vapor diffusion method by equilibrating the protein solution against 100 nL of reservoir solution (40% PEG 300, 0.1 M Na citrate-phosphate, pH 4.2) at 293 K. X-ray diffraction data for MIC5 and MIC7 crystals were collected at the P14 beamline at the PETRAIII, Hamburg, Germany synchrotron radiation source, and for MIC10 crystals at the X06SA beamline at the SLS, Villigen, Switzerland radiation source, using an EIGER2 16M detector (Johnson et al. (2014), JINST, 9, C05032.). All collected diffraction images were processed using autoPROC with the STRAANISO option for anisotropic data processing (Tickle, et al. (2018), STARANISO (http: / / staraniso.globalphasing.org / cgi-bin / staraniso.cgi), Cambridge, United Kingdom: Global Phasing Ltd.). The structure was solved by molecular replacement using the program PHASER from the CCP4 program suite (Collaborative Computational Project, Number 4 (1994), "The CCP4 Suite: Programs for Protein Crystallography". Acta Cryst. D50, 760-763), an in-house VHH structure from another Merck research program. Crystallographic refinement was performed using Buster 2.11.8 (Bricogne et al. (2017), BUSTER version 2.11.7. Cambridge, United Kingdom: Global Phasing Ltd.). Model building was performed in Coot (Emsley, P. & Cowtan, K. (2004), Acta Cryst. D60, 2126-2132.) The final model was obtained after several cycles of rebuilding in Coot and subsequent runs in Buster.Crystallographic data processing and refinement statistics for all three structures can be found in Table 9.

[0246] [Table 9]

[0247] In the following, consecutive numbering is used based on the parental VHH wild-type sequence to indicate the amino acid exchanges (Figure 29).

[0248] Structural elucidation and interactions between MIC5 and the E3 ligase binding domain VH032 Interestingly, in the MIC5-GNE987P co-crystal structure, the E3 ligase moiety is trapped between the CDR3 loops, which fold back toward the VHH body. The primary interactions between the ligand and VHH occur through hydrogen bonds with the main chains of CDR3 residues Gly108, Gln109, and Ile111, as well as through van der Waals interactions with the side chains of CDR3 residues Ala99, Pro104, Ile111, and Leu113. All interactions between the VH032 moiety and the MIC5 CDR3 loops are detailed in Table 10.

[0249] [Table 10]

[0250] Structural elucidation and interaction between MIC7 and the E3 ligase binding domain VH032 The same E3 ligase moiety capture was also found in the MIC7-GNE987P co-crystal structure. The primary interactions between the ligand and VHH occur through hydrogen bonds with the main chains of CDR3 residues Ile111 and Tyr113, and through van der Waals interactions with the side chains of CDR3 residues Ile99, Val107, and Arg112. All interactions between the VH032 moiety and the MIC7 CDR3 loops are detailed in Table 11.

[0251] [Table 11]

[0252] Structural elucidation and interaction between MIC10 and the E3 ligase binding domain VH032 Again, similar to the two previous structures, in the MIC10-GNE987P co-crystal structure, the E3 ligase moiety is trapped between the CDR3 loops that fold back towards the VHH body.

[0253] Here, the crystallographic asymmetric unit consisted of two copies of the VHH(MIC10)-VH032 complex, hereafter referred to as monomers A and B.

[0254] All interactions between the VH032 moiety and the MIC10 CDR3 loop are detailed in Tables 12 and 13 for Monomer A and Monomer B of the co-crystal structure.

[0255] [Table 12]

[0256] [Table 13]

[0257] All interactions were calculated according to the criteria specified in Bissantz et al. (2010), J. Med. Chem. 53 (14), 5061-8.

[0258] We were able to obtain a well-defined electron density map of MIC5, MIC7, and MIC10 in complex with GNE987P (the linker and BRD4-binding moieties could not be clearly defined in the electron density map). Therefore, only the amide-modified ligase-binding domain VH032 ligand was incorporated into the structure (Figure 1A).

[0259] In co-crystallization studies, the structures of the MIC5, MIC7, and MIC10 VHHs were refined to 1.52 Å, 1.55 Å, and 1.84 Å resolution, respectively. All three VHHs adopt the standard fold of an immunoglobulin variable domain: nine conserved antiparallel β-strands combined with three hypervariable regions (Figure 30). Surprisingly, all three co-crystal structures of MIC5, MIC7, and MIC10 revealed that of the three CDRs, only CDR3 was found to be involved in PROTAC binding (Figure 30). Furthermore, the structures revealed an unexpected conformational arrangement in which the ligase-binding portion of the PROTAC is buried under CDR3 like a clasp, which is likely the reason for the stable complexes formed by PACs designed based on MIC5, MIC7, and MIC10.

[0260] Comparing all three VHHs, they were found to overlap well as the protein backbones maintained similar conformations for all three VHHs. Although the CDR3 loop conformations showed some slight differences, all three VHHs showed very good binding to VH032 and VH032-based PROTACs (Figure 31).

[0261] 7.8.8 Determining the influence of CDR1 and CDR2 on the binding of VHH MIC7 To further elucidate the binding patterns of the screened VHHs, all amino acids in CDR1 and CDR2 of VHH MIC7 were stepwise mutated to lysines, and their binding ability was examined by nano-differential scanning fluorimetry (nano-DSF) in a thermal shift assay to determine whether the VHHs were stabilized or destabilized by the ligand molecules. Nano-DSF measurements were performed using a Prometheus NT.Plex system from NanoTemper (Munich, Germany). For all titration experiments, the VHH MIC7 protein and MIC7 mutants and the respective VHL-ligand compounds were formulated in 30 mM HEPES; 150 mM NaCl; pH 7.4, optionally supplemented with 0.01% Tween 20. The final assay concentrations of proteins were 0.2–0.4 mg / ml, and compounds were 50 μM in the buffer. All buffers were adjusted to a final concentration of 1% (v / v) DMSO. The sample was loaded into a high-sensitivity capillary tip and sealed with capillary sealing paste. Thermal unfolding was recorded at a temperature interval of 20 °C to 110 °C with a gradient of 1 °C / min. Fluorescence changes were monitored at 330 nm, and the unfolding transition was fitted to a two-state model using PR. Stability analysis software (NanoTemper) to determine the melting temperature, T. M The T of the DMSO control was determined. M T in the presence of compound VH032 by subtracting M The change in T is calculated. Table 14 shows the melting temperatures for the parental MIC7 as well as the T observed in the presence of ligand VH032. M Change in (ΔT M(リガンド) ) The parental MIC7 VHH exhibits a clear shift in melting temperature (ΔT M(リガンド) = 14.0 °C), indicating a very strong stabilizing interaction. These results support the observed K values ​​of at least low single-digit nM measured by isothermal titration calorimetry (ITC) performed as described in Section 7.8.10, except that 50 μM of VHH was used for the measurements. DThis was further confirmed by (Table 15). As expected, all generated lysine mutants of CDR1 and CDR2 of MIC7 showed a significant improvement in the measured ΔT M(リガンド) The VHHs exhibited specific binding, as indicated by the ITC values. This was further confirmed by exemplary ITC data for two CDR2 mutants (Table 15). The small effect on the observed binding properties can be explained by the resulting changes in the CDR1 or CDR2 loop structure, which also affected the observed melting temperatures of the VHHs alone.

[0262] [Table 14]

[0263] [Table 15]

[0264] 7.8.9 Examining the Effects of Solvent-Exposed Residues and Other Point Mutations in CDR3 of MIC7 Therefore, solvent-exposed amino acids in the CDR3 loop were exchanged for lysine or glutamic acid, and the resulting mutants were tested for their effect on melting temperature and its change in the presence of the ligand VH032 (Table 16). As expected, T M and ΔT M(リガンド) No significant differences were found, providing the possibility of amino acid substitutions at the mutation sites in CDR3 without perturbing the binding behavior of the single domain antibody. These positions do not directly interfere with VH032, providing space for amino acid exchange at these positions.

[0265] [Table 16]

[0266] Furthermore, additional point mutations based on rational design were generated and tested for their binding behavior by nanoDSF (Table 17). All three variants showed a ΔT within the range of the parent MIC7 VHH. M(リガンド) Values ​​were found, indicating that these variants were able to bind equally strongly to VH032.

[0267] [Table 17]

[0268] 7.8.10 Humanization of VHHs To reduce immunogenicity, which can lead to reduced efficacy due to rapid drug clearance or neutralization, or toxicity due to cross-reactivity with proteins in the body, VHH humanization was performed. To this end, six mutants of MIC7 were designed and generated in a bispecific format based on gemtuzumab as the parent antibody with IgG1 PG-LALA as the scaffold. The original sequences were stepwise aligned to the most similar germline sequence. Because alterations of residues at critical structural positions can affect the (bioactive) conformation of the CDRs and potentially affect production yield, stability, and binding affinity, we first mutated residues not located at critical structural positions (Vernier positions, Hallmark positions). Furthermore, additional variants were designed using amino acid residues introduced (from the closest germline sequence) into critical structural positions. The designed versions were named as follows: Illustratively, a CD33-targeting antibody in which a newly generated humanized version of one of the MIC7 VHHs is fused to the C-terminus of each heavy chain via the above linker would be designated CD33xhMIC7_1.1, and the next stepwise engineered bispecific antibody would then be designated CD33xMIC7-VHH1.2, etc.

[0269] The resulting variants were first tested for their binding ability by isothermal titration calorimetry (ITC). ITC measurements were performed using a VP-ITC microcalorimeter from MicroCal / Malvern Panalytical (UK). For all titration experiments, PAX and each VHL-ligand compound were formulated in 30 mM HEPES, 150 mM NaCl, pH 7.4, optionally supplemented with 0.01% Tween 20. The final protein concentration in the injection syringe was 25 μM. A 10 mM compound stock solution in DMSO was diluted with buffer to a 5 μM concentration and loaded into the sample cell. All buffers were adjusted to a final concentration of 1% (v / v) DMSO. ITC titrations were performed at a constant temperature of 303 K. ITC data analysis was performed using Origin7-based calorimetry customization (OriginLab Cooperation, Northampton, USA) supplied as standard instrument software by MicroCal / Malvern Panalytical (UK). The integrated thermal data were fitted to a one-site binding model to determine apparent values ​​for binding affinity, enthalpy, and stoichiometry. The binding parameters of a reference ligand were monitored, and the binding stoichiometry was used as a reference point to normalize the concentrations of various protein batches. As predicted, all newly designed variants of MIC7 were found to bind to the VH032 E3-ligase binding moiety of multiple VHL-targeting PROTACs with affinities comparable to those of the parent antibody CD33xMIC7 (Tables 1A and 18). Furthermore, the ability to bind two VH032 ligands was assessed, and the binding stoichiometries of all novel fusion proteins were found to be 2 (Table 18). In all cases, an enthalpy-driven binding process was observed.

[0270] [Table 18]

[0271] To verify the extent to which PAX technology can mediate cell-selective targeting of cells based on their cell surface receptor expression, we investigated whether these novel variants could still perform in in vitro assays (Figure 32 and Table 19).

[0272] [Table 19]

[0273] 7.9 Fabrication of VHH-based PAX VHH antibody fragments were fused to the heavy and light chains of an IgG antibody to enable delivery of PROTACs to various disease-related cell lines. Fusions were constructed as follows: The antibody heavy or light chains, or heavy and light chains, were extended at the C-terminus with a linker (GSGGGSGGSGGGGSG) (SEQ ID NO: 14), followed by a VHH sequence (e.g., YU734-F06(MIC7)). In this way, bispecific antibodies capable of recognizing cell surface receptors and simultaneously binding to the VHL-ligand of a PROTAC can be constructed as shown in Figure 3. The linker-VHH (linker: GSGGGSGGSGGGGSG) (SEQ ID NO: 14) sequence can be fused multiple times to the HC, LC, or both as a repeat unit (e.g., linker-VHH-linker-VHH, i.e., two repeats). Up to three linker-VHHs per chain were fused to either the HC, LC, or both. The nomenclature is as follows: a CD33-targeting antibody in which one MIC7 VHH is fused to the C-terminus of each heavy chain via the linker described above is named CD33xMIC7. This is the only exception to the nomenclature used for all other constructs, where the generalized formula is: CD33xMIC7 NHML where N and M are 2, 4, and 6. "H" indicates that the fusion is made with the heavy chain, and "L" indicates fusion to the LC. If the VHH is not fused to either the HC or the LC, the respective letter is missing. For example, a CD33-targeting antibody in which two MIC7 VHHs (linker-VHH-linker-VHH, i.e., two repeats) are fused to the C-terminus of each heavy chain and the C-terminus of each light chain is designated CD33xMIC7.4H4L The antibody scaffolds and scaffold modifications used to create bispecific fusion proteins are presented in Table 20. Table 21 shows the nomenclature of PAX targeting CD33 as an example.

[0274] [Table 20-1]

[0275] [Table 20-2]

[0276] Antibody generation was performed as described in section 7.2. Conjugation to generate PAX was performed as follows: 10 μM (final) antibody-VHH fusion was mixed with VHL-based PROTAC in DMSO in PBS pH 7.4 to achieve the desired loading (Table 3). PAX determined for in vivo application was conjugated at an antibody concentration of 68.8 μM.

[0277] For partitioning, 5% Tween-20 in PBS pH 7.4 solution was added to a final concentration of 0.3%. Samples were incubated for 3 hours at 25°C on a ThermoMixer with shaking at 650 rpm.

[0278] [Table 21]

[0279] Furthermore, the effect of fusing the MIC7 VHH to the N-terminus instead of the C-terminus of the heavy chain was investigated using the same linker defined above. For N-terminally conjugated VHHs, an "N" was incorporated before "2H" in the nomenclature. The nomenclature for these antibodies is as follows: CD33-targeting antibodies with two VHHs attached to the N-terminus of the heavy chain via the above linkers are designated CD33xMIC7 [N-2H] It is called (Figure 33.

[0280] Furthermore, we engineered the strand-exchange engineered domain (SEED) platform to generate asymmetric, bispecific antibody-like molecules, potentially expanding the therapeutic applications of natural antibodies. This novel protein-engineered platform is based on the exchange of structurally related sequences of immunoglobulins within the conserved CH3 domain. Alternating sequences from human IgA and IgG in the SEED CH3 domain generate two asymmetric but complementary domains, designated AG and GA. The SEED design allows for the efficient generation of AG / GA heterodimers while disfavoring homodimerization of the AG and GA SEED CH3 domains (Muda et al., Therapeutic assessment of SEED: a new engineered antibody platform designed to generate mono- and bispecific antibodies. Protein Eng Des Sel. 2011 May;24(5):447-54. doi: 10.1093 / protein / gzq123. PMID: 21498564).

[0281] Using SEED technology, we isolated EGFR with either two MIC7 VHHs attached to the normal heavy chain or only one MIC7 VHH attached to one heavy chain (EGFRxMIC7 [S2H] and EGFRxMIC7 [S1H] ) were generated. For SEED antibodies, an "S" was introduced before "2H" in the nomenclature. At the same time, the same technique was applied to generate antibodies that retain one or two MIC7 VHHs but have only one EGFR-binding arm (oaEGFRxMIC7 [S1H] and oaEGFRxMIC7 [S1H] ) were generated. These formats are called SEED PAX. The same linker defined above was utilized. Figure 34 contains a graph of the SEED molecule.

[0282] Furthermore, the effect of fusing the MIC7 VHH C-terminus to the heavy chain was investigated using a cathepsin B-cleavable linker (GSGGVAGGSGGGVAGGS). To this end, a VHH antibody fragment was fused to a heavy IgG antibody as described at the beginning of this chapter, where the glycine-serine linker (GSGGGSGGSGGGGSG) was replaced by a cathepsin B-cleavable linker. For cathepsin B-cleavable constructs, an additional "CL" was introduced in the nomenclature. The nomenclature for these antibodies is as follows: A TROP2-targeting antibody with two VHHs attached to the C-terminus of the heavy chain via the cathepsin B-cleavable linkers described above is called TROP2xMIC7. [2H_CL] It is called.

[0283] To further demonstrate the modular approach of utilizing VHH fragments for antibody-mediated targeted delivery of PROTACs via PAX, we investigated the enzymatic bioconjugation of PROTAC-binding VHHs to glutamine 295 (Q295) of the heavy chain of a native IgG backbone using microbial transglutaminase (MTG) to generate bispecific antibodies for the PAX approach (Figure 36). For nomenclature, when two VHHs are attached to Q295, a "C2" is added. Illustratively, the EGFR-targeting cetuximab as a parent antibody, in which two MIC7 VHHs are conjugated to Q295 of each heavy chain via mTG, is designated EGFRxMIC7. [C2]Specifically, MTG-mediated antibody coupling was performed using the EGFR-targeting antibody cetuximab at a final antibody concentration of 5 mg / mL, 10 molar equivalents of VHH, and 10 U / mL of MTG in 24 mM HEPES pH 7.0. MTG was generated in-house, and enzyme activity was determined by the photometric assay ZediXclusive (Zedira). VHH MIC7, equipped with an N-terminal GS-linker motif and a transglutaminase recognition tag (three glycine residues), was selected for gene fusion to provide an acyl-acceptor substrate. The reaction mixture was incubated at 30°C and 550 rpm for 24 hours and either terminated with 1 nM of final MTG blocker (Zedira) or purified directly by preparative size-exclusion chromatography (SEC). Purification was performed on an HPLC 1260 system (Agilent Technologies) using a Superdex 200 10 / 300 GL increase column and running buffer PBS pH 7.0. Subsequently, concentration was performed using an Ultra-15 centrifugal filter device (50 kDA molecular weight cutoff (MWCO), Amicon) and sterile filtered (0.2 μm). Protein concentration was measured at 280 nm using a NanoDrop One UV-Vis spectrophotometer (Thermo Fisher) and calculated using the extinction coefficient based on the amino acid sequence. Purified samples or quenched reaction mixtures were analyzed by size exclusion chromatography (SEC) and SDS-PAGE (Invitrogen), and the VHH-to-antibody ratio (VAR) was determined by hydrophobic interaction chromatography (HIC), RP-HPLC, and LC-MS. HIC analysis was performed using a TSKgel® Butyl-NPR column (4.6 × 100 mm, 2.5 μm, Tosoh bioscience) heated to 35 °C. A 40-80% gradient over 40 min was used, moving from 0.025 M Tris HCl pH 7.5 to 20% isopropanol, 0.025 M Tris HCl, 2 M ammonium sulfate pH 7.5 at 0.45 mL / min. Samples were previously adjusted to 0.5 M ammonium sulfate, and 42.5 μg was injected. Signal recording was performed at a wavelength of 280 nm.The final VHH ratio for antibody was 1.90 (EGFRxMIC7. [C2] ) and 1.76(DIGxMIC7 [C2] ) and determined by HIC analysis.

[0284] 7.10 PAX Loading To investigate the loading of PAX with PROTACs and understand the time point at which full PAX loading can be achieved, a kinetic assessment of PAX loading was performed using native size-exclusion liquid chromatography-native mass spectrometry (native SEC / MS). For analysis, a SCIEX (Ontario, Canada) eksigent M5 MicroLC system connected to a quadrupole time-of-flight (QTOF) mass spectrometer (MS) (QTOF X500B, SCIEX, Ontario, Canada) was used. Protein samples were separated using a PolyHYDROXYETHYL A capillary (150 x 0.30 mm, 3 μm, 1000 A). The column temperature was set to 25°C, and an isocratic gradient using 100 mM ammonium acetate, pH 6.8, was run at 10 μL / min for 10 minutes. A positive time-of-flight (TOF) MS method was used for analysis. The electrospray voltage of the X500B QTOF instrument was set to 5500 V, and the temperature was 400 °C. Full MS scans were acquired over a mass-to-charge range of 5000–7000, using an accumulation time of 1 s and a total of 120 time bins. Ion source 1 and 2 gases were set to 60 psi. The declustering potential was set to 20 V. Data were manually evaluated using the Explorer tool in SCIEX OS software, version 1.5.0.23389. For this purpose, a multiply charged spectrum was acquired by summing the appropriate time range of the eluting peaks in the total ion current chromatogram (TIC). The five most intense multiply charged peaks were marked and deconvoluted using the Bio Toolkit in Explorer. The input spectrum isotope resolution was set to 1000 (very low). The mass step was 1 Da, and the charge agent was set to H+. The mass range to be calculated was set between 170,000 and 190,000 Da.

[0285] To assess complex formation, CD33xMIC7 [2H] was mixed with a two-fold excess of GNE987 PROTAC at a final antibody concentration of 6 mg / mL. The DAR of the resulting PAX was measured at the following time points: 0, 13, 26, 39, 52, 120, and 360 minutes, and 1 and 7 days. Note that at 0 minutes, the PROTAC was added to the antibody followed by direct injection into the device. Fully complexed PAX (DAR 1.93) was already observed at 0 minutes, and the DAR did not change over the 7-day time course, indicating rapid complexation kinetics and stability of PAX (Figure 37).

[0286] 7.11 Cellular characterization of VHH-based PAX A general description of cell viability assays can be found in Section 7.11.5.

[0287] 7.11.1 Cellular Profiling of VHL-Based PROTAC-Binding Antibody Fragments as Fusions of Cetuximab and Gemtuzumab Gemtuzumab- and cetuximab-based VHH fusions were generated by genetically attaching the VHH to the C-terminus of the respective antibody HC via a linker (GSGGGSGGSGGGGSG) (SEQ ID NO: 14), as described in Section 7.9. After expression and purification, the antibody fusion proteins were loaded with GNE987 at a 1:1 ratio (50% loading). Thus, 10 VHH antibody fragments could be characterized for their ability to induce cell killing in target-positive cells or prevent nonselective uptake into non-targeted cells (as described in Section 7.11.5). The results are presented in Table 22. A selectivity index was introduced as a metric of selectivity. First, the potency of the gemtuzumab-based construct was divided by the potency of the cetuximab-based construct, resulting in a selectivity index that allows for comparison of constructs in the same cellular context. In this case, clones MIC5–MIC8 had the highest selectivity index, demonstrating that these constructs bound to the PROTAC during the 3-day incubation period in cell culture medium. Second, the potency of the cetuximab-based constructs against EGFR-negative HEPG2 cells was divided by the potency of the same construct against EGFR-positive MDAMB468 cells, a measure of selectivity mediated by receptor expression-dependent uptake. In this case, constructs MIC7–MIC10 yielded the highest selectivity index. One additional parameter demonstrating high selectivity is the potency of the PROTAC-loaded fusion protein against HEPG2 cells, where cytotoxicity was not predicted. The PROTAC alone had an IC50 value of 2.6 nM in HEPG2 cells. This indicates that the PROTAC is already released outside the cells, leading to potent cell killing. In contrast, the VHH-based construct led to a strong detoxification effect with a potency of >100 nM. Overall, clones MIC5, MIC7 and MIC10 showed promising profiles, especially when tropism (section 7.8.6) was taken into account.

[0288] [Table 22]

[0289] 7.11.2 Cell binding of scaffold antibodies is unaffected by PAX loading in VHH fusions and PROTACs The effect of VHH fusions via the linker (GSGGGSGGSGGGGSG) (SEQ ID NO: 14) described in Section 7.9 on cell binding to either the CD33-binding antibody gemtuzumab (IgG4-PG-SPLE) or the EGFR-binding antibody cetuximab was investigated using flow cytometry. Therefore, binding of CD33xMIC5 and CD33 Abs without VHH MIC5 fusion to CD33-expressing MV411 cells was assessed. 1 x 10 5 MV411 or MDAMB468 cells were seeded into round-bottom 96-well plates and washed three times with PBS pH 7.4 containing 1% (w / v) bovine serum albumin (BSA). Subsequently, cells were incubated with the respective antibodies on ice for 30 minutes. Cells were then washed three times with PBS, pH 7.4, 1% (w / v) BSA and incubated with the fluorescently labeled secondary antibody Alexa Fluor 488 AffiniPure Fab fragment goat anti-human IgG (H+L) for 30 minutes on ice. Cells were then washed three times with PBS, pH 7.4, 1% (w / v) BSA. Cells were analyzed by flow cytometry using an Intellicyt iQue3 Screener and analyzed via IntelliCyt ForeCyt Enterprise Client Edition 8.0 (R3) software. Cells were cultured in RPMI-1640 + 10% FCS. Direct comparison demonstrated that the addition of VHH MIC5 did not affect cell binding (Figure 38).

[0290] We then investigated the extent to which loading of antibody-VHH fusions with PROTACs affected cell binding behavior. Therefore, cell binding to HL60, MOLM13, MV411, RAMOS, and U937 was determined for the CD33-binding antibody-VHH fusion CD33xMIC7. Binding of the same molecule loaded with 90% GNE987 was assessed. A digoxigenin-binding antibody was used as a non-isotype binding control. CD33xMIC7 was incubated with a 1.8-fold molar excess of GNE987 dissolved in 5% DMSO with 1% FCS in PBS for 3 hours at 25°C with shaking at 650 rpm before use for cell staining. The non-isotype binding control was also analyzed upon incubation with 5% DMSO. From each cell line, 200,000 cells per condition were harvested, centrifuged, and incubated with the respective antibody, antibody-VHH fusion, or PAX condition at a concentration of 10 μg / ml in 200 μl of PBS with 1% FCS for 45 minutes at 4°C. After quenching and one washing step with PBS with 1% FCS, the samples were then treated with fluorescein-labeled anti-human antibody #607 (1:50) for another 45 minutes. After quenching with PBS with 1% FCS, the cells were resuspended in PBS containing 1 μg / mL propidium iodide (PI) stain, which stains dead cells and should be excluded during analysis performed on a Becton Dickinson FACSCalibur flow cytometer. Quantitative analysis was performed using Flowing software from Turku Bioscience. Cells were cultured in RPMI-1640 + 10% FCS.

[0291] Loading of antibody-VHH fusions with PROTACs had no effect on binding, as demonstrated in Figure 39. The isotype control antibody showed strongly reduced binding to the cell line panel.

[0292] 7.11.3 CD33 PAX (with pHAb dye) is internalized into CD33-positive cells To elucidate whether PAX uptake is mediated by receptor-mediated endocytosis, CD33xMIC7 was loaded with the pH-responsive VH032-pHAb dye (Figure 40). The VH032-pHAb dye is a pH sensor that exhibits minimal fluorescence at pH >7 but significantly enhanced fluorescence at acidic pH. Therefore, upon receptor-mediated internalization, transport of CD33xMIC7 + VH032-pHAb dye into acidic endosomal and lysosomal vesicles should result in enhanced fluorescent signal. Prior to use for cell staining, CD33xMIC7 was incubated in the dark for 2 hours at 25°C and 650 rpm in PBS with a 1.8-fold molar excess of VH032-pHAb dye dissolved in 5% DMSO. CD33-positive MOLM13, MV411, U937, and receptor-negative RAMOS cells were treated with the resulting PAX or with VH032-pHAb dye alone as a control. From each cell line, 200,000 cells per condition were harvested, centrifuged, and incubated with PAX at a concentration of 10 μg / ml in 200 μL of PBS with 1% FCS for 6 hours at 37°C with shaking at 650 rpm in the dark. As a control, cells were treated with an equimolar concentration of VH032-pHAb dye alone. After one washing step with PBS with 1% FCS, cells were resuspended in 400 μl of PBS with 1% FCS, and cytometric analysis was performed on a Becton Dickinson FACSCalibur flow cytometer. To eliminate interference with the VHL-pHAb dye, dead cell staining with propidium iodide (PI) was not performed. Quantitative analysis was performed using FlowJo (BD Biosciences). Cells were cultured in RPMI-1640 + 10% FCS.

[0293] Enhanced fluorescence signals were observed in all CD33-positive cell lines treated with the CD33xMIC7+VH032-pHAb dye compared to the control, whereas no enhanced mean fluorescence intensity was observed in receptor-negative RAMOS cells (Figure 41). These results demonstrated that PAX is internalized via receptor-mediated antibody internalization.

[0294] 7.11.4 CD33xMIC7+GNE987 PAX induces BRD4 degradation in targeted cells To demonstrate that PAX can mediate target protein degradation in targeted cells, a BRD4 Western blot degradation assay was performed (Figures 42 and 43). Therefore, CD33-positive MV411 cells were treated with CD33xMIC7+GNE987 or DIGxMIC7+GNE987 as a non-binding PAX control at different concentrations, and with GNE987 alone as a control. MV411 cells were cultured in RPMI-1640 medium supplemented with 10% FCS and penicillin-streptomycin. MV411 cells were seeded in 12-well plates at 1 million cells / ml in 2 ml of culture medium and cultured overnight in a cell incubator at 37°C and 5% CO2. Complex formation was performed as described in Section 7.4. GNE987 alone was preincubated under the same conditions but in the absence of antibody. Cells were then treated with the respective concentrations of either CD33xMIC7+GNE987 (1:1), DIGxMIC7+GNE987 (1:1), or GNE987 via nanodrop dispensing using a Tecan dispenser. All conditions were standardized to 0.0005% (v / v) DMSO and 3.0E-5% Tween 20. Cells were incubated for 24 hours in a cell incubator at 37°C and 5% CO2. Treated cells were harvested and washed with PBS, and cell pellets were lysed in cell lysis buffer (20 mM TRIS pH 7.4, 100 mM NaCl, 1 mM EDTA, 0.5% Triton-100) supplemented with Roche Complete protease inhibitor mixture. After incubation on ice for 10 min, the crude lysate was cleared by centrifugation at 15,000 x g and 4 °C, and the cleared lysate was precipitated with acetone. The dried pellet was dissolved in SDS-loading buffer. Protein concentration was determined using a Mettler Toledo UV5 Nano photometer. Samples (50 μg / lane) were applied to a 4-12% Bis-Tris SDS-PAGE gel (Thermo Fisher Scientific).After the gel was run, the samples were transferred to nitrocellulose membranes (Sigma Aldrich), and the membranes were blocked with 5% non-fat milk in 0.1% TBS-Tween 20 and incubated with the indicated primary antibodies (Table 23).

[0295] [Table 23]

[0296] After incubation with the corresponding HRP-conjugated anti-rabbit / mouse secondary antibody (GE-Healthcare) at a 1:10,000 dilution, the blots were detected using ECL solution (Advansta) and X-ray film (GE-Healthcare). Results were analyzed using ImageJ software (version 1.53K, NIH). Background-subtracted BRD4 signals were normalized to the corresponding actin signals, and the normalized BRD4 signal of the solvent control was set to 100%.

[0297] As expected, BRD4 degradation was observed with treatment with GNE987 alone. Furthermore, concentration-dependent BRD4 degradation was observed with CD33xMIC7+GNE987, whereas no degradation was observed with the non-binding PAX control DIGxMIC7+GNE987 at any concentration (Figures 42 and 43). These results demonstrated that PAX can mediate concentration-dependent, cell type (target receptor)-selective degradation of intracellular proteins of interest.

[0298] 7.11.5 CD33xMIC5+GNE987 PAX induces a cytotoxic effect that is dependent on receptor expression and PAX loading Several cell viability experiments were performed according to the procedures described below.

[0299] Cells were seeded in various media in white cell culture-treated, flat, clear-bottom multiwell plates, followed by overnight incubation at 37°C and 5% CO2 in a humidified chamber. Complexation was performed as described in section 7.4. More specifically, all cell viability assays were performed according to the same procedure: cancer cells were seeded in various media and densities in white cell culture-treated, flat, clear-bottom multiwell plates (corning®), followed by overnight incubation at 37°C and 5% CO2 in a humidified chamber. PAX complexation with PROTAC at a final concentration of 10 μM was performed as described in section 7.9. Serial dilutions of PAX solution were added to the cells using nanodrop dispensing and a Tecan D300e digital dispenser. All wells were standardized to the same volume using a solution of 0.3% Tween-20, pH 7.4, and DMSO in PBS, resulting in final concentrations of 0.003% Tween-20 and 0.05% DMSO, respectively. The treated cells were then incubated at 37°C and 5% CO2. After 3 days, assays were performed using the CellTiter-Glo Luminescent Cell Viability Assay as described in the manufacturer's protocol. Briefly, the plate was equilibrated to room temperature for 30 minutes. Then, 100 mL of CellTiter-Glo buffer was added to the CellTiter-Glo substrate flask and mixed thoroughly. Then, 25 μL of the reagent was transferred to each well of the cell culture plate. After 3 minutes of incubation at room temperature with shaking at 550 rpm, the plate was incubated for an additional 30 minutes at room temperature. Luminescent signals were read using a Perkin Elmer Envision reader. Vehicle alone and staurosporine (10 μM) served as 100% and 0% viability controls, respectively. Raw data were converted to percent cell viability with vehicle and staurosporine set to 100% and 0% viability, respectively. IC50 value calculations were performed using GraphPad Prism software with a variable slope sigmoidal response fitting model.

[0300] Solutions were added to cells based on antibody concentration using a Tecan D300e digital dispenser with nanodrop dispensers. All wells were standardized to the same volume using 0.3% TW20 and DMSO in PBS pH 7.4, with a final solvent concentration of 0.05% DMSO and 0.003% TW20. Incubations were performed at 37°C and 5% or 10% CO2, depending on the medium. Assays were performed after 3 days, unless otherwise noted, using the CellTiter-Glo Luminescent Cell Viability Assay as described in the manufacturer's protocol. Briefly, plates were equilibrated to room temperature for 30 minutes. 100 mL of CellTiter-Glo buffer was added to the CellTiter-Glo substrate flask and mixed thoroughly. 30 μL of reagent was transferred to each well. After 3 minutes of incubation at room temperature with shaking at 550 rpm, the plate was incubated at room temperature for an additional 10 minutes. Luminescence was read on a Perkin Elmer Envision reader. Vehicle alone and bortezomib (1.0E-05M) served as high (100% viability) and low (0% viability) controls, respectively. Raw data were converted to percent cell viability relative to high and low controls, which were set to 100% and 0%, respectively. IC50 calculations were performed using GraphPad Prism software with a variable slope sigmoidal response fitting model using 0% viability as the lower constraint and 100% viability as the upper constraint. Concentrations correspond to the concentration of PROTAC in PAX.

[0301] We investigated whether and to what extent PAX technology can mediate cell-selective targeting of cells based on their cell surface receptor expression. Therefore, we generated CD33-targeted PAX by genetically fusing MIC5 to HC and subsequently loading it with the PROTAC GNE987. PAX was then tested on CD33-positive and CD33-negative cells.

[0302] Treatment of CD33-positive MV411 and MOLM13 cells and CD33-negative RAMOS cells with serial dilutions of CD33xMIC5 preloaded with 50% GNE987 led to a decrease in viable cells after 3 days of incubation in the case of MV411 and MOLM13 cells, but not RAMOS cells (Figure 44). This demonstrates that CD33xMIC5 mediates uptake of GNE987 PROTAC into receptor-positive cells but prevents uptake into receptor-negative cells, and therefore, PAX was able to selectively deliver the PROTAC to targeted cells.

[0303] The cytotoxicity of the CD33xMIC5 construct can potentially be modulated by increasing or decreasing loading with the PROTAC, GNE987 (Figure 45). The cytotoxicity of CD33xMIC5 in combination with GNE987 against MV411 cells can be increased when loading is increased from 25% to 50% or even to 75%. This demonstrates that it is possible to tailor the cell-killing properties of PAX by adjusting the amount of PROTAC loaded as desired.

[0304] 7.11.6 Complexation of CD33xMIC5 with GNE987P can improve the cell-killing efficacy of the PROTAC GNE987P alone Previously, we found that PAX technology could be utilized to deliver the PROTAC GNE987 to target cells, but it was unclear whether it was also suitable for other PROTACs. Therefore, we selected another PROTAC, GNE987P, for further study and investigated whether PAX technology could also mediate selectivity for this PROTAC.

[0305] Therefore, CD33-positive MV411 and MOLM13 cells and CD33-negative RAMOS cells were treated with 25-75% loading of CD33xMIC5+GNE987P and the PROTAC GNE987P alone (Figure 46). For both CD33-positive cell lines, the CD33xMIC5+GNE987P combination was more potent than the PROTAC GNE987P alone. The potency of CD33xMIC5+GNE987P varied depending on the loading, with higher loadings resulting in greater efficacy. Overall, no toxicity was observed up to 150 nM for CD33-negative cells. We conclude that the concept of using antibody-conjugation to facilitate targeted delivery of a PROTAC may be more generalizable, as a second PROTAC was selectively delivered to CD33-expressing cells. Furthermore, we observed that this technique offers the potential to further improve the efficacy of certain PROTACs through targeted delivery to cells.

[0306] 7.11.7 CD33xMIC5 can be used to deliver FLT3 degraders to CD33-positive cells To extend the PAX approach to other intracellular targets, we investigated whether FLT3 degrading agents could be specifically delivered to CD33-expressing cells using antibody-PROTAC conjugates. Therefore, CD33-positive MOLM13 and CD33-negative RAMOS cells were treated with CD33xMIC5+FLT3d1 at 75% loading and with the PROTAC FLT3d1 alone as a control (Figure 47). Cytotoxicity was observed with CD33xMIC5+FLT3d1 in CD33-positive MOLM13 cells. No cytotoxicity was observed in CD33 receptor-negative RAMOS cells. This demonstrated the feasibility of using CD33xMIC5 to deliver FLT3 degrading agents to CD33-positive cells. The assay was performed according to the procedure described in Section 7.11.5 with the slight modification that cells were treated with either PROTAC or PAX for 6 days. The experiment again demonstrated that PAX can deliver PROTACs to target cells depending on their receptor status. Furthermore, the experiments demonstrated that the PAX technology could be transferred to another PROTAC, degrading FLT3, further supporting the versatility of the PAX approach.

[0307] 7.11.8 Complexation of the PROTAC GNE987 with CD33xMIC5 Can Be Achieved by Separate Application of the Antibody and PROTAC to Cells In another experiment, we investigated whether pre-complexing of CD33xMIC5 with the PROTAC GNE987 is necessary for cell target receptor-dependent cell killing. Therefore, CD33xMIC5 was complexed with GNE987 for 3 hours to reach 75% loading and added to CD33-positive MV411 and CD33-negative RAMOS cells. Furthermore, CD33xMIC5 was added to the cells, followed by separate addition of GNE987 to reach 75% loading. Interestingly, separate addition of CD33xMIC5 and GNE987 produced the same results as pre-complexed CD33xMIC5 + GNE987 PAX with the same loading (Figure 48).

[0308] These results demonstrated that precomplex formation of PAX is not a prerequisite for cell surface receptor-dependent cell killing by PAX.

[0309] 7.11.9 PROTAC loading of PAX can be increased by increasing the number of VHH PROTAC binders fused to the targeting antibody To increase the number of PROTACs that can be delivered into target cells by a single PAX molecule, the number of fused PROTAC-binding VHH units attached to IgG-targeting cells was increased. More specifically, different numbers of MIC7 PROTAC-binding VHHs were fused to the C-terminus of a CD33-targeting antibody on either the heavy chain, the light chain, or a combination of both. The antibody fragments are separated from the IgG heavy chain and any additional fragments by a short linker. For further details, see Section 7.9. We then investigated how genetic fusion of VHH fragments to various sites on the targeting antibody affects the efficacy of the resulting PAX.

[0310] VHH-antibody fusions complexed with GNE987 or GNE987P at the complexation ratios shown in Table 24 were examined in CD33-positive MV411 and CD33-negative RAMOS cells according to the cell viability assay procedure described in 7.11.5. In the case of combination with GNE987P, the same antibody was loaded with different ratios of GNE987P, and thus treatment concentrations were related to antibody concentration. All fusions demonstrated cell surface receptor-dependent cytotoxicity. Even some VHH-antibody fusions loaded with GNE987P showed enhanced potency in positive MV411 cells and reduced cytotoxicity in receptor-negative RAMOS cells compared to PROTAC alone. This further demonstrated the versatility of this approach to generate multiple PAXs with complex properties.

[0311] [Table 24]

[0312] Furthermore, EGFR and CD33-based VHH antibody fusions were complexed with GNE987 or GNE987P at different molar ratios and investigated in CD33-positive MV411 or CD33-negative RAMOS cells, or EGFR-positive A431 and MDAMB468, and EGFR-negative HEPG2 cells (Table 25 and Table 25 & Table 26, respectively). Different loadings are represented by the PROTAC-to-antibody ratio (PAR). Additionally, a non-targeting anti-digoxigenin-MIC7 fusion (DIGxMIC7) was used as an isotype control for cell proliferation assays.

[0313] Because the fusion proteins were loaded with various ratios of each PROTAC, treatment concentrations were related to antibody concentration. All EGFR-based fusions demonstrated receptor-dependent cytotoxicity. All EGFR-based fusions loaded with GNE987P demonstrated potency at least equivalent to that of the CD33xMIC7 variant loaded with one PROTAC. All variants were found to further enhance potency against receptor-positive cell lines compared to free PROTAC. Furthermore, higher PARs resulted in higher potency against receptor-positive A431 and MDAMB468 cells.

[0314] [Table 25]

[0315] [Table 26-1]

[0316] [Table 26-2]

[0317] 7.11.10 CLL1xMIC7-PROTAC Conjugates Induce Selective Cytotoxicity Dependent on CLL1-Mediated Uptake The scope of the present invention has so far been limited to CD33-targeting antibodies, and therefore we investigated whether it is possible to deliver PROTACs in a cell-selective manner using antibodies other than CD33-targeting antibodies.

[0318] To target PROTACs to cells expressing CLL1, a fusion protein was constructed using a CLL1-binding antibody and the PROTAC-binding clone MIC7. Therefore, the HC of the CLL1-binding antibody was extended at the C-terminus by a linker followed by the sequence of MIC7, resulting in CLL1xMIC7. Additionally, a digoxigenin antibody was modified in the same manner to obtain an isotype control. CLL1-positive MOLM13 and U937 cells and CLL1-negative K562 cells were treated with CLL1xMIC7+GNE987P or DIGxMIC7+GNE987P at 75% loading, or with the PROTAC GNE987P alone as a control. Assays were performed according to the procedure described above. Cytotoxicity was observed in both CLL1-positive cell lines with CLL1xMIC7+GNE987P, but not with DIGxMIC7+GNE987P (Figure 49). This again demonstrated that targeting PAX to the desired cells mediated uptake in receptor-positive cells, and further demonstrated that this technology could be applied to different antibody scaffolds, highlighting the versatility of this approach.

[0319] In additional experiments, CLL1-positive MV411 and U937 or CLL1-negative RAMOS and K562 cells were treated with CLL1xMIC7+GNE987, CLL1xMIC7+GNE987P, or CLL1xMIC7+SIM1 at 75% loading, or with GNE987, GNE987P, or SIM1 alone as controls. Assays were performed according to the procedures described above. Cytotoxicity was observed in both CLL1-positive cell lines with the CLL1xMIC7 PROTAC combination, but significantly less or no cytotoxicity was observed in CLL1-negative K562 and RAMOS cells (Figure 50). This again demonstrated that uptake into receptor-positive cells is mediated by targeting PAX to the desired cells. Furthermore, this demonstrated that this technology can be applied to different antibody scaffolds, confirming the versatility of the present invention. Furthermore, a total of three different PROTACs were delivered to CLL1-expressing cells using the PAX approach, demonstrating that the choice of PROTAC is highly flexible.

[0320] 7.11.11 B7H3xMIC7-PROTAC Conjugates Induce Selective Cytotoxicity Dependent on B7H3-Mediated Uptake To further broaden the scope of this invention, we investigated whether it is possible to deliver PROTACs in a cell-selective manner using B7H3-targeting antibodies. To target PROTACs to cells expressing B7H3, a fusion protein was constructed using a B7H3-binding antibody and the PROTAC-binding clone MIC7. Thus, the HC of the B7H3-binding antibody was extended at the C-terminus by a linker followed by the sequence of MIC7, resulting in B7H3xMIC7. B7H3-positive MV411, U937, and MOLM13 cells, as well as B7H3-negative RAMOS cells, were treated with B7H3xMIC7+GNE987P and B7H3xMIC7+SIM1 at 75% loading, and with the PROTACs GNE987P and SIM1 alone as controls. The assay was performed according to the procedure described above. Cytotoxicity was observed for all B7H3xMIC7 PROTAC combinations in all B7H3-positive cell lines (Figure 51). While no cytotoxicity was observed for any of the B7H3xMIC7 PROTAC combinations in B7H3 receptor-negative RAMOS cells, treatment with the PROTAC alone resulted in the highest, but not cell type-specific, cytotoxicity. This demonstrated that B7H3xMIC7 mediated the uptake of GNE987P and SIM1 into receptor-positive cells but prevented their uptake into receptor-negative cells. The experiment also demonstrated that PAX can deliver PROTACs to target cells depending on the receptor status. Furthermore, the experiment demonstrated that PAX technology may be transferable to other antibody scaffolds, further highlighting the versatility of this approach.

[0321] In a separate experiment, B7H3-positive MV411, U937, and MOLM13 cells and B7H3-negative RAMOS cells were treated with B7H3xMIC7+GNE987 or DIGxMIC7+GNE987 at 75% loading and the PROTAC GNE987 alone as a control. The assay was performed according to the procedure described above. Cytotoxicity was observed for all B7H3xMIC7+GNE987 constructs in all B7H3-positive cell lines, but significantly less toxicity was found for the isotype control DIGxMIC7+GNE987 (Figure 52). In B7H3 receptor-negative RAMOS cells, less cytotoxicity was found for B7H3xMIC7+GNE987 compared to PROTAC alone. This further demonstrated that B7H3xMIC7 mediated the uptake of GNE987P and SIM1 PROTACs into receptor-positive cells but prevented uptake into receptor-negative cells.

[0322] Whether targeted delivery of the BRD4 PROTAC GNE987P to the solid tumor cell line A2780, which expresses the receptor B7H3, is feasible using PAX with more VHHs remains to be investigated. Therefore, as described in Section 7.9, the B7H3 antibody Omburtamab was used to C-terminally fuse three copies of the MIC7 VHH to the heavy chain, creating B7H3xMIC7. [6H]Similarly, a digoxigenin antibody was modified to provide a non-binding control. Both antibody-based constructs were loaded with PROTAC GNE987P at a DAR of 4.5. PAX, unloaded antibody, and PROTAC GNE987P were tested on A2780 B7H3-expressing cells in a 3-day cell viability assay. Results can be seen in Figure 53. Treatment with the PROTAC-antibody conjugate B7H3xMIC7[6H]+GNE987P (DAR 4.5) resulted in efficacy in the subnanomolar range, comparable to that of free PROTAC, while the non-binding control strongly reduced cytotoxicity in the double-digit nanomolar range. This suggests that the effect was driven by delivery of the PROTAC to target cells, a conclusion that can be drawn since the antibody itself had no effect on cell viability up to 100 nM.

[0323] 7.11.12 EGFRxMIC7-PROTAC Conjugates Induce PROTAC Loading-Dependent and EGFR-Mediated Cell Type-Selective Cytotoxicity To target PROTACs to cells expressing EGFR, a fusion protein was constructed using the EGFR-binding antibody cetuximab and the PROTAC-binding clone MIC7. Thus, the HC of cetuximab was extended at the C-terminus by a linker followed by the sequence of MIC7, resulting in EGFRxMIC7. Additionally, a digoxigenin antibody was modified in the same manner to obtain an isotype control. Highly EGFR-expressing OVCAR3 and SKOV3 cells and lowly EGFR-expressing HEPG2 cells were treated with EGFRxMIC7 and DIGxMIC7 loaded with 50% PROTAC, GNE987, GNE987P, or SIM1, as described above, to determine the efficacy of these constructs (Table 28). The efficacy of the PROTACs alone is summarized in Table 27. While all DIGxMIC7 + PROTAC combinations had IC50s >100 nM, EGFRxMIC7 in combination with the PROTACs GNE987 and SIM1 induced cell death with IC50 values ​​in the single-digit nM range. The toxicity of EGFRxMIC7 in combination with all PROTACs in EGFR-low expressing HEPG2 was reduced for all constructs (double- to triple-digit nM range) compared to EGFR-high expressing OVCAR3 and SKOV3. Overall, the GNE987P combination was inactive. This experiment again demonstrated that PAX can deliver PROTACs to target cells depending on receptor status.

[0324] [Table 27]

[0325] [Table 28]

[0326] In a separate experiment, EGFRxMIC7 was separately loaded with the PROTACs ARV771, GNE987, GNE987P, and SIM1 at 75% loading to treat a variety of cells with varying EGFR expression levels (Table 29). Overall, the non-binding control DIGxMIC7 loaded with 75% GNE987, GNE987P, and SIM1 was not as potent as the EGFR-binding EGFRxMIC7 loaded with the same PROTACs.

[0327] [Table 29]

[0328] In conclusion, we demonstrated that PAX can also target PROTACs to solid tumor cells. Uptake was found to vary with EGFR receptor expression levels and, therefore, to be driven by active uptake via EGFR binding by the EGFRxMIC7+PROTAC complex, followed by internalization and PROTAC release. Cell selectivity driven by active EGFR-mediated uptake was also demonstrated by testing the non-binding isotype control PAX, which showed significantly less cytotoxicity.

[0329] 7.11.13 NAPI2BxMIC7 complexes with GNE987, GNE987P, and SIM1 exhibit cell-selective cytotoxicity To target PROTACs to cells expressing NAPI2B, a fusion protein was constructed using the NAPI2B-binding antibody XMT1535 and the PROTAC-binding clone MIC7. Thus, the HC of the NAPI2B-binding antibody was C-terminally extended with a linker followed by the sequence of MIC7, resulting in NAPI2BxMIC7. NAPI2B-positive OVCAR3 and NAPI2B-negative SKOV3 cells were treated with 50% loading of NAPI2BxMIC7+GNE987, NAPI2BxMIC7+GNE987P, NAPI2BxMIC7+SIM1, or DIGxMIC7+GNE987, DIGxMIC7+GNE987P, or DIGxMIC7+SIM1, as well as the PROTACs GNE987, GNE987P, and SIM1 alone as controls. The assay was performed according to the procedure described above. Cytotoxicity was observed for all NAPI2BxMIC7 PROTAC combinations in NAPI2B-positive OVCAR3 cells (Figure 54). While no cytotoxicity was found for all NAPI2BxMIC7 PROTAC combinations in NAPI2B receptor-negative SKOV3 cells, treatment with PROTAC alone resulted in the highest observed cytotoxicity across all cell lines, independent of NAPI2B receptor expression status. This demonstrated that NAPI2BxMIC7 mediated the uptake of GNE987, GNE987P, and SIM1 into receptor-positive cells but prevented their uptake into receptor-negative cells. This experiment also demonstrated that PAX can deliver PROTACs to target cells depending on receptor status. Furthermore, this experiment demonstrated that PAX technology may be transferable to other antibody scaffolds, further highlighting the versatility of this approach.

[0330] To further highlight the versatility of the invented technology, we investigated whether targeted delivery of BRD4 PROTAC to NAPI2B-expressing OVCAR3 and NCIH1437 cells is feasible, enabled by the active uptake of PAX, which binds to NAPI2B, allowing for the complexation of more PROTACs per antibody. Therefore, we used the NAPI2B antibody upifitamab as a scaffold and fused the MIC7 sequence C-terminally three times to the heavy chain of the antibody, as described in Section 7.9. This resulted in the construction of NAPI2BxMIC7. [6H] This resulted in an antibody fusion protein carrying a total of six VHHs per antibody, termed MIC7. Similarly, a digoxigenin-binding antibody (DIGxMIC7) was generated that does not bind to expressed surface antigens and therefore cannot bind to cells. [6H] ) was generated and used as a non-binding isotype control.

[0331] The antibodies were complexed with the BRD4 PROTAC GNE987 or GNE987P at DARs of 3 and 4.5, respectively, and tested for their efficacy in cell viability assays. The results of the cell viability assays on OVCAR3 and NCIH1437 cells are presented in Figure 55. Overall, NAPI2B-targeted PAX was found to be effective, with potency in the single-digit nanomolar range, while the non-binding isotype control PAX was found to be less potent than DIGxMIC7. [6H] +GNE987P DAR4.5 combination did not induce cytotoxic effects up to 100 nM, and DIGxMIC7 [6H] The +GNE987P DAR4.5 combination had an IC50 of 36 nM, yet was 22-fold less potent than the NAPI2B-binding NAPI2BxMIC7[6H]+GNE987P DAR4.5 complex (Figure 55).

[0332] Overall, in the case of GNE987, the potency of PAX was found to be similar to that of the PROTAC alone. In the case of GNE987P, the potency of NAPI2BxMIC7[6H]+GNE987P DAR4.5 was improved by 19-fold in OVCAR3 cells and 3-fold in NCIH1437 cells. Furthermore, unloaded NAPI2BxMIC7 [6H] The antibody, DIGxMIC7 [6H] The antibody also had no effect on cell viability up to 100 nM, demonstrating that cellular efficacy was dependent on the PROTAC, not the antibody. The data clearly demonstrated that complexing a PROTAC with an antibody bearing the MIC7 can improve the cellular cell-killing efficacy of the PROTAC.

[0333] 7.11.14 HER2xMIC7 in combination with the PROTACs GNE987, GNE987P, and SIM1 demonstrates HER2-dependent cytotoxicity To target PROTACs to HER2-expressing cells, a fusion protein was constructed using the HER2-binding antibody trastuzumab and the PROTAC-binding clone MIC7. Thus, the HC of the HER2-binding antibody was extended at the C-terminus by a linker followed by the sequence of MIC7, resulting in HER2xMIC7. HER2-positive SKBR3 and NCIN87 cells and HER2-negative MDAMB468 cells were treated with 75% loading of HER2xMIC7+GNE987, HER2BxMIC7+GNE987P, HER2xMIC7+SIM1, and the PROTACs GNE987, GNE987P, and SIM1 alone as controls. The assay was performed according to the procedure described above. Cytotoxicity was observed for all HER2xMIC7 PROTAC combinations in HER2-positive SKBR3 and NCIN87 cells (Table 30). While no cytotoxicity was observed for any of the HER2xMIC7 PROTAC combinations in HER2 receptor-negative MDAMB468 cells, treatment with the PROTAC alone resulted in clear cytotoxicity in all cell lines, independent of HER2 receptor expression status. This demonstrated that HER2xMIC7 mediated the uptake of GNE987, GNE987P, and SIM1 into receptor-positive cells but prevented their uptake into receptor-negative cells. This experiment also demonstrated that PAX can deliver PROTACs to target cells depending on receptor expression status. Furthermore, the experiment demonstrated the potential for transferring PAX technology to other antibody scaffolds, further highlighting the versatility of this approach.

[0334] [Table 30]

[0335] 7.11.15 TROP2-based PAX mediates TROP2-dependent cytotoxicity To further broaden the scope of this invention, we investigated whether it is possible to deliver PROTACs in a cell-selective manner using a TROP2-targeting antibody. To target PROTACs to cells expressing TROP2, a fusion protein was constructed using the TROP2-binding antibody sacituzumab and the PROTAC-binding clone MIC7. Thus, the HC of the TROP2-binding antibody was extended at the C-terminus by a linker followed by the sequence of MIC7, resulting in TROP2xMIC7. TROP2-positive A431, SKBR3, MDAMB468, NCIN87, and SKOV3 cells and TROP2-negative SW620 cells were treated with TROP2xMIC7+GNE987 at 75% loading or with the PROTAC GNE987 alone as a control. The assay was performed according to the procedure described in Section 7.11.5. Cytotoxicity was observed for TROP2xMIC7+GNE987 in all TROP2-positive A431, SKBR3, MDAMB468, NCIN87, and SKOV3 cells (Table 31). Reduced cytotoxicity was found in TROP2-negative SW620 cells for TROP2xMIC7+GNE987 compared to GNE987 alone. These results demonstrated that TROP2xMIC7 mediated GNE987 uptake into receptor-positive cells, but reduced its uptake into receptor-negative cells. This experiment also demonstrated that PAX can deliver PROTACs to target cells depending on receptor expression status. Furthermore, this experiment demonstrated that PAX technology may be transferable to other antibody scaffolds, further highlighting the versatility of this approach.

[0336] [Table 31]

[0337] Furthermore, different molecular forms of PAX were investigated for their cytotoxic effects on cells in vitro. Thus, the anti-TROP2 antibody sacituzumab was engineered with three MIC7 VHHs per HC, as described in Section 7.9, to generate TROP2xMIC7 VHHs capable of binding up to six PROTACs.[6H] The molecules were investigated in TROP2-expressing cells to study the selectivity of the cytotoxic effect. Therefore, A431 and SNU840 cells were treated with the TROP2-targeting TROP2xMIC loaded with GNE987 and GNE987P at different DARs. [6H] and the non-binding isotype control DIGxMIC7 [6H] (Figure 56 and Table 32). Additionally, cells were tested with PROTACs GNE987 and GNE987P alone, as well as the antibody without the PROTAC to account for the effects driven by the antibody alone.

[0338] TROP2xMIC7 [2H] +GNE987 (DAR1.5) and TROP2xMIC7 [6H] +GNE987 (DAR 4.5) potently inhibited the proliferation of SNU840 cells, while the non-binding control DIGxMIC7 [2H] +GNE987(DAR1.5) and DIGxMIC7 [6H] +GNE987 (DAR 4.5) had little effect on cancer cell viability, indicating PAX-mediated TROP2 expression-dependent cell killing. Identical results were observed when the antibody was loaded with PROTAC GNE987P.

[0339] In the case of GNE987P, PROTAC GNE987P TROP2xMIC7 [6H] It was observed that the potency could be improved from 1.17E-08 M to 1.29E-10 M by conjugation to the antibody TROP2xMIC7. [2H] , TROP2xMIC7 [6H] , DIGxMIC7 [2H] and DIGxMIC7 [6H] Treatment with either alone had no effect on cell viability. A similar picture was obtained when the same molecules were tested on A431 cells.

[0340] Overall, conjugation of PROTACs with antibodies significantly improved their potency, and the cytotoxic effect was dependent on receptor binding ability.

[0341] [Table 32]

[0342] 7.11.16 PSMAxMIC7 PSMA-Dependent Cytotoxicity in Complex with GNE987, GNE987P, and SIM1 The heavy chain of the prostate-specific membrane antigen (PSMA)-binding antibody MLN591 antibody is extended at the C-terminus with a linker followed by the sequence of MIC7, PSMAxMIC7 [2H] Additionally, a digoxigenin antibody was modified in the same manner to obtain a non-binding isotype control, resulting in DIGxMIC7. [2H] The resulting fusion proteins were complexed with the BRD4-degrading PROTAC GNE987 or GNE987P at DARs of 1 and 1.5. Their PAX was studied in PSMA-expressing LNCAP cells. To assess the selectivity of the constructs, a non-binding isotype control (DIGxMIC7) complexed with the same PROTAC and DAR was used. [2H] ) was investigated. PROTAC alone was used as a positive control. Overall, PAX exhibited potency in the subnanomolar range for both PROTACs, which was comparable to the potency of the PROTAC alone. At the same time, the potency of the non-binding control PAX was low in the single- to double-digit nanomolar range. The data can be found in Table 33.

[0343] [Table 33]

[0344] Furthermore, a construct capable of carrying up to six PROTACs was created by fusing a total of six VHHs (three per chain, all linked via glycine-serine linkers) to the heavy chain of an anti-PSMA antibody. Construct PSMAxMIC7[6H] The non-binding control antibody DIGxMIC7 was also loaded with PROTAC GNE987P or SIM1 at a DAR of 4.5. [6H] The constructs were tested alongside free PROTAC in PSMA-expressing LNCAP cells. The potency of PROTAC GNE987P was 3.5 nM, and PAX PSMAxMIC7 [6H] +GNE987P (DAR4.5) has an IC50 of 5.5E-11M, which can be improved 63-fold by targeted delivery. At the same time, the non-binding control had only a weak antiproliferative effect at the highest tested concentration of 100 nM. The results can be seen in Figure 57. The results clearly demonstrate the feasibility of using PSMA-targeted PAX for targeted PROTAC delivery.

[0345] 7.11.17 cMETxMIC7+GNE987 / GNE987P mediates cMET-dependent cytotoxicity We then tested whether targeted delivery of the BRD4 PROTAC to EBC1 cells via PAX-mediated active delivery and uptake via cMET receptor binding was feasible. Therefore, the cMET antibody telisotuzumab was extended at the C-terminus with a linker followed by the sequence of MIC7, resulting in cMETxMIC7. [2H] Additionally, a digoxigenin antibody was modified in the same manner to obtain a non-binding isotype control, resulting in DIGxMIC7. [2H] The antibodies were loaded with BRD4 PROTACs GNE987 or GNE987P at a DAR of 1 (GNE987 or GNE987P) and a DAR of 1.5 (GNE987P). Additionally, as a negative control, a non-binding isotype control antibody (DIGxMIC7 [2H]) were loaded with the same PROTAC at the same DAR. PROTAC alone served as a positive control. The results of the cell viability assay on EBC1 cells are presented in Figure 58. Overall, cMET-targeted PAX was found to be more potent in the single-digit nanomolar concentration range and more effective than the non-binding control PAX, which did not induce cytotoxic effects up to 100 nM. Furthermore, in the case of GNE987P, higher efficacy of PAX was found compared to free PROTAC for both DARs. The results clearly demonstrate the feasibility of using cMET-targeted PAX for targeted PROTAC delivery.

[0346] 7.11.18 CLDN18.2xMIC7-PROTAC Conjugates Induce PROTAC Loading-Dependent and CLDN18.2-Engineered Cell Type-Selective Cytotoxicity Furthermore, we investigated the cellular effects that could be induced by delivering a BRD4-targeting PROTAC into cells via a claudin-18 (CLDN18.2)-targeting antibody. Therefore, three copies of the MIC7 antibody were C-terminally fused to the heavy chain of a CLDN18.2-binding antibody using the linkage described in Section 7.9. The resulting fusion protein, CLDN18.2xMIC7, [6H] was complexed with either 1.5 or 3 GNE987 PROTACs. Additionally, the non-binding isotype control antibody DIGxMIC7 [6H] Additionally, the same antibody constructs were complexed with 3 or 4.5 SIM1 or GNE987P PROTACs per antibody.

[0347] PAX was studied in comparison to the PROTAC GNE987 alone in BXPC3 cells recombinantly engineered to express high levels of CLDN18.2 (Figure 59). PAX relied on receptor-mediated delivery to induce cell killing, as CLDN18.2-targeted PAX exhibited IC50 values ​​in the single- to double-digit nanomolar range, whereas nonbinding DIG-targeted PAX was not found to be effective up to 100 nM. Furthermore, the potency of CLDN18.2 PAX was found to be DAR-dependent for all three PROTACs tested. In the case of GNE987P, active uptake into cells mediated by the CLDN18.2 antibody further enhanced the potency of the PROTAC by 9.6-fold for a DAR of 4.5.

[0348] To exclude that the additional cell killing effect was mediated by the antibody, antibodies without a PROTAC were also tested, but as expected, these did not induce any cellular cytotoxicity up to 100 nM.

[0349] This data clearly demonstrated that delivery of multiple PROTACs to BXPC3 engineered to express CLDN18.2 on its surface via CLDN18.2-targeted PAX is feasible and that cell killing efficacy can be tuned by the applied DAR of the antibody-PROTAC conjugate.

[0350] 7.11.19 CD33xMIC7[N-2H]+GNE987P induces cytotoxic effects dependent on receptor expression Next, the effect of fusing the MIC7 VHH to the heavy chain at the N-terminus instead of the C-terminus was investigated, utilizing the same linker defined in section 7.9. For N-terminally conjugated VHHs, an additional "N-" was introduced in the nomenclature. This resulted in CD33xMIC7 [N-2H] To exclude any non-specific uptake effects mediated by the novel molecular structure, a non-binding antibody with the same fusion was generated (DIGxMIC7 [N-2H]Both antibodies were loaded with GNE987P at a DAR of 1 to form PAX. PAX and unloaded antibodies and PROTAC GNE987P alone were tested on MV411 cells expressing CD33. CD33xMIC7 [N-2H] +GNE987P(DAR1)PAX efficiently killed MV411 cells with a subnanomolar IC50, which was comparable to that of the free PROTAC GNE987P, whereas the unbound PAX DIGxMIC7 [N-2H] +GNE987P(DAR1) had only a weak anti-proliferative effect in the double-digit nanomolar concentration range. Antibody alone without PROTAC loading had no effect on cell viability up to 100 nM. The results are presented in Figure 60.

[0351] This data clearly demonstrated the possibility of attaching the MIC7 VHH to various locations while maintaining the ability to generate functional PAX capable of selectively delivering PROTACs to cells, further showcasing the versatility of the PAX approach.

[0352] 7.11.20 SEED PAX Cell Profiling Using SEED technology, we isolated EGFR with either two MIC7 VHHs attached to the normal heavy chain or only one MIC7 VHH attached to one heavy chain (EGFRxMIC7 [S2H] and EGFRxMIC7 [S1H] ) were generated. At the same time, the same technology was applied to generate antibodies that retain one or two MIC7 VHHs but have only one EGFR-binding arm (oaEGFRxMIC7 [S1H] and oaEGFRxMIC7 [S1H] ) were generated. These formats are called SEED PAX. They utilized the same linker defined above.

[0353] Antibody constructs were loaded with 0.5 or 1.0 of the PROTAC GNE987 when the antibodies carried one or two VHHs, respectively. The constructs were investigated in EGFR-expressing A431 tumor cells. The potency of all constructs was similar in the double-digit nanomolar concentration range, but the efficacy of PAX loaded with 0.5 PROTAC was reduced compared to the efficacy of a construct carrying 1.0 PROTAC per antibody. The results are presented in Figure 61 (top). Furthermore, we investigated whether the antibody itself had an antiproliferative effect. Overall, only minimal antibody-mediated antiproliferative effects on A431 cells were observed. At the highest dose tested (100 nM), the bivalent antibody induced a 25% reduction in cell viability (Figure 61 (bottom)). In conclusion, the activity of PAX was found to be significantly more potent than that of the antibody alone, suggesting that the observed cell-killing effect is mediated by antibody-mediated delivery of the PROTAC to targeted cells. This further broadens the applicability of the invented PAX technology to SEED-based antibodies and highlights the possibility of applying it to multispecific binding antibodies for cell surface antigens.

[0354] 7.11.21 Cellular Profiling of Cathepsin B-Cleaved PAX Furthermore, the effect of fusing a MIC7 VHH to the C-terminus of the heavy chain using a cathepsin B cleavable linker as defined in section 7.9 was investigated. For cathepsin B cleavable antibodies, an additional "CL" was introduced in the nomenclature. The nomenclature for these antibodies is as follows: A TROP2-targeting antibody in which two VHHs are attached to the C-terminus of the heavy chain via the cathepsin B cleavable linker described above is designated TROP2xMIC7 [2H_CL] To exclude any nonspecific uptake effects mediated by the novel molecular structure, a non-binding antibody with the same fusion was generated (DIGxMIC7 [2H_CL]Both antibodies were loaded with PROTAC GNE987, GNE987P, or SIM1 at a DAR of 1.5 to form PAX. PAX, unloaded antibodies, and PROTACs alone were tested in TROP2-positive SNU840 and TROP2-negative SW620 cells. All TROP2xMIC7 antibodies at a DAR of 1.5 [2H_CL] PAX-based PAX potently inhibited the proliferation of SNU840 cells, while the non-binding control DIGxMIC7 at DAR 1.5 was significantly lower than that of the control. [2H_CL] Antibody TROP2xMIC7 had little effect on cancer cell viability, indicating TROP2 expression-dependent cell killing mediated by PAX (Figure 64). [2H_CL] , and DIGxMIC7 [2H_CL] Treatment with either alone had no effect on cell viability. Furthermore, in the case of GNE987P, efficacy was significantly higher than that of the TROP2xMIC7 PROTAC GNE987P. [2H_CL] It was observed that the potency could be improved 66-fold from 9.04E-09 M to 1.35E-10 M by complexation with PROTAC SIM1. [2H_CL] It was observed that conjugation to an antibody could improve the potency of PROTACs by a factor of 5, from 6.29E-10 M to 1.37E-10 M. Overall, conjugation of PROTACs to antibodies matched or even significantly improved the potency of PROTACs alone against receptor-positive cells, and the cytotoxic effect was found to be dependent on receptor binding affinity.

[0355] 7.11.22 cPAX Cell Profiling To further demonstrate the modular approach of utilizing VHH fragments for antibody-mediated targeted delivery of PROTACs via PAX, we investigated the enzymatic bioconjugation of PROTAC-binding VHHs to an IgG backbone using microbial transglutaminase (MTG) to generate bispecific antibodies for the PAX approach. Conjugated bispecific antibody production, complex formation to generate conjugated PAX (cPAX), and cell experiments were performed as described in Section 7.9. For cell viability assays, CTG readouts were performed after 6 days of treatment. EGFR receptor-positive A431 and MDAMD468 cells were treated with EGFR-targeted cPAX (DAR1), isotype control cPAX (DAR1), or GNE987P alone. Overall, cPAX displayed a 10- to 100-fold increased potency compared to treatment with PROTAC alone (Table 34, Figure 65). At the same time, the potency of the non-binding control PAX was reduced by two orders of magnitude in the nanomolar to micromolar range. This clearly demonstrated that conjugated bispecific antibodies based on PROTAC-binding VHHs can be used for cell-specific targeted delivery in the context of PAX.

[0356] [Table 34]

[0357] 7.11.23 Comparable Cytotoxicity of PAX and PROTAC-ADC To understand the extent to which PAX technology is comparable to covalently linked PROTAC-ADCs, the EGFR-IgG1-L328C-GNE987 PROTAC-ADC (DAR 1.62) described in Section 7.5.1 was investigated alongside EGFRxMIC5 loaded with 50% GNE987 in EGFR-positive MDAMB468 and EGFR-negative HEPG2 cells. For better comparison, treatment concentrations were correlated with antibody concentration. It was observed that both constructs had identical potency in MDAMB468 cells and exhibited a reduced but comparable cytotoxic effect on HEPG2 cells (Figure 66). This demonstrated that, although the PROTAC is only non-covalently associated, PAX can enable selective cell killing comparable to that of a covalently linked PROTAC-ADC. Furthermore, this effect can be achieved at a lower DAR compared to the PROTAC-ADC.

[0358] 7.11.24 STEAP1xMIC7-PROTAC conjugates induce STEAP1-mediated cell type-selective cytotoxicity We investigated further cellular effects that could be induced by delivering a BRD4-targeting PROTAC into cells via the six transmembrane epithelial antigens of the prostate (STEAP1)-targeting antibody. Therefore, using the linkage described in Section 7.9, one copy of the MIC7 antibody was C-terminally fused to the heavy chain of the STEAP1-binding antibody MSTP2109A. The resulting fusion protein, STEAP1xMIC7 [2H] was complexed with either GNE987 or GNE987P at a 1:1 or 1:1.5 ratio, respectively. Additionally, the non-binding isotype control antibody DIGxMIC7 [2H]The resulting PAX was compared to PROTACs GNE987 and GNE987P alone in PC3 cells recombinantly modified to express high levels of STEAP1 and in unmodified PC3 cells that do not express STEAP1 as a negative cell line (Table 35). PAX relied on receptor-mediated delivery to induce cell killing, as STEAP1-targeted PAX exhibited IC50 values ​​in the single- to double-digit nanomolar range, whereas nonbinding DIG-targeted PAX was not found to be effective up to 100 nM. To exclude that the additional cell killing effect was mediated by the antibody, antibodies without a PROTAC were also tested. However, as expected, these did not induce any cellular cytotoxicity up to 100 nM.

[0359] [Table 35]

[0360] The data clearly demonstrated that delivery of PROTACs via STEAP1-targeted PAX to PC3 cells engineered to express STEAP1 on their surface is feasible, further highlighting the modular nature of the PAX approach.

[0361] 7.12 Conjugation of PROTACs with anti-PROTAC antibodies can significantly improve their pharmacokinetic profiles We investigated whether and to what extent complexation of the PROTAC GNE987 with the PROTAC-binding antibody MIC2 affects the overall pharmacokinetic profile of the PROTAC. Therefore, a pharmacokinetic study was performed as follows.

[0362] Female SCID beige mice (n = 9, combined profile) received a single tail vein intravenous (iv) bolus injection of 0.4 mg / kg PROTAC alone (GNE987) in 2% (v / v) DMSO / 20% (v / v) (hydroxypropyl β-cyclodextrin) kleptose in water at a dose volume of 5 mL / kg. For MIC2 + GNE987 (1:2 antibody-drug ratio), female SCID beige mice (n = 12, combined profile) received a single tail vein intravenous (iv) bolus injection of the PROTAC shuttle at an equivalent dose of 0.4 mg / kg GNE987 and 30 mg / kg MIC2 in 5% (v / v) DMSO in PBS at a dose volume of 5 mL / kg.

[0363] For PROTAC alone, under sublingual isoflurane anesthesia, serial blood samples (n=3) were collected at 0.1 (Group 1), 0.5 (Group 2), 1 (Group 3), 2 (Group 1), 4 (Group 3), 6 (Group 2), and 24 hours (Group 3) after iv administration using ethylenediaminetetraacetic acid (K3-EDTA) as an anticoagulant and further processed to obtain plasma.

[0364] For MIC2+GNE987, serial blood samples (n=3) were collected at 0.1 (G(group)1), 0.5 (G2), 1 (G3), 2 (G4), 6 (G2), 24 h (G1, G3), 30 (G3), 48 (G3, G4), 72 (G1, G2), and 96 h (G2) after iv administration as described above and further processed to obtain plasma.

[0365] For sample preparation, 10 μL of plasma was diluted with 10 μL of methanol in a LowBind (protein) plate and precipitated with 80 μL of acetonitrile containing labetalol s internal standard (2.5 μg / mL). After 1 min of shaking / vortexing, the sample was filtered (polypropylene filter, 0.45 μm pore size, Captiva filtration), and 120 μL of methanol:water (1:1, v / v) was added to the filtrate. The sample was stored at 4°C until analysis and placed in the autosampler prior to injection. Analysis was performed on an LC-MS / MS system consisting of a UPLC coupled to a QTRAP 6500+ (Sciex) mass spectrometer. Mobile phase A was water with 0.1% formic acid, and mobile phase B was methanol with 0.1% formic acid. The gradient started at 10% B, reached 95% B in 1.5 min, and held at 95% B for 2 min. Then, it decreased to 10% B in 0.5 min and held at 10% B for 2 min. Chromatography was performed on an Agilent Technologies Poroshell 120 EC-C18 column, 2.7 μm particles, 3 × 50 mm. The flow rate was 0.6 mL / min, and the cycle time (injection to injection) was approximately 6 min. The sample injection volume was 10 μL. The MRM transitions for GNE987 were 548.788 (m / z, z=2) → 779.2 (m / z, z=1) and 329.101 (m / z, z=1) → 91 (m / z, z=1) for labetalol (IS). The calibration curve for quantification was based on standards ranging from 0.5 (lower limit of quantification) to 10,000 (upper limit of quantification) ng / mL, had a minimum of five calibration points, and ensured that a minimum of 75% of the calibration standards were within ±20% of their nominal value.

[0366] Total antibody concentrations were determined by a ligand binding assay (LBA) based on Mesoscale Diagnostics Technology (MSD, LLC., Rockville, MD). All incubation steps were performed at 22°C with gentle agitation. All washing steps (200 μL / well) were performed with PBS-T containing PBS pH 7.4 and 0.01% Tween 20 using a plate washer ELx405 (BioTek Instruments Inc., Winooski, VT). First, 2.5 μg / mL biotin-SP-conjugated AffiniPure goat anti-human IgG, Fcγ fragment-specific (Jackson ImmunoResearch Europe Ltd., JIR, Cambridgeshire, UK, #109-065-098) was coated onto an MSD GOLD 96-well streptavidin QUICKPLEX plate (MSD, #L55SA) for 2 hours. The plate was then washed three times. Plasma samples, standards, and quality controls were serially diluted in a dilution buffer consisting of PBS pH 7.4, 0.05% Tween 20, and 3.0% (w / v) BSA and incubated on the plate for 1 hour. The plate was washed again and incubated for 1 hour with 0.6 μg / mL mouse anti-human IgG, F(ab')2 fragment specific (JIR, #209-005-097) pre-labeled with MSD GOLG SULFO-tag (MSD, #R31AA-1) according to the manufacturer's instructions. After a final washing step, 150 μL of 2x MSD Read Buffer T with surfactant (MSD, #R92TC) was added to each well, and the plate was read on a MESO Quickplex SQ120 plate reader (MSD). The total mAb concentrations in plasma samples were calculated using the software Watson LIMS (version 7.5, ThermoFisher Scientific Inc.) by fitting the standard curve with the 5PL (Marquart) equation and a weighting factor of 1 / Y2. The lower limit of quantification (LLOQ) was 50 ng / mL.

[0367] The half-life of PROTAC GNE987 was determined to be 5.8 hours, which is within the same range as the half-life reported in the literature (2.8 hours, Pillow, TH et al., ChemMedChem 15 (2020) 17-25). Complexation of PROTAC GNE987 with MIC2 led to a half-life of PROTAC GNE987 of 14.7 hours in mice, which corresponds to a 2.5-fold half-life improvement.

[0368] Additionally, PAX CD33xMIC5+GNE987 and CD33xMIC7+GNE987 with 100% theoretical loading were generated and investigated in a PK study conducted in C57BL / 6N inbred mice (N = 2 males and 2 females per group) provided by Charles River Laboratories Italia, Calco, Italy. Seven- to eight-week-old mice received a single dose of 30 mg / kg (equivalent to 0.38 mg / kg of PROTAC) of CD33xMIC5+GNE987 and CD33xMIC7+GNE987, CD33 antibody alone, CD33xMIC5, or CD33xMIC7 intravenously injected into the tail vein. Samples were serially collected from all animals using microsampling techniques (20 mL for each blood draw). Two blood samples were collected on day 1 after administration, followed by seven over the following three weeks. Each sample was collected in a pre-chilled (0–4°C) Minivette POCT EDTA tube, transferred to a Microvette CB300 EDTA tube, and centrifuged at 2500 × g for 10 min at 4°C. The resulting plasma was transferred to a new vial and immediately stored at -80°C until further analysis. PK studies, animal handling, and experimental methods were performed in accordance with the Italian Directive 2014 / 26 and Directive 2010 / 63 / EU. The study was conducted at the Instituto di Ricerche Biomediche Antoine Marxer, Colleretto Giacosa, Italy. This laboratory is fully accredited by the Italian Ministry of Health.

[0369] Total antibody concentrations were determined by a ligand binding assay (LBA) based on Mesoscale Diagnostics Technology (MSD, LLC., Rockville, MD). All incubation steps were performed at 22°C with gentle agitation. All washing steps (200 μL / well) were performed with PBS-T containing PBS pH 7.4 and 0.01% Tween 20 using a plate washer ELx405 (BioTek Instruments Inc., Winooski, VT). First, 2.5 μg / mL biotin-SP-conjugated AffiniPure goat anti-human IgG, Fcγ fragment-specific (Jackson ImmunoResearch Europe Ltd., JIR, Cambridgeshire, UK, #109-065-098) was coated onto an MSD GOLD 96-well streptavidin QUICKPLEX plate (MSD, #L55SA) for 2 hours. The plate was then washed three times. Plasma samples, standards, and quality controls were serially diluted in a dilution buffer consisting of PBS pH 7.4, 0.05% Tween 20, and 3.0% (w / v) BSA and incubated on the plate for 1 hour. The plate was washed again and incubated for 1 hour with 0.6 μg / mL mouse anti-human IgG, F(ab')2 fragment specific (JIR, #209-005-097) pre-labeled with an MSD GOLG SULFO-tag (MSD, #R31AA-1) according to the manufacturer's instructions. After a final wash step, 150 μL of 2x MSD Read Buffer T with surfactant (MSD, #R92TC) was added to each well, and the plate was read on a MESO Quickplex SQ120 plate reader (MSD). The total mAb concentrations in plasma samples were calculated using the software Watson LIMS (version 7.5, ThermoFisher Scientific Inc.) by fitting the standard curve with the 5PL (Marquart) equation and a weighting factor of 1 / Y2. The lower limit of quantification (LLOQ) was 50 ng / mL.

[0370] The concentration of MSC2734242 was determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using a SCIEX 5500 triple quadrupole equipped with a Turbo ion spray source (ITS) in positive mode (SCIEX, Redwood City, CA, USA). Chromatographic separation was achieved using a Waters ACQUITY UPLC BEH (C18, 2.1 × 50 mm, 1.7 μm) column mounted on a Waters ACQUITY I-Class UPLC system (Milford, MA, USA) configured with a 100 μL extended loop. At a flow rate of 0.350 mL / min, the chromatographic gradient used for phases A (HO:ACN 95:5, 0.1% formic acid) and B (ACN:HO 95:5, 0.1% formic acid) was an isocratic gradient of 100% A in 0.25 min, followed by a 2.25 min gradient to 100% B and a subsequent 0.75 min wash step at 100% B and a 2.5 min readjustment at initial conditions.

[0371] Extraction of MSC2734242 from C57BL / 6N mouse plasma samples was performed by protein precipitation. On a Phenomenex Impact protein precipitation plate (Phenomenex, Torrance, CA, USA, CE0-7565), 3 μL of plasma sample was precipitated in 100 μL of acetonitrile containing 50 ng / mL of MSC2737500, used as an internal standard. After 5 minutes of shaking (900 rpm), all wells were filtered by vacuum and collected into a clean 96-well plate. The plate was then diluted with 100 μL of an aqueous solution containing 2.5% formic acid and submitted for LC-MS / MS analysis. All reagents were LC-MS grade or equivalent.

[0372] The total MSC2734242 concentration in plasma samples was calculated by fitting the standard curve with linear regression and area ratio (analyte signal / internal standard signal) with a weighting factor of 1 / X2 using the software Watson LIMS (version 7.5, ThermoFisher Scientific Inc.). The lower limit of quantification (LLOQ) was 5 ng / mL, and the full range of quantification was 5–2000 ng / mL.

[0373] Primary PK parameters were estimated by non-compartmental analysis (NCA) using Phoenix WinNonlin version 8.3.4 (Pharsight Corporation, USA). Pharmacokinetic parameters were obtained or calculated from the individual plasma concentrations of total antibody and MSC2734242 analytes versus time post-dose.

[0374] Individual plasma concentration-time profiles were used for parameter estimation. Concentrations of all PK samples calculated below the limit of quantification (BQL) were considered missing values ​​to better estimate AUC, clearance, and volume of distribution. Terminal half-life (t) and λ (the first-order rate constant associated with the terminal log-linear portion of the curve) values ​​were calculated only if at least three time points were quantifiable in the terminal phase of the linear regression. Values ​​below the BQL were considered 0 ng / mL for descriptive statistics. Overall, complexation of GNE987 with either CD33xMIC5 or CD33xMIC7 led to significantly longer half-lives of the PROTACs in mice, 29.6 and 105 h, respectively (Figure 67). Impressively, GNE987 concentrations were still on average 69 ng / mL in mouse plasma even after 21 days (504 hours) in the group receiving CD33xMIC7+GNE987, but PROTAC GNE987 concentrations had already fallen below the LLOQ after 25 hours. This result illustrates that antibody-conjugation can potently increase tumor cell exposure to PROTACs, for example, by complexing the PROTAC with an antibody.

[0375] Antibody clearance in PK studies was compared to determine whether the generation of VHH fusions and the loading of each fusion protein with the PROTAC GNE987 altered the clearance rate (Figure 68). The clearance of the unmodified CD33 antibody and its fusion with the VHL-PROTAC-binding VHHs MIC5 and MIC7 (CD33xMIC5 / 7) was similar, suggesting a minimal effect of the VHH fusion on clearance. Furthermore, loading the antibody-VHH fusion proteins CD33xMIC5 and CD33xMIC7 with the PROTAC GNE987 did not affect antibody clearance. In conclusion, the addition of VHL-PROTAC-binding VHHs and complex formation with the PROTAC did not affect the PK profile.

[0376] The pharmacokinetic parameters of the latter study are summarized in Table 36.

[0377] [Table 36]

[0378] Additionally, up to four PROTACs, CD33xMIC7 [4H] , CD33xMIC7 [4L] and CD33xMIC7 [2H2L] Studies were conducted on antibodies capable of retaining the MIC7-dependent pharmacokinetic (PK) profile. Therefore, PROTAC GNE987 was used to generate PAX with 50 or 100% theoretical loading, resulting in DAR2 or DAR4. PK studies were performed in C57BL / 6N inbred mice (N=9 females per group) provided by Charles River Laboratories Italia, Calco, Italy. 9-11 week old mice were administered 30 mg / kg (equivalent to 0.76 or 0.38 mg / kg PROTAC, respectively) of CD33xMIC7. [4H] , CD33xMIC7 [4L] and CD33xMIC7 [2H2L] was given as a single dose, which was injected intravenously into the tail vein.

[0379] Blood was collected sublingually under inhalation anesthesia (isoflurane). Animals were not fasted before blood sampling. At 0.1, 3, 6, 24, 72, 168, 336, 480, 600, 720, 840, and 960 hours, 50 μl of blood was collected and placed in pre-cooled (approximately 4°C) EDTA tubes (Microvette® CB 300 K2E Sarstedt AG, Numbrecht). After blood collection, the blood samples were kept refrigerated and centrifuged (2500 g / 10 min at 4°C) within 30 minutes. Two plasma aliquots (approximately 12 μL each) were prepared and immediately stored at -80°C. After the final blood sampling for each designated animal, the animals were sacrificed anesthetized using isoflurane followed by carbon dioxide anesthesia. PK studies, animal handling and experimental procedures were performed in accordance with the German Animal Protection Act (Article 8a).

[0380] Plasma samples were analyzed as described above. Biochemical analysis measurements of each antibody and pharmacokinetic evaluation of the antibodies were performed at the Istituto di Ricerche Biomediche "Antoine Marxer" - RBM SpA1, Via Ribes, 1, 10010 - Colleretto Giacosa (TO), Italy. Plasma samples were analyzed by using a specialized method for quantification of PROTAC GNE987 in mice. The results of this study can be found in Table 37.

[0381] [Table 37]

[0382] Doubling the antibody-complexed PROTAC resulted in a longer half-life of the PROTAC, as can be seen by the data for all PAX containing four VHHs and loaded with four PROTACs, and CD33xMIC7 containing two VHHs. [2H]The PAX containing four VHHs and loaded with two PROTACs had half-lives of 134-159 hours compared to 105 hours for CD33xMIC7. Interestingly, the half-lives of the PAX containing four VHHs and loaded with two PROTACs were also found to be extended for some formats (95-156 hours). All PAX containing four VHHs and loaded with four PROTACs were significantly more potent than CD33xMIC7. [2H] (AUC 0-inf =231000h*ng / mL), demonstrating comparable or even enhanced exposure (AUC 0-inf =181000~546000h*ng / mL). The most promising molecule is CD33xMIC7 [4H] +GNE987(DAR4), which is CD33xMIC7 [2H] +GNE987(DAR2). Attaching more VHHs per antibody as well as loading two or four PROTACs has minimal effect on antibody PK properties. This clearly demonstrates that PAX with a higher number of VHHs is superior in its pharmacokinetic properties compared to PAX with only two VHHs per antibody, demonstrating that CD33xMIC7 [4H] presented the most favorable characteristics.

[0383] 7.13 CD33xMIC7+GNE987 PROTAC-antibody conjugate is effective in the MV411 mouse xenograft model Human leukemia MV411 cells were xenografted into immunodeficient mice. Three million MV411 cells were injected subcutaneously into the left flank of naive female CB17 SCID mice. Randomization of animals into the various treatment groups and initiation of treatment occurred when the mean tumor size was 45 mm. 2The treatment started after reaching 100%. The control group was treated with vehicle. The test group was treated once (day 1) with 0.38 mg / kg GNE987 and 30 mg / kg CD33xMIC7+GNE987 (loaded with 0.38 mg / kg GNE987). In addition, two groups were treated twice: with 30 mg / kg CD33xMIC7+GNE987 (loaded with 0.38 mg / kg GNE987) on day 1, followed by either 0.38 mg / kg GNE987 on day 8 or 0.38 mg / kg GNE987 on days 1 and 8. Additionally, one group received 30 mg / kg of CD33xMIC5+GNE987 (loaded with 0.38 mg / kg of GNE987) once (day 1), and another group received antibody control 30 mg / kg of CD33xMIC7 without PROTAC once (day 1). Individual groups were divided into groups based on tumor size (maximum tumor size, 225 mm 2 ) was stopped before reaching (Figure 69).

[0384] While the antibody alone had no significant relative effect compared to the vehicle control, the PROTAC GNE987 induced an antitumor effect. However, at approximately day 4, tumors began to progress again, exhibiting the same growth rate as the vehicle control. In the group dosed with GNE987 on days 1 and 8, re-dosing with GNE987 again induced an antiproliferative effect until approximately day 3 after re-dosing, at which point tumors began to grow again. A single dose of CD33xMIC7+GNE987 led to tumor growth inhibition until day 15, after which tumors progressed. In the group dosed with 30 mg / kg CD33xMIC7+GNE987 (loaded with 0.38 mg / kg GNE987) on day 1, followed by treatment with 0.38 mg / kg GNE987 on day 8, re-dosing led to sustained tumor growth inhibition until approximately day 23. Treatment with CD33xMIC5+GNE987 induced tumor growth delay compared to vehicle, but the effect was much less pronounced compared to CD33xMIC7+GNE987.

[0385] Overall, the antitumor efficacy of GNE987-loaded CD33xMIC7 was superior to that of PROTAC alone at comparable PROTAC doses. Interestingly, the antitumor efficacy of CD33xMIC7 + GNE987 could be further enhanced by simply re-administering GNE987 on day 8. This demonstrates a clear benefit of additional administration of GNE987 alone to the CD33xMIC7 + GNE987 group and the potential of CD33xMIC7 to capture the PROTAC GNE987 from serum and accumulate it at the tumor site. These results pave the way for pretargeting tumors by administering a bispecific antibody that binds to a tumor-specific antigen and a PROTAC, followed by administration of an unconjugated PROTAC (e.g., an orally applicable one). This approach allows for targeting of separately administered PROTACs to desired tissues without the need for pre-manufacturing PAX ex vivo.

[0386] Comparison of the anti-PROTAC clones MIC5 and MIC7 reveals that CD33xMIC7+GNE987 induces a stronger anti-tumor effect than CD33xMIC5+GNE987. CD33 binding does not affect tumor growth as demonstrated by treatment with CD33xMIC7, confirming that the anti-tumor effect is driven by loading CD33xMIC7 with the PROTAC GNE987.

[0387] 7.14 Improved PAX tumor growth inhibition by increasing DAR and demonstrating targeted delivery Human leukemia MV411 cells were xenografted into immunodeficient mice. Three million MV411 cells were injected subcutaneously into the left flank of naive female CB17 SCID mice. Randomization of animals into the various treatment groups and initiation of treatment occurred when the mean tumor size was 150 mm. 3 All treatments were given intravenously once.

[0388] The control group was treated with vehicle. Test groups were treated once with 0.38 mg / kg GNE987P and 30 mg / kg CD33xMIC7+GNE987P(DAR2) (loaded with 0.38 mg / kg GNE987P). Additionally, mice were treated with 30, 10, or 3 mg / kg CD33xMIC7. [2H] +GNE987(DAR2) (equivalent to 0.38 mg / kg, 0.125 mg / kg, or 0.038 mg / kg GNE987). As a control for nonspecific uptake and effects mediated by improved PROTAC exposure, mice were given 30.1 mg / kg DIGxMIC7 [2H] +GNE987(DAR2) (equivalent to 0.38 mg / kg GNE987). It is important to note that two versions of CD33xMIC7+GNE987[2H](DAR2) with different IgG-based antibody scaffolds were investigated. Additionally, mice were given either 35.1 or 17.53 mg / kg CD33xMIC7. [4H] +GNE987(DAR4) (equivalent to 0.76 mg / kg or 0.38 mg / kg GNE987, respectively), and each non-binding control received 35.2 mg / kg DIGxMIC7 [4H] +GNE987 (DAR4) (equivalent to 0.76 mg / kg GNE987). Individual groups were randomly assigned to receive 1000x1000x1000 tumors at the maximum tumor size (1600 mm 3 Note that the growth was stopped before reaching . The resulting tumor growth curves are shown in Figure 70.

[0389] Xenograft tumor growth inhibition experiments showed that DAR was significantly increased from DAR2 PAX (CD33xMIC7 + GNE987, 30.1 mg / kg) to DAR4 PAX (CD33xMIC [4H]It was clearly demonstrated that tumor growth inhibition could be significantly improved by increasing the dose of PAX (17.53 mg / kg + GNE987) while delivering the same amount of PROTAC to the target cells. It should be noted that the dose was chosen relative to the molecular weight, so that the molar concentration of the antibody was half, but the total amount of PROTAC injected was the same in both groups. At the same time, the improved growth inhibition indeed depends on the ability of PAX to engage with, or more precisely, bind to, tumor cells. This is due to the CD33-binding PAX CD33xMIC7 [2H] +GNE987(DAR2) and CD33xMIC7 [4H] +GNE987(DAR4) were compared with their respective isotype counterparts (DIGxMIC7[2H] +GNE987 and DIGxMIC7) that are unable to bind to MV411 cells. [4H] This was observed by comparing the doses of CD33xMIC7 and CD33xMIC7 (+GNE987). Note that the dosages were selected relative to molecular weight, so that the molar concentrations of the antibody and PROTAC were identical for CD33-targeting and their respective DIG-targeting counterparts, PAX. Furthermore, the backbone of the antibody (IgG4 PG-SPLE vs. IgG1 PG-LALA) had a dependent effect on tumor growth inhibition, which was observed for CD33xMIC7. [2H] +GNE987 (DAR2) (IgG4 PG-SPLE backbone) and CD33xMIC7 [2H] +GNE987 (DAR2) (IgG1 PG-LALA backbone).

[0390] CD33xMIC7, as dose reduction from 30 mg / kg to 10 and 3 mg / kg led to decreased antitumor activity. [2H] The dose of +GNE987(DAR2) played an important role. Interestingly, this effect was not observed in CD33xMIC7 at 35.1 or 17.53 mg / kg. [4H] This was not observed when +GNE987(DAR4) was administered.

[0391] The PROTAC GNE987P (MSC2764898) had no significant effect on tumor growth. [2H] A slight improvement was achieved by complexation with (DAR2). The differences between targeted delivery of GNE987 or GNE987P using CD33xMIC7[2H] could be caused by differences in PROTAC potency, potential reduction in bystander killing, or differences in PK, which were not further evaluated.

[0392] All treatments were generally well tolerated as can be seen in FIG.

[0393] 7.15 Conjugation of PROTACs with anti-PROTAC antibodies can significantly improve pharmacodynamic biomarker modulation We were able to demonstrate that PAX can induce antitumor effects in vivo and that these effects are pronounced compared to PROTAC treatment alone. To better understand what causes the antitumor effects, the degradation of BRD4, the target protein of GNE987, was investigated in an in vivo pharmacodynamic (PD) biomarker study.

[0394] Therefore, CD33-expressing MV411 cells were expanded and 3x1E+06 cells / 0.1 mL (100% Matrigel) were implanted subcutaneously into the left flank of naive female CB17 SCID mice. 3Upon reaching 100 mg / kg, animals were assigned to treatment groups by a stratified randomization procedure. Treatment then began with an IV injection of either 30 mg / kg of the CD33-binding CD33xMIC7+GNE987(DAR2) PROTAC-antibody conjugate (corresponding to 0.38 mg / kg of conjugated PROTAC) or 0.38 mg / kg of GNE987 alone. Additionally, groups of animals received the same vehicle as PAX or the PROTAC. Tumors from GNE987 and PROTAC vehicle-treated animals were sampled at 1, 6, 24, and 48 hours. Tumors from CD33xMIC7+GNE987(DAR2) and PROTAC-antibody conjugate vehicle-treated animals were sampled at 6, 24, 48, and 168 hours. For PROTAC alone, a shorter observation time was chosen based on the short half-life and rapid clearance observed in PK studies of the free PROTAC compared to the PROTAC-antibody conjugate of the same PROTAC. The tissue was then pulverized to generate lysates, and BRD4 and HSP90 levels were assessed using simple Western analysis. BRD4 levels were normalized to HSP90 levels. A similar study was performed by implanting A431 cells and treating them with an EGFR-targeted PAX called EGFRxMIC7+GNE987 DAR2 and the corresponding PROTAC GNE987 alone. The results are presented in Figure 72.

[0395] Both CD33- and EGFR-targeted PAX loaded with GNE987 induced stronger and longer-lasting BRD4 degradation compared to equivalent doses of PROTAC alone. Furthermore, this effect was dependent on the receptor density of each cell line model used. Furthermore, the antitumor effects observed in the CD33 efficacy study may be due to the stronger and longer-lasting degradation of the essential protein BRD4, demonstrating not only the potential of complexed PROTAC-antibody to degrade proteins of interest (POIs) in vivo, but also further illustrating that the potential for PROTAC degradation in vivo may be improved by applying the technology of the present invention.

[0396] 8. Summary The present invention discloses an unprecedented delivery technology that enables targeted delivery of PROTACs via noncovalent PROTAC-antibody conjugates (PAX). It is important to note that PAX technology, unlike other methods in the field of noncovalent drug delivery, in which active drug substances are typically modified with haptens, enables targeted delivery of unmodified, active PROTACs. This invention encompasses the delivery of PROTACs to multiple cell types according to their cell surface receptor expression. These receptors include, but are not limited to, CD33, CLL1, TROP2, HER2, EGFR, NAPI2B, cMET, CLDN18.2, STEAP1, PSMA, and B7H3. With respect to PROTACs, the versatility of the present invention is demonstrated by the selective delivery of various structurally distinct PROTACs (GNE987, ARV771, SIM1, GNE987P, SIM1, and FLT3d1). This platform has also been demonstrated to offer the possibility of delivering up to 12 PROTAC molecules using a single antibody by leveraging a modular antibody-engineering strategy. One of the strengths of the technology is the ease and speed of molecular assembly compared to traditional antibody-drug conjugation methods. The PROTAC only needs to be mixed with the antibody, and complex formation is completed within minutes, enabling the effortless generation of PROTAC-antibody complexes and, consequently, the selection of suitable drug candidates in a data-driven manner. Furthermore, the present invention demonstrates that antibody-conjugation can significantly improve the exposure of target cells to PROTACs. Finally, the inventors were able to validate the PAX technology in several in vivo xenograft models, demonstrating not only excellent tumor growth inhibition but also protein knockdown. Table 38 provides an overview.

[0397] [Table 38]

Claims

1. An isolated antibody capable of binding via its VHH portion to a degron of the VHL ligand VH032 of a PROTAC or a derivative thereof.

2. The antibody of claim 1, which is a monospecific antibody.

3. 3. The antibody of claim 1, which is a bispecific antibody and whose second binding possibility is for a target protein.

4. a) a monospecific bivalent antibody consisting of two full-length antibody heavy chains and two full-length antibody light chains, each chain containing only one variable domain; b) two or more variable heavy chain single domain (VHH) antibodies, each consisting of one antibody variable domain, and optionally c) a peptide-linker connecting the C-terminus of portion (a) and the N-terminus of portion (b). The antibody of claim 3, comprising:

5. The antibody of claim 4, wherein the N-terminus of the two or more heavy chain single domain (VHH) antibodies of portion (b) and the C-terminus of the monospecific bivalent antibody of portion (a) are connected via a peptide linker.

6. The antibody of claim 5 , wherein the peptide linker is cleavable or non-cleavable.

7. The antibody of claim 4, 5 or 6, wherein the number of heavy chain single domain (VHH) antibodies in part (b) is between 2 and 12.

8. 8. The antibody of claim 7, wherein each of the two heavy and light chains of portion (a) carries up to three heavy chain single domain (VHH) antibodies of portion (b) fused to the C-terminus of the antibody chain of portion (a).

9. The antibody of claim 8 , wherein each of the two heavy chains of portion (a) carries one heavy chain single domain (VHH) antibody of portion (b).

10. 10. The antibody of any one of claims 4 to 9, wherein the variable domain of portion (a) is capable of binding to the target protein and the variable domain of portion (b) is capable of binding to a degron of the VH032 ligand of the PROTAC or a derivative thereof.

11. The antibody of any one of claims 1 to 10, wherein only CDR3 of said VHH antibody is involved in binding of said VH032 or a derivative thereof.

12. The VH032 derivative has formula I: 【Chemistry 1】 [In the formula, R １ or R ２ one of which is a linker connected to the warhead, provided that: R ２ is a linker, then R １ is acetyl, and R １ is a linker, then R ２ is methyl, R ３ is H, OH, cyano, F, Cl, amino or methyl, R ４ is H or methyl, R ５ , R ６ is H or OH, with the proviso that R ６ If is H, then R ５ is OH, and R ５ If is H, then R ６ is OH] 12. The antibody of claim 10 or 11, which corresponds to:

13. R １ However, PB-Q-(CH ２ -CH ２ -O) ｎ - (CH ２ -CH ２ -CH ２ -O) ｍ - (CH ２ ) ｐ -(C=O)-, During the ceremony, PB is protein-binding warhead; Q is NH, C═O or absent; n and m are independently 0, 1, 2, 3, or 4; p is 0 to 10; R ２ is methyl, R ３ , R ４ , R ５ and R ６ The antibody of claim 12, wherein:

14. R １ However, PB-Q-(CH ２ -CH ２ -O) ｎ - (CH ２ -CH ２ -CH ２ -O) ｍ - (CH ２ ) ｐ -(C=O)-, During the ceremony, PB is protein-binding warhead; Q is NH, C═O or absent; (xi) n, m, and p are 1; or (xii) n is 3 or 4, m is 0, and p is 1, or (xiii) n is 1, m is 0, and p is 2, or (xiv) n is 2, m is 0, and p is 2, or (xv) n and m are 0, and p is 6, 7, 8, 9, or 10; R ２ is methyl, R ３ , R ４ , R ５ and R ６ The antibody of claim 13, wherein the antibody is as described in claim 12.

15. R １ is acetyl, R ２ PB-NH-(CH ２ ) ｐ -S-, where PB is a protein-binding warhead and p is 1, 2, 3, 4, 5, or 6; R ３ , R ４ , R ５ and R ６ The antibody of claim 12, wherein:

16. 13. The antibody of claim 12, wherein the PROTAC is selected from the PROTACs shown in Figures 8(a) and 8(b).

17. The antibody of any one of claims 1, 3 to 16, wherein the target protein is a cell surface protein.

18. The antibody of claim 17, wherein the cell surface protein is a tumor antigen.

19. 19. The antibody of claim 18, wherein the cell surface protein is Her2, CD33, CLL1, TROP2, NAPI2B, B7H3, STEAP1, PSMA, cMET, CLDN18.2, or EGFR.

20. said VHH antibody having the following CDR3 sequence (SEQ ID NO: 1): CDR3: X １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ X １２ X １３ X １４ X １５ X １６ X １７ X １８ X １９ X ２０ X ２１ 、 Including, Here, X １ is A, S or T, and X ２ is A, V or I, and X ３ is S, A, I or D, and X ４ is any amino acid, and X ５ is any amino acid, and X ６ is V, S, L or T, and X ７ is any amino acid, and X ８ is S, A, C or P, and X ９ is T, A, K, S or N, and X １０ is any amino acid, and X １１ is absent, V or A, and X １２ is any amino acid or is absent, and X １３ is V, G or P, and X １４ is any amino acid, and X １５ is any amino acid, and X １６ is T, V, K or R, and X １７ is R, I or Y, and X １８ is Y, Q, F or A, and X １９ is V or L, and X ２０ is any amino acid, and X ２１ The antibody of any one of claims 1 to 19, wherein is V, Y or A.

21. X １ is A, S or T, and X ２ is A, V or I, and X ３ is S, A, I or D, and X ４ is K, T, Y, R or A, and X ５ is E, R, Y or G, and X ６ is V, S, L or T, and X ７ is K, L, G, S or C, and X ８ is S, A, C or P, and X ９ is T, A, K, S or N, and X １０ is K, P, I, V or D, and X １１ is absent, V or A, and X １２ is E, D, S, R or absent, and X １３ is V, G or P, and X １４ is K, D, T, G or R, and X １５ is K, Q, I, T or R, and X １６ is T, V, K or R, and X １７ is R, I or Y, and X １８ is Y, Q, F or A, and X １９ is V or L, and X ２０ is K, E, P or D, and X ２１ is V, Y or A.

22. X １ is A or S, and X ２ is V or A, and X ３ is A or I, and X ４ is K, T or Y, and X ５ is E, G or R, and X ６ is L or S, and X ７ is K, C or S, and X ８ is P or C, and X ９ is A, K or S, and X １０ is K, V or D, and X １１ is absent or V, and X １２ is E, R or absent, and X １３ is G or P, and X １４ is K, T or G, and X １５ is K, Q or I, and X １６ is T, K or R, and X １７ is R, I or Y, and X １８ is F or A, and X １９ is L and X ２０ is K, E or D, and X ２１ is V or Y.

23. the CDR3 is SAIYRLSCSVVRPTIRYALDY (SEQ ID NO: 2), SAIYRLSCSVVRPTIKYALDY (SEQ ID NO: 3), SAIYRLSCSVVRPTITYALDY (SEQ ID NO: 4), SAIYRLSCKVVRPTIRYALDY (SEQ ID NO: 5), SAIKRLSCSVVRPTIRYALDY (SEQ ID NO: 6), SAIYELSCSVVRPTIRYALDY (SEQ ID NO: 7), SAIYRLKCSVVRPTIRYALDY (SEQ ID NO: 8), SAIYRLSCSKVRPTIRYALDY (SEQ ID NO: 9), SAIYRLSCSVVEPTIRYALDY (SEQ ID NO: 10), SAIYRLSCSVVRPKIRYALDY (SEQ ID NO: 11), SAIYRLSCSVVRPTKRYALDY (SEQ ID NO: 12), SAIYRLSCSVVRPTIRYALKY (SEQ ID NO: 13) 21. The antibody of claim 20, selected from:

24. 24. In vitro use of the monospecific antibody of any one of claims 1 to 23 for detecting, quantifying or purifying PROTACs.

25. 24. A conjugate (PAX) of the bispecific antibody and a PROTAC of any one of claims 1, 3-23, wherein the bispecific antibody binds to a degron of the VHL ligand VH032, or a derivative thereof, of the PROTAC.

26. 26. The conjugate (PAX) of claim 25, wherein the VHL ligand and the linker of the PROTAC are as defined in any one of claims 8 to 12.

27. 28. A pharmaceutical composition comprising the conjugate of claim 25 or 27 and one or more further pharmaceutically acceptable ingredients.

28. 30. Use of the conjugate of claim 25 or 27 to deliver a PROTAC to a target cell expressing a target protein for degradation.

29. 28. A method of treating a disease by administering the conjugate of claim 25 or 27 to a patient in need thereof, wherein the disease would benefit from the degradation of a protein targeted for degradation by the PROTAC.

30. 30. The conjugate (PAX) of claim 25 or 27 for use in the treatment of a disease that benefits from degradation of a target protein of the PROTAC.

31. 28. The conjugate (PAX) of claim 25 or 27 for use in treating a disease that benefits from degradation of a PROTAC degradation target protein, wherein the PAX is administered first, followed by subsequent administration of the PROTAC portion of the PAX alone.

32. DEGRADATION OF PROTACs. The conjugate (PAX) of claim 25 or 27 for use in treating a disease that benefits from degradation of a target protein, wherein the antibody portion of the PAX is administered first and the PROTAC portion of the PAX is administered subsequently.

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