Antibody killer scavengers

Abstract

The present invention provides a conjugate comprising a first protein that is capable of binding to a protein of interest, a second protein that is bound to the first protein and that comprises an endocytosis-triggering peptide that is capable of binding to an endocytic receptor of a tumor cell, and a cargo, preferably a drug that is toxic for tumor cells, and that is bound to the first protein via an intracellular cleavable or degradable linker.

Description

[0001] ANTIBODY KILLER SCAVENGERS

[0002] TECHNICAL FIELD

[0003] The present invention relates to the field of biomedicine, and specifically to a lysosome targeting chimera and its conjugation to antibody drug conjugates.

[0004] BACKGROUND ART

[0005] Cancer has become the second largest global health threat, accounting for approximately 10.0 million deaths in 2020. For decades, chemotherapy based on cytotoxic agents has been the main approach for the treatment of a wide range of cancers. However, most of these chemotherapeutic agents have a low therapeutic index, with severe side effects generally attributed to non-specific drug exposure to off-target tissues. To address this issue, scientists have been working on the development of novel cancer therapeutics with higher targeting ability.

[0006] The emergence of monoclonal antibodies (mAbs) transformed the landscape of cancer therapy. They offer unparalleled precision, targeting surface antigens on the tumor cells, blocking significant signaling pathways for tumor progression and survivability and triggering antibody dependent cellular cytotoxicity. However, monotherapy with mAbs is often insufficient. This is potentially due to their reduced lethality against cancer cells compared to chemotherapeutic agents. To address these shortcomings, a novel concept was conceived: the antibody-drug conjugate (ADC). This merging of mAbs and chemotherapeutic agents improved the therapeutic effectiveness of both treatments while reducing off-target toxicity. ADCs consist of a tumor targeting mAb conjugated to a cytotoxic payload through a sophisticatedly designed chemical linker, enabling the ability of precise targeting and potent effectiveness simultaneously. Moreover, due to the conjugation to a large hydrophilic antibody, the antigen-independent uptake of cytotoxic payloads in antigen-negative cells is limited, contributing to widening the therapeutic index of ADCs.

[0007] The delivery of ADCs cytotoxic payloads depends on the internalization of the formed ADC-cell surface antigen complex via clathrin-mediated endocytosis, caveolae-mediated endocytosis and pinocytosis. Once internalized, the ADC-antigen complex is transported first to endosomes and then to lysosome where the payload is released. The trafficking of the complex may be a limiting step for the effectivity of ADCs, since the payload may not be released in early endosomes and / or be shuttled back to the cell surface due to receptor recycling, thereby limiting the effectiveness of the ADCs on delivering their payload. Thus, improved intracellular transport and degradation systems are needed. The targeted protein degradation (TPD) technology is a new and highly effective technology that specifically identifies target proteins (target proteins) and directly degrades them by utilizing the intrinsic protein degradation pathways in cells.

[0008] The first TPD applications were developed to target undruggable intracellular proteins that were extremely difficult to target by traditional therapeutic approaches. Protein degradation platforms such as Proteolysis-targeting Chimaeras (PROTACs) and molecular glues were among the first described TPD strategies. These can form a ternary complex between the target protein and an E3 ligase, which induces the ubiquitination of the target protein and signals its degradation by the proteasome. Lysosome-dependent TPD strategies are novel strategies for targeting and degrading extracellular and membranebound proteins through the lysosomal degradation pathway. Extracellular and mem- brane-bound proteins play a critical role in the development of significant diseases, including cancer, aging-related diseases, and autoimmune disorders. It is therefore imperative to develop novel methods for targeted degradation of these proteins, as this could have a significant impact on human health and open up new avenues of research for the treatment of these diseases. LYTACs (Lysosomal Targeting Chimeras) were the first of the lysosome-dependent TPD strategies described (Banik SM, et al., 2020, Nature. 584(7820), p.p:291-297). LYTACs are bifunctional molecules capable of capturing extracellular proteins and inducing their degradation in the lysosomes. LYTACS consist of i) a ligand of the extracellular target protein, which can be a small molecule, an antibody or an aptamer; ii) an optional linker; and iii) an agonist ligand of an endocytic membrane receptor, which upon binding to the receptor triggers the internalization and sorting of the LYTAC and its associated cargo to the lysosomes (Ahn G. et al., 2021 , Nature Chem Biology. 17(9): 937-946; Zhou, Y et al., 2021 , ACS Cent Sci, 7(3): p.p. 499-506).

[0009] Initial evidence for lysosomal targeted degradation demonstrated the feasibility of promoting targeted degradation of extracellular and membrane-associated proteins using conjugates that bind to the cation-independent mannose 6-phosphate receptor (Cl- M6PR), a cell surface lysosome shuttling receptor and the extracellular domain of a target protein (Banik SM, et al., 2020. Nature. 584(7820):291-297). These bifunctional molecules consist of a small molecule or antibody that recognizes the target protein, linked to a glycopeptide that acts as an agonist ligand of the CI-M6PR. Subsequently, novel LTDs designed to bind to the asialoglycoprotein receptor present in hepatocytes were reported (Ahn G et al., 2021 , Nature Chem Biology 17(9):937-946). Since the development of LYTACs new lysosome-dependent TPD strategies have been reported in the scientific literature. In many of these new strategies the recognition of the lysosome-targeting receptors (LTR) and the target proteins is mediated by the use of antibodies as recognition molecules. However, it is technically difficult to construct an antibody-based method that preferentially targets tumor cells while achieving an effective degradation rate of the target protein. Moreover, the synthesis of the LTR ligands, such as mannose-6-phosphate analogs present technical problems such as long production cycles, high cost, and it is time-consuming and labor-intensive.

[0010] The present invention aims to address the drawbacks associated to both LYTAC and ADC conjugates and provide an improved conjugate for cancer therapies.

[0011] BRIEF DESCRIPTION OF DRAWINGS

[0012] Fig. 1 : Comparation of Anti IL1 b ADC and AKS toxicity (mean data ± s.d of n=3 independent experiments). HeLa cells were treated for 24h with 100nM of one the following drugs: Ab MMAE (Anti IL1 b MMAE ADC), Ab MMAE R12 (Anti IL1 b MMAE R12 AKS), Ab MMAF (Anti IL1 b MMAF ADC) or Ab MMAF R12 (Anti IL1 b MMAF R12 AKS). Cells treated with Ab MMAE and Ab MMAF ADCs showed no significant toxicity compare with controls (Vehicle). Cells treated with Ab MMAE R12 and Ab MMAF R12 ADKs showed significant reduction in cell viability compared with the controls (**) and its respective ADC (*).

[0013] Fig. 2: Comparation of Anti IL1 b ADC and AKS toxicity (mean data ± s.d of n=3 independent experiments) and internalizing peptide selectivity toward lung cancer cell line NCI-H1299. HeLa and NCI-1299 cells were treated for 48h with 100nM of one the following drugs: @MMAE (Anti IL1 b MMAE ADC), @MMAE+R12 (Anti IL1 b MMAE R12 AKS), @MMAE+MGS4 (Anti IL1b MMAE MGS4 AKS), @MMAF (Anti IL1b MMAF ADC), @MMAF+R12 (Anti IL1 b MMAF R12 AKS) or @MMAF+MGS4 (Anti IL1 b MMAF MGS4 AKS). HeLa and NCI-H1299 cells treated with @MMAE and @MMAF ADCs showed no significant toxicity compare with controls (Vehicle). Both cell lines when treated with @MMAE+R12 and @MMAF+R12 ADKs showed significant reduction in cell viability compared with the controls (**) and their respective ADC (*). HeLa cells showed no significant toxicity when treated with either @MMAE+MGS4 or @MMAF+MGS4. However, NCI-H1299 cells showed significant toxicity when treated with @MMAE+MGS4 or @MMAF+MGS4 AKSs compared with the controls (*) and their respective ADCs (**). Fig. 3: a) Uptake ofILI b by HeLa cells after being treated for 24h with 100nM of IL1 b and 100 nM of Anti IL1 b ADCs or ADKs in absence or presence of Bafilomycin A1 (100nM) (+baf), b) Uptake of Anti I L1 b antibody by HeLa cells after being treated for 24h with 100nM of Anti I L1 b ADCs or ADKs in absence or presence (+baf) of Bafilomycin A1 (100nM). @ (Anti 111b antibody), @ HC (IgG Heavy Chain), @ LC (IgG Light Chain), @+MMAE (Anti I L1 b MMAE ADC), @+MMAF (Anti I L1 b MMAF ADC), @+R12+MMAE (Anti IL1 b MMAE R12 AKS), @+R12+MMAF (Anti IL1 b MMAF R12 AKS), ctrllLIb (IL1 b), ctrl@ (Anti I L1 b antibody).

[0014] Fig. 4 : Phase contrast images of HeLa cells after being treated for 24h with Anti I L1 b ADCs / ADKs: a) control, b) Anti IL1 b MMAE ADC, c) Anti IL1 b MMAE R12 AKS, d) Anti IL1 b MMAE R12 AKS + 100nM of Bafilomycin A1.

[0015] Fig. 5: Uptake of anti IL1 b IgG antibody by a) HeLa, b) A549 cells and c) NCI-H1299 cells after being treated for 24h with 100nM of Anti 111 b ADC or 100 nM of Anti IL1 b ADKs in absence or presence of Bafilomycin A1 (100nM) (+b). @ (Anti 111b antibody), @+MMAE (Anti I L1 b MMAE ADC), @+MMAF (Anti I L1 b MMAF ADC), @+MMAE+R12 (Anti IL1 b MMAE R12 AKS), @+MMAE+MGS4 (Anti IL1b MMAE MGS4 AKS), @+MMAF+R12 (Anti I L1 b MMAF R12 AKS), @+MMAF+MGS4 (Anti I L1 b MMAF MGS4 AKS) Ctrl (anti IL1 b IgG antibody), Ctrl@ (anti IL1 b IgG antibody) @HC (IgG Heavy Chain), @ LC (IgG Light Chain).

[0016] Fig. 6: Uptake of AntilLI b by NCI-H1299 cells mediated by MGS4 and R12 AKSs. Cells were treated for 24h with 100nM of 111b and 100 nM of AntilH b ADCs or 100 nM of Anti IL1 b ADKs in absence or presence of Bafilomycin A1 (100nM) (+b), Anti@ (Anti 111b antibody), @+MMAE (Anti IL1b MMAE ADC), @+MMAF (Anti IL1b MMAF ADC), @+MMAE+R12 (Anti IL1 b MMAE R12 AKS), @+MMAE+MGS4 (Anti IL1 b MMAE MGS4 AKS), @+MMAF+R12 (Anti IL1 b MMAF R12 AKS), @+MMAF+MGS4 (Anti IL1 b MMAF MGS4 AKS) Ctrl (Anti IL1 b IgG antibody), ctrllLI b (IL1b).

[0017] Fig. 7 : Effect of Atezolizumab-based AKS constructs on PC9 and HCC827 cell viability after 24 h treatment. Cell viability was measured relative to untreated control (C). Treatments included the free cytotoxic payload (MMAE), a conventional Atezolizumab ADC(AE), and the Atezolizumab-based AKS construct Atezo-R12(AE). The AKS constructs induced a stronger reduction in cell viability compared to the free payload or the conventional ADC, with Atezo-R12(AE) showing the greatest cytotoxic effect. Statistical analysis Two-way ANOVA (compared to control. ** - p<0.01 ; *** p<0.001. Fig. 8. Effect of Atezolizumab-based AKS constructs the expression of PD-L1 in HCC827 cell line after 24 h treatment with antibody only (Atezo), antibody-peptide conjugate (Atezo-R12), a conventional Atezolizumab ADC(Atezo-AE), and the Atezolizumab-based AKS construct ( Atezo-AE-R12). Both the antibody-peptide conjugate and the AKS construct induced a stronger reduction in the PD-L1 expression that ADC.

[0018] Fig. 9. Effect of Bevacizumab-based AKS constructs on H1299 (upper panel) and HeLa (lower panel) cell viability after 24 h treatment. Cell viability was measured relative to untreated controls (C). Treatments included the free cytotoxic payload (MMAF), a conventional Bevacizumab antibody-drug conjugate (Beva ADC(AF)), and the Bevacizumab-based AKS constructs Beva-R12(AF) and Beva-MGS4(AF). Both AKS constructs, particularly Beva-R12(AF), induced a stronger reduction in cell viability compared to the free payload or the conventional ADC. Statistical analysis Two-way ANOVA (compared to control ** - p<0.01 ; *** p<0.001 , **** p<0.0001.

[0019] Fig. 10. Effect of 100nM bevacizumab alone (Beva) and Bevacizumab-MGS4 conjugate (Beva_MGS4) on the internalization of VEGF in H1299 cells upon 24h treatment. Some wells were treated with E-64, which is a potent, irreversible inhibitor of cysteine proteases.

[0020] Fig. 11. Cytotoxic effects of Cetuximab-based AKS constructs on A549 and H1975 cell viability after 24 h treatment, evaluated by crystal violet assay and bright field microscopy. Cell viability was measured relative to untreated control. Treatments included free cytotoxic payload (MMAE), Cetuximab-R12 (Cetuximab-LYTAC), a conventional Cetuximab antibody-drug conjugate (Cetuximab ADC), and the Cetuximab- R12-based AKS construct (Cetuximab AKS). In both A549 and H1975 cells, the Cetuximab AKS induced a significantly stronger reduction in viability than MMAE or Cetuximab ADC. Bright field microscopy images further revealed apoptotic phenotypes upon AKS treatment, more pronounced than those observed with control or ADC treatments. *** p<0.001 , **** p<0.0001.

[0021] Fig. 12. Effect of 100nM Cetuximab alone (Ctx), Ctx with toxic load (Ctx-AE), Ctx-MGS4 or Ctx-R12 conjugates, Ctx-MGS4 or Ctx-R12 conjugates with toxic load (Ctx-AE-R12 or Ctx-AE-MGS4) on the total EGFR in H1299 cells upon 24h treatment. Some wells were treated with bafilomycin (baf), which is a potent lysosome inhibitor. Fig. 13 Toxicity of atezolizumab conjugates in PC9 cells. Atezo - atezolizumab; AtezoMMAE - atezolizumab, conjugated with MMAA; AtezoMMAE_R12- atezolizumab conjugated with MMAE and R12 peptide AtezoMMAE_R12_Mod- atezolizumab conjugated with MMAE and modified R12 peptide.**, *** - paired t-test treatments vs. control (p<0.01 and p<0.0001 respectively), $$$ - paired t-test treatments vs. AtezoMMAE, p<0.0001

[0022] GENERAL DEFINITIONS

[0023] It must be noted that, as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Further, unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0024] If the term "about" is used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. For instance, the term “about” means the indicated value ± 1 % of its value, or the term “about” means the indicated value ± 2% of its value, or the term “about” means the indicated value ± 5% of its value, the term “about” means the indicated value ± 10% of its value, or the term “about” means the indicated value ± 20% of its value, or the term “about” means the indicated value ± 30% of its value; preferably the term “about” means exactly the indicated value (± 0%).

[0025] As used herein, the conjunctive term "and / or" between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by "and / or", a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term "and / or" as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term "and / or." Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or "including" or sometimes when used herein with the term "having". Any of the aforementioned terms (comprising, containing, including, having), whenever used herein in the context of an aspect or embodiment of the present invention may be substituted with the term "consisting of', though less preferred.

[0026] When used herein "consisting of' excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0027] "Nucleic acids," "nucleic acid molecules," "oligonucleotide," and "polynucleotide" are used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix.

[0028] As used herein, the term "gene" or "coding sequence" refers to a polynucleotide sequence in vitro or in vivo that encodes a gene product. In some instances, the gene consists or consists essentially of coding sequence, that is, sequence that encodes the gene product.

[0029] The terms “sequence identity” or “percent identity” in the context of two or more nucleotide sequences, polypeptide sequences or proteins sequences refers to two or more sequences or subsequences that are the same (“identical”) or have a specified percentage of nucleotide or amino acid residues that are identical (“percent identity”) when compared and aligned for maximum correspondence with a second molecule, as measured using a sequence comparison algorithm, preferably BLAST alignment tool, or alternatively, by visual inspection. The “sequence identity” or “percent identity” can be determined by calculating the number of identical nucleotides or amino acids at the same positions in a nucleic acid, polypeptide or protein. Calculation of percent identity includes determination of the optimal alignment between two or more sequences. Alignment can take into account insertions and deletions (i.e. “gaps”) in each of the sequences to be tested, such as, without limitation, in the non-coding regions of nucleic acids and truncations or extensions of polypeptide sequences. Computer programs and algorithms such as the Basic Local Alignment Search Tool (BLAST) may be used to determine the percent identity. BLAST is one of the many resources provided by the U.S. National Center for Biotechnology Information. Because the genetic code is degenerate, and more than one codon can encode a given amino acid, coding regions of nucleic acids are considered identical if the nucleic acids encode identical polypeptides. Thus, percent identity could also be calculated based on the polypeptide encoded by the nucleic acid. Percent identity could be calculated based on full length consensus genomic sequences or on a fraction of the genomic sequence, such as for example without limitation on individual open reading frames (ORFs).

[0030] "Percent (%) amino acid sequence identity" with respect to proteins or polypeptides described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence (i.e., the protein or polypeptide from which it is derived), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the sequences being compared.

[0031] The term “fusion protein” is synonymous to “chimeric protein” and refers to a single polypeptide having at least two domains. The term “domain” is referred herein to a part of a protein that has a specific function in the protein and that, usually, folds independently.

[0032] The term "antibody" refers to a molecule comprising at least one immunoglobulin domain that binds to, or is immunologically reactive with, a particular target. The term includes whole antibodies and any antigen binding portion or single chains thereof and combinations thereof; for instance, the term “antibody” in particular includes bivalent antibodies and bivalent bispecific antibodies. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, bivalent antibody fragments (such as F(ab')2), multispecific antibodies such as bispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and any other modified immunoglobulin molecule comprising an antigen binding site.

[0033] An antibody can be of any the five major classes (isotypes) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses thereof (e.g. lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as therapeutic agents or diagnostic agents to form immunoconjugates.

[0034] A typical type of antibody comprises at least two heavy chains ("HO") and two light chains ("LG") interconnected by disulfide bonds. Each "heavy chain" comprises a "heavy chain variable domain" (abbreviated herein as "VH") and a "heavy chain constant domain" (abbreviated herein as "CH"). The heavy chain constant domain typically comprises three constant domains, CH1 , CH2, and CH3. Each "light chain" comprises a "light chain variable domain" (abbreviated herein as "VL") and a "light chain constant domain" ("CL"). The light chain constant domain (CL) can be of the kappa type or of the lambda type. The VH and VL domains can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions ("CDR"), interspersed with regions that are more conserved, termed "framework regions" ("FW").

[0035] Each VH and VL is composed of three CDRs and four FWs, arranged from aminoterminus to carboxy-terminus in the following order: FW1 , CDR1 , FW2, CDR2, FW3, CDR3, FW4. The present disclosure inter alia presents VH and VL sequences as well as the subsequences corresponding to CDR1 , CDR2, and CDR3.

[0036] A person skilled in the art would understand that the sequences of FW1 , FW2, FW3 and FW4 are equally disclosed. For a particular VH, FW1 is the subsequence between the N-terminus of the VH and the N-terminus of H-CDR1 , FW2 is the subsequence between the C-terminus of H-CDR1 and the N-terminus of H-CDR2, FW3 is the subsequence between the C-terminus of H-CDR2 and the N-terminus of H-CDR3, and FW4 is the subsequence between the C-terminus of H-CDR3 and the C-terminus of the VH. Similarly, for a particular VL, FW1 is the subsequence between the N-terminus of the VL and the N-terminus of L-CDR1 , FW2 is the subsequence between the C-terminus of L- CDR1 and the N-terminus of L-CDR2. FW3 is the subsequence between the C-terminus of L-CDR2 and the N-terminus of L-CDR3, and FW4 is the subsequence between the C- terminus of L-CDR3 and the C-terminus of the VL.

[0037] The variable domains of the heavy and light chains contain a region that interacts with a binding target, and this region interacting with a binding target is also referred to as an “antigen-binding site” or “antigen binding site” herein. The constant domains of the antibodies can mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Exemplary antibodies of the present disclosure include typical antibodies, but also bivalent fragments and variations thereof such as a F(ab’)2.

[0038] The term “antigen-binding fragment” or “Fab” refers to an antibody fragment comprising one constant and one variable domain of each of the heavy and light chain. A Fab fragment may be obtained by digesting an intact monoclonal antibody with papain.

[0039] The term “single-chain antigen-binding fragment” or “scFab” refers to a fusion protein comprising one variable and one constant domain of the light chain of an antibody attached to one variable and one constant domain of the heavy chain of an antibody, wherein the heavy and light chains are linked together through a short peptide.

[0040] The term “single-chain variable fragment” or “scFv” refers to a fusion protein comprising the variable domains of the heavy chain and light chain of an antibody linked to one another with a peptide linker. The term also includes a disulfide stabilized Fv (dsFv). Methods of stabilizing scFvs with disulfide bonds are disclosed in Reiter et a / ., 1996. Nat Biotechnol. 14(10): 1239-45.

[0041] The term “anticalin” refers to a protein that is derived from lipocalin and that been engineered to bind to a specific target.

[0042] The term “fynomer” refers to a protein that is derived from the SH3 domain of human Fyn kinase that has been engineered to bind to a specific target (see Bertschinger et al., 2007. Protein Eng Des Sei. 20(2):57-68).

[0043] The term “VNAR” refers to the variable domain of immunoglobulin new antigen receptor (IgNAR) naturally found in sharks, which contains autonomous function as a singledomain antibody (see Greenberg et al., 1995, Nature, 374, pp. 168-173). The term “nanobody” refers to a protein comprising the soluble single antigen-binding V- domain of a heavy chain antibody, preferably a camelid heavy chain antibody (see Bannas etal., 2017. Front Immunol. 8:1603). The term “repebody” refers to a protein that is derived from a leucine-rich repeat module and that has been engineered to bind to a specific target (see Lee et al., 2012. PNAS. 109(9): 3299-3304).

[0044] The term “peptide aptamer” refers to a short, 5-20 amino acid residue sequence that can bind to a specific target. Peptide aptamers are typically inserted within a loop region of a stable protein scaffold (see Reverdatto et al., 2015. Curr Top Med Chem. 15(12):1082- 101).

[0045] "Specific binding” or “specifically binds” refer to an antibody, or a targeting moiety, which recognizes and binds with a binding partner (e.g., a stimulatory tumor antigen) protein present in a sample, but which antibody or moiety does not substantially recognize or bind other molecules in the sample. A targeting moiety or antibody that specifically binds to a protein of interest binds to that target with a greater affinity than any other target. For example, a targeting moiety that specifically binds to PD-L1 binds to PD-L1 with a greater affinity than to any other target. The skilled person is clearly aware of various experimental procedures that can be used to test binding and binding specificity. Some cross-reaction or background binding may be inevitable in many protein-protein interactions; this is not to detract from the "specificity" of the binding between antibody and epitope. The term "directed against" is also applicable when considering the term "specificity" in understanding the interaction between antibody, ora targeting moiety, and epitope.

[0046] The term “cancer” refers to a group of diseases, which can be defined as any abnormal benign or malignant new growth of tissue that possesses no physiological function and arises from uncontrolled usually rapid cellular proliferation and has the potential to invade or spread to other parts of the body. A “antigen-positive” cancer, including a “antigen - positive” cancerous disease, is one comprising cells that have increased expression levels of a given antigen, such as PDL-1 , IL-1 B, EGFR, VEGF-A, IL-6, relative to a control cell.

[0047] By “cancer cell” is meant a cell exhibiting a neoplastic cellular phenotype, which may be characterized by one or more of, for example, abnormal cell growth, abnormal cellular proliferation, loss of density-dependent growth inhibition, anchorage-independent growth potential, ability to promote tumor growth and / or development in an immunocompromised non-human animal model, and / or any appropriate indicator of cellular transformation. “Cancer cell” may be used interchangeably herein with “tumor cell”, “malignant cell,” “neoplastic cell,” or “cancerous cell”, and encompasses cancer cells of a solid tumor, a semi-solid tumor, a hematological malignancy (e.g., a leukemia cell, a lymphoma cell, a myeloma cell, etc.), a primary tumor, a metastatic tumor, and the like.

[0048] The terms “individual”, “patient” or “subject” are used interchangeably in the present application to designate a human being and are not meant to be limiting in any way. The “individual”, “patient” or “subject” can be of any age, sex and physical condition. The term “patient in need thereof” usually refers to a patient who suffers from a CD1a-positive cancer.

[0049] As used herein, "pharmaceutically acceptable carrier" or “pharmaceutically acceptable diluent” means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and, without limiting the scope of the present invention, include: additional buffering agents; preservatives; co-solvents; antioxidants, including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers, such as polyesters; salt-forming counterions, such as sodium, polyhydric sugar alcohols; amino acids, such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactitol, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur containing reducing agents, such as glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [alpha]-monothioglycerol, and sodium thiosulfate; low molecular weight proteins, such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; and hydrophilic polymers, such as polyvinylpyrrolidone. Other pharmaceutically acceptable carriers, excipients, or stabilizers, such as those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) may also be included in a pharmaceutical composition described herein, provided that they do not adversely affect the desired characteristics of the pharmaceutical composition.

[0050] As used herein, the term “conjugate” refers to a molecule comprising two or more components that are covalently or non-covalently linked to form a single functional entity. Such components may include, for example, a therapeutic agent, a targeting moiety (e.g., an antibody, receptor ligand, or peptide), a detectable label, a carrier molecule, or any combination thereof.

[0051] As used herein, the term “cargo” refers to a moiety carried or delivered by the conjugate of the invention. The cargo preferably comprises a biologically active agent, such as a cytotoxic compound, toxin, radioactive isotope, enzyme, or other therapeutic or diagnostic molecule, that is linked directly or indirectly (e.g., via a linker) to the conjugate.

[0052] As used herein, the terms “IL-1 b,” “IL-1 B,” “IL1 b,” and “IL-1 P” or similar are considered synonymous and are used interchangeably to refer to interleukin-1 (IL-1 P).

[0053] DESCRIPTION OF THE EMBODIMENTS

[0054] The inventors have developed a conjugate that is capable of 1) acting like a lysosomal targeted degrader and 2) acting as a targeted chemotherapy for treating cancer cells. This has been achieving by designing a conjugate that comprises four main elements: first, at least one protein that is capable of binding to target tumor protein that is present in the extracellular environment of a tumor or in the cell membrane of a tumor-associated cell; second, at least another protein or peptide that will bind to an endocytic receptor of a tumor cell and will trigger the internalization of the conjugate and the tumor-associated protein; third a drug that is toxic for the cancer cell; and fourth, a linker that is cleavable intracellularly and that will release the drug inside the cancer cell. This way, the tumor is attacked from two different mechanisms: the capture and internalization of target extracellular and cell membrane proteins associated with tumor progression and survivability, and the internalization of a drug that is toxic for tumor cells.

[0055] As proof of concept, the inventors developed an antibody against 111b, a soluble cytokine that has pleiotropic effects in cancer, including neoangiogenesis, cancer cell proliferation, migration and metastasis among others. This antibody is conjugated to MMAE or MMAF, which are cytotoxic drugs. The conjugation is performed by means of a lysosomally cleavable peptide, that will be digested in the lysosomal compartment of the target cell, thereby releasing the cytotoxic drug. Lastly, this conjugate is also linked to an endocytosis triggering peptide, either R12 or MGS4, which will drive the internalization of the conjugate into the tumor cells. This conjugate is also called herein AKS conjugate. As shown in the Examples of the present invention, a significant reduction in cell viability was only observed in HeLa cells treated with the Anti IL-1 b MMAE R12 and Anti I L-1 b MMAF R12 Aks (65,4 % and 62,1 % respectively) (Figure 2 a)). Further, NCI-H1299 cells showed a significant reduction in cell viability when treated with either set of AKSs the ones that bore the CPP R12 (anti-111 b MMAE R12 53,7%, anti-111 b MMAF R12 54,5%) and the ones that bore the lung cancer specific peptide MGS4 (anti-111 b MMAE MGS4 12 % 46%, anti-111 b MMAF 12 55,2%), while no reduction in viability was observed in those cells treated with the MMAE or MMAF ADCs (Figure 2 b)). This supports a selective internalizing mechanism of AKSs driven by tumor specific peptides, which could improve both safety and efficiency in delivering cytotoxic drugs to specific cancer cell lines.

[0056] To test whether the endocytosis triggering peptide worked in the conjugate, the inventors investigated the targeted protein degradation of extracellular targets mediated by AKSs. When cells are treated with AKSs, either MMAE R12 or MMAF R12, in presence of bafilomycin A1 (lysosomal inhibitor) there was a significative increase of intracellular levels of I L1 b. This indicates an active internalization of the I L1 b mediated by the AKSs and intracellular accumulation when the lysosomal degradation mechanisms are inhibited (Figure 3 a)). Accordingly, levels of intracellular Anti IL1 b antibody were only detected in those cells treated with AKSs with a significant accumulation in cells treated with bafilomycin A1 (Figure 3 b)). All this evidence supports an active internalization and degradation of I L1 b mediated by AKSs in a LYTAC-like fashion coupled to the delivery of cytotoxic payloads to tumoral cell in an ADC-like fashion. Furthermore, the differential internalization and toxicity of MGS4 AKSs on NCI-H 1299 cells over HeLa cells prove that tumor-selective AKSs can target a soluble protein for its degradation, in this case the IL1b cytokine, while delivering a chemotherapeutic agent (MMAE / MMAF) selectively. This directly overcomes the ADC limitation of targeting only cell membrane proteins.

[0057] Figure 4 also shows the different morphology of cells treated with the AKS of the invention in comparison to control cells or bafilomycin-treated cells.

[0058] Lastly, in order to test whether a specific tumor delivery could be achieved, the inventors tested the specificity of MGS4, a tumor homing peptide, to deliver the AKS of the invention to lung tumor cells. Intracellular levels of the Anti 111 b antibody were positively detected in those cells treated with R12 AKS, independently of their lineage (Figure 6 a)). Moreover, the inhibition of the lysosomal degradation pathways correlated with the detection of higher intracellular levels of the Anti I L1 b antibody, supporting the thesis of R12 AKSs lysosomal shuttling and degradation. Interestingly, for MGS4 ADKs solely in A549 cells significant intracellular levels of Anti IL1 b antibody were observed (Figure 6 b)). This evidence es the versatility of the conjugate of the invention, and its specificity for tumor cells when a tumor homing peptide is used.

[0059] In view of the above results, a first aspect, the present invention provides a conjugate, also referred herein to as “the conjugate of the invention”, comprising at least a first protein, at least a second protein, and at least a cargo of interest.

[0060] In an embodiment, the first protein is bound to the cargo via at least a cleavable or degradable linker, and the second protein is bound to the first protein. Thus, preferably, the first protein is bound to both the cargo and to the second protein. Preferably, the second protein is bound to the first protein via a linker.

[0061] In an embodiment, the conjugate of the invention is represented by the Formula (I): Formula (I):

[0062] (cargo - cleavable or degradable linker)n- first protein - (linker - second protein)m, where “n” represents the number of linker-cargo units linked to the first protein and “m” represents the number of second protein-linker units linked to the first protein. Preferably, n is 1 , 2, 3, 4, 5, 6, or more, more preferably 1. Preferably, m is 1 , 2, 3, 4, 5, 6, or more, more preferably 1. In an embodiment, the first protein and the second protein are part of a single fusion protein.

[0063] Each of the above elements will be defined in detail below. It is understood that all embodiments describing each of those elements can be combined among them.

[0064] The first protein

[0065] The first protein, also called herein the “first protein of the invention”, is capable of binding, preferably specifically binding, to a molecule of interest. Preferably, the molecule of interest is a protein of interest (POI). Preferably, the first protein comprises a targeting moiety that is able to bind a POI, preferably specifically bind a POI. In an embodiment, the POI is a membrane protein or extracellular protein of interest, or a fragment thereof. Preferably, the membrane protein or extracellular protein of interest is present in the extracellular media of a tumor microenvironment. In an embodiment, the POI promotes tumor growth, progression, immunoescape and / or survivability. In an embodiment, the POI promotes or contributes to the individual’s cancer. In an embodiment, the POI is a protein that, when removed from the tumor microenvironment, the tumor decreases in size, malignancy, and / or growth. In an embodiment, the first protein comprises at least one targeting moiety that is configured to bind, preferably specifically bind to, a membrane protein or extracellular protein of interest.

[0066] In another embodiment, the POI is a membrane protein or an extracellular protein of interest that is present when there is tumor growth, progression, immunoescape and / or survivability. Preferably, the POI is a protein that is indicative of the presence of a tumor, or of tumor progression and malignancy. In other embodiments, the POI is a protein that is indicative of the presence of a tumor, and thus it has diagnostic and theragnostic utility.

[0067] In certain embodiments, the membrane protein of interest is a membrane receptor or the ectodomain of a membrane receptor. Membrane receptors of interest include, but are not limited to, stem cell receptors, immune cell receptors, growth factor receptors, cytokine receptors, hormone receptors, receptor tyrosine kinases, a receptor in the epidermal growth factor receptor (EGFR) family (e.g., HER2 (human epidermal growth factor receptor 2), etc.), a receptor in the fibroblast growth factor receptor (FGFR) family, a receptor in the vascular endothelial growth factor receptor (VEGFR) family, a receptor in the platelet derived growth factor receptor (PDGFR) family, a receptor in the rearranged during transfection (RET) receptor family, a receptor in the Eph receptor family, a receptor in the discoidin domain receptor (DDR) family, and a mucin protein (e.g., MUC1).

[0068] The membrane protein of interest may be an immune inhibitory receptor. As used herein, an “immune inhibitory receptor” is a receptor present on an immune cell that negatively regulates an immune response.

[0069] The membrane protein of interest may optionally be a ligand of an immune inhibitory receptor, preferably a receptor known to be overexpressed in cancer cells. The membrane protein of interest may also be an immune checkpoint molecule including immune checkpoint proteins and ligands. As explained above, the first protein comprises a targeting moiety that binds a membrane or extracellular protein of interest. In some embodiments, the targeting moiety binds an extracellular protein of interest.

[0070] The extracellular protein of interest may be a ligand for a membrane receptor. Membrane receptor ligands of interest include, but are not limited to, growth factors (e.g., epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and the like), cytokines (e.g., an interleukin, an interferon, a transforming growth factor p (TGF- P), including any particular subtypes of such cytokines), hormones, and the like.

[0071] The extracellular protein of interest may be an antibody, such as an antibody that binds, preferably specifically binds, to a membrane protein or a different extracellular protein.

[0072] The extracellular protein of interest may be a secreted protein, including, but not limited to, secreted growth factors, extracellular matrix-degrading proteinases, cell motility factors and immunoregulatory cytokines or other bioactive molecules. The extracellular protein may also be a mutated protein, or a fragment thereof.

[0073] In an embodiment, the protein of interest may be present on a cancer cell or produced by a tumor or cancer cell. In some embodiments, the protein of interest is a tumor- associated antigen or a tumor-specific antigen. In an embodiment, the protein of interest is Interleukin ip (IL1 b). In an embodiment, the protein of interest is Epithelial Growth Factor Receptor (EGFR). In an embodiment, the protein of interest is Programmed Death-ligand 1 (PD-L1). In an embodiment, the protein of interest is Interleukin 6 (IL6) In an embodiment, the protein of interest is Vascular Endothelial Growth Factor A (VEGF- A). Preferably, the targeting moiety binds, preferably specifically binds, to IL-1 b, IL-6, EGFR, PD-L1 or VEGF-A, or any combination thereof.

[0074] In an embodiment, the targeting moiety comprised in the first protein is an antibody, anticalin, repebody, monobody, scFv, Fab, scFab, affibody, fynomer, DARPin, nanobody, vNAR, or peptide aptamer that specifically binds to POI. Preferably, the first protein is an antibody or a fragment thereof. In this case, the first protein, when bound to the cargo, and if the cargo is a toxic drug, results in an ADC conjugate. Thus, in an embodiment, the protein conjugate of the invention comprises an ADC component that is formed by an antibody that binds, preferably specifically binds, to a POI and that is bound to a toxic drug. Preferably, the first protein is or comprises an antibody, and it binds, preferably specifically binds, to a protein that promotes tumor growth, progression, immunoescape and / or survivability. In certain embodiments, the antibody binds, preferably specifically binds, to a ligand of an immune inhibitory receptor. In certain embodiment, the antibody binds, preferably specifically binds, to one or more immunodominant epitope(s) within a ligand of an immune inhibitory receptor. In certain embodiments, the antibody binds, preferably specifically binds, to an immune checkpoint molecule. In certain embodiments, the antibody binds, preferably specifically binds, to one or more immunodominant epitope(s) within an immune checkpoint molecule. In certain embodiments, the antibody binds, preferably specifically binds, to the ectodomain of a membrane protein that promotes tumor growth, progression, immunoescape and / or survivability. In certain embodiments, the antibody binds, preferably specifically binds, to I L1 b. In certain embodiments, the antibody binds, preferably specifically binds, to EGFR. In certain embodiments, the antibody binds, preferably specifically binds, to PD-L1. In certain embodiments, the antibody binds, preferably specifically binds, to VEGF-A. In certain embodiments, the antibody binds, preferably specifically binds, to IL-6. Preferably, the first protein comprises or consists of Atezolizumab, Bevacizumab, Canakinumab, Siltuximab and / or Cetuximab.

[0075] In a preferred embodiment, the first protein is an evolved biomolecule, such as an antibody or its derivatives, which may have enhanced functions such as paratopemasking, increased affinity for the epitope and / or pH-modulated affinity, which can change the affinity of the antibody towards the epitope from the endosomal to the lysosomal pH, allowing the AKS to release the target protein at acidic pHs . This may potentially allow for the partial recycling of the AKS through the antibody Fc , even after the loss of its payload in the lysosomes, which may allow the recycled AKS to still direct the target protein for its degradation. Thus, the teachings herein can be applied to create novel AKSs using monoclonal antibodies having properties of interest, such as, for example, antibodies with established therapeutic efficacy.

[0076] In an embodiment, the first protein comprises a targeting moiety comprising a VL domain and a VH domain, wherein: the VH domain comprises, consists, or consists essentially of SEQ ID NO: 3, or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 3; and the VL domain comprises, consists, or consists essentially of SEQ ID NO: 4, or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 4.

[0077] In an embodiment, the first protein comprises a targeting moiety comprising a VL domain and a VH domain, wherein: the VH domain comprises, consists, or consists essentially of SEQ ID NO: 5, or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 5; and the VL domain comprises, consists, or consists essentially of SEQ ID NO: 6, or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 6.

[0078] In an embodiment, the first protein comprises a targeting moiety comprising a VL domain and a VH domain, wherein: the VH domain comprises, consists, or consists essentially of SEQ ID NO: 7, or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 7; and the VL domain comprises, consists, or consists essentially of SEQ ID NO: 8, or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 8.

[0079] In an embodiment, the first protein comprises a targeting moiety comprising a VL domain and a VH domain, wherein: the VH domain comprises, consists, or consists essentially of SEQ ID NO: 9, or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 9; and the VL domain comprises, consists, or consists essentially of SEQ ID NO: 10, or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 10.

[0080] In an embodiment, the first protein comprises a targeting moiety comprising a VL domain and a VH domain, wherein: the VH domain comprises, consists, or consists essentially of SEQ ID NO: 11 , or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 11 ; and the VL domain comprises, consists, or consists essentially of SEQ ID NO: 12, or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO: 12. The second protein

[0081] The second protein, also called herein “the second protein of the invention”, is characterized in that it comprises an endocytosis-triggering peptide which is capable of binding to an endocytic receptor of a tumor cell and trigger the internalization of the receptor along the conjugate of the invention bound to the POI. Thus, the binding of the endocytosis-triggering peptide to the endocytic receptor of a tumor cell causes the endocytosis or internalization of the conjugate and the POI inside the tumor cell. The second protein acts as a shuttle protein for the internalization of the conjugate of the invention and the POI bound to thereto.

[0082] In an embodiment, the endocytic receptor of the tumor cell delivers the conjugate of the invention, and the POI bound thereto to the lysosomal compartment of the tumor cell, wherein the components of the conjugate and the POI are degraded by lysosomal proteolytic enzymes. This process also releases the cargo bound to the conjugate of the invention, as will be explained in detail below.

[0083] Thus, in an embodiment, the endocytic receptor is a lysosomal-shuttling receptor, that will internalize the POI and the conjugate of the invention and sorts them into the lysosomal compartment of the tumor cell. In view of this, it can be considered that the second protein comprises a lysosomal-targeting chimera component, that directs the internalization of the POI and the conjugate into a tumor cell, and promotes its degradation, preferably its lysosomal degradation. Preferably, the endocytic receptor is exclusively present or highly overexpressed on the surface of cancer cells and will deliver the conjugate of the invention and the POI bound thereto to the lysosomal compartment of said tumor cells. In the context of the present invention, by “highly overexpressed” is referred herein to expression levels that are at least 1.2, 1.3, 1.5, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2-fold change higher in said tumor cell in comparison to a healthy, control cell. Preferably, the healthy, control cell is the same cell type as the tumor cell. The expression levels of a protein can be measured by techniques known in the art, such as flow cytometry or western blot.

[0084] The second protein can also possess other desirable capabilities such as selective toxicity towards cancer cells, receptor signal blockage, targeting specific phenotypes of tumor cells and / or their environment (e.g. tumor neovasculature), enhanced tumor penetration, enhanced tumor retention, etc. Thus, increasing the concentration and improving the distribution of the conjugate within the tumor. In certain embodiments, the second protein is a cell-penetrating peptide (CCP), a tumor penetrating-peptide (TPP), a tumor-homing peptide (THP), a tissue-specific homing peptide (TSHP) , and RGD polypeptide, an internalizing-RGD (iRGD) polypeptide, a Lyp- 1 polypeptide, a cripto-1 binding polypeptide, a somatostatin receptor binding polypeptide, a prohibitin binding polypeptide, a NGR polypeptide, an iNGR polypeptide, a molecular guidance system (MGS) peptide, an activatable cell penetrating peptide (ACPP) comprised of a polycationic cell CPP (e.g. R12) connected via a cleavable linker to a neutralizing polyanion, or the combination thereof.

[0085] In some embodiments, the second protein comprises a targeting moiety, an antibody or an antigen-binding antibody fragment. In an embodiment, the second protein comprises a targeting moiety against EFGR and / or PD-L1. In an embodiment, the second protein comprises or consists of atezolizumab or cetuximab.

[0086] In an embodiment, the second protein comprises or consists of SEQ ID NO: 1 , or an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to SEQ ID NO: 1. In an embodiment, the second protein comprises or consists of SEQ ID NO: 2, or an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to SEQ ID NO: 2. In an embodiment, the second protein comprises or consists of SEQ ID NO: 13, or an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to SEQ ID NO: 13. In an embodiment, the second protein comprises or consists of SEQ ID NO: 14, or an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to SEQ ID NO: 14.

[0087] Linker between the first and the second proteins.

[0088] In an embodiment, the second protein further comprises a linker or connector that binds the second to the first protein. Thus, second protein is preferably characterized by: i. being bound to the first protein of the invention, and ii. comprising an endocytosis-triggering peptide, as explained above. In an embodiment, the linker between the first and the second protein is a linker peptide. “Linker peptide” as used herein is a short peptide sequence that is located between the two monomers of the fusion dimer. Linker peptides are placed to provide the two monomers comprised in the fusion dimer with movement flexibility. In the context of the present invention, the linker peptide has at least one amino acid residue, preferably at least two consecutive amino acid residues, optionally 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues. The linker peptide includes flexible linkers, rigid linkers, and in vivo cleavable linkers. Thus, preferably, the first and the second protein are part of a single polypeptide, i.e., a fusion protein that has been created through the joining of the first and second proteins. Linker peptides can provide and be selected to increase protein stability, folding, expression and biological activity, modify pharmacokinetics, enable targeting, etc. Examples of Linker peptides include, but are not limited to, (GGGGS)n, (GGGS)n, (GGS)n, (G3)n, (GS)nG, GSAGSAAGSGEF, (EAAAK)n, A(EAAAK)n, A(EAAAK)nA, ALEA(EAAAK)n, A(EAAAK)nALEA((EAAAK)nA, AEAAAKE- AAAKA, (AP)n, (PAPAP)n, (P)n, (PPG)n, (XP)n (where X= A, K, E), (lEALEGK)n, (IE- TVEK)n, LEAGCKNFFPRSFTSCGSLE, KESGSVSSEQLAQFRSLD, GEWTYDDAT- KTFTVTE, (DEDEG)n, (EEEEK)n, EGKSSGSGSESKST, LVPRGS, ENLYFQ, RVLAEA, PLGLWA, GGIEGRGS, TRHRQPRGWE, VSQTSKLTRAETVFPDV, EDVVCCSMSY, AGNRVRRSVG, RRRRRRRRR, (GFLG)n, (LE)n, SS, CRRRRRREAEAC. Wherein n is greater than or equal to 1. In one embodiment, n is between 1 and 20, inclusive. In one embodiment, n=l. In one embodiment, n=2. In another embodiment, n=3. In another embodiment, n=4. In another embodiment, n=5. In yet another embodiment, n=6. In another embodiment, n=7. In yet another embodiment, n=8. In another embodiment, n=9. In yet another embodiment, n=10.

[0089] The linkers are preferably chemically stable to conditions outside the cell, and may be designed to cleave, immolate and / or otherwise specifically degrade inside the cell or in specific extracellular milieus such as a tumor microenvironment.

[0090] In an embodiment the linker between the first and the second protein is a chemical linker. The linker can provide and be selected to generate spatial distance and certain degree of flexibility between the first and second protein to avoid steric hindrances and facilitate the ternary complex formation. The linker may have a valency from 1 to 15 or be a flexible linker molecule such as PEG. The selection of the linker is based on properties such as its biocompatibility, solubility in organic and aqueous media, and low immunogenicity. In a preferred embodiment, the linker provides a length enough between the first and second protein, which allows simultaneous engagement of both the cell surface endocytic receptor and the protein of interest.

[0091] In a more preferred embodiment, the linker is selected from polyethylene glycol linkers, polypropylene glycol liners, polyethylene glycol-co-polypropylene glycol oligomers, polyamino acid liners, alkane linkers, cycloalkane linkers or heterocyclic linkers.

[0092] When a chemical linker is used, the sequence of the second protein can be modified by the addition or substitution of at least one amino acid in its sequence, to introduce a new chemoselective moiety in the peptide structure. A chemoselective moiety is chemical moiety that reacts selectively to a functional group or groups in presence of others. The addition / substitution may be done either internally or in one of the ends of the amino acid sequence of the second protein. In a more preferred embodiment, when the second protein is a peptide, its sequence is modified by the addition of an extra amino acid, whether it is canonical, non-canonical or unnatural (for example, cysteine or azido- Lysine), at the N or / and C terminal end. For the present disclosure, modifications are made to introduce specific chemical groups that would ensure orthogonality and chemoselectivity in the synthesis of the AKS. The modified peptides for the second protein may include modifications at both N and C terminal ends. Peptides can also include unnatural amino acids or substitute in their sequence L amino acids for D amino acids to increase their stability against host proteases.

[0093] In a more preferred embodiment, the linker is selected from polyethylene glycol linkers, polypropylene glycol linkers, polyethylene glycol-co-polypropylene glycol oligomers, polyamino acids linkers, alkane chain linkers, cycloalkanes linkers or heterocyclic linkers. Preferably, the linker comprises maleimide, Polyethylene glycol (PEG), and succinimide, or any derivative thereof. Preferably, the PEG is PEGn, wherein “n” is a natural number, preferably 1 , 2, 3 4, 5, 6, 7, 8, 9, 10 or more than 10. Preferably, “n” is any number between 1-50, preferably 1-25, more preferably 5-20. Also preferably, “n” is 4, 8, 12, or 24. Most preferably, “n” is 6.

[0094] Preferably, the linker comprises maleimide-PEGe-N-hidroxysuccinimide, or a derivative thereof. Preferably, the linker comprises Maleimide-PEGe-succinimidyl ester or maleimide-PEG6-N-Hydroxysuccinimidyl ester. Preferably, the linker comprises Maleimide-PEGe-succinimidyl ester and comprises or consists of the formula (II): Formula (II):

[0095] Another example is the use of heterobifunctional linkers that bear an azide group or alkyne moiety (e.g.2-propynyl) in one end of the linker, to ligate preferably to the second protein through a CuAAC (copper (l)-catalyzed alkyne-azide cycloaddition) reaction, and, in the other end of the linker, an amine reactive ester, such as Succinimidyl or Tetrafluorophenil esters, or a thiol reactive moiety, such as a maleimide ring, to ligate preferably with the first protein.

[0096] When one of the moieties of the AKS is a peptide or a polypeptide (such an antibody), a chemoselective conjugation of the first and second proteins can be achieved targeting specific amino acids that bear a chemoselective group. Specific amino acids may be canonical, non-canonical or unnatural origin and can be located or introduced in the N- terminal or C-terminal ends or internally to the protein. Amino acids bearing chemoselective groups for protein ligation can be introduced during the protein synthesis either chemically, enzymatically or by genetic expression.

[0097] Preferred non-canonical and unnatural amino acids bearing a chemical group for orthogonal bioconjugation are 6-azido-L-lysine, N-e-propargyloxicarbonyl-L-lysine, 3- azido-L-alanine, 4-azido-L-homoalanine, 4-azido-L-phenylalanine, (S)-2-aminohex-5- y,noic acid, p-acetylphenylalanine, 4-propargyloxy-L-phenylalanine, L-pyrrolysine, L- selenocysteine, 3-(6-acetylnaphtalen-2-yl-amino)-2-aminopropanoic acid, N-e-(cyclooct- 2-yn-1-yloxy)carbonyl)-L-lysine, N-e-acryllysine or cyclopropane-L-lysine.

[0098] The selection of a specific chemical group, to direct the chemoselective ligation, defines the generation of the specific connector moieties as part of the AKS. Multiple strategies have been developed for the orthogonal bioconjugation of peptides and polypeptides into other molecules. Examples of chemoselective reactions, but not limited to, are copper- catalyzed alkyne-azide cycloaddtion chemistry, strain-promoted alkyne-azide cycloadditions, copper free click chemistry, copper catalyzed click chemistry, strain- promoted alkyne-nitrone cycloaddition, amine-active ester coupling, thiol-maleimide, thiol-haloacetamide additions, alkyne hydrothiolation, imine-establishing reactions (oximes and hydrazones), Staudinger ligation, tetrazine ligation, hydrazine-iso-Pictet- Spengler ligation, etc.

[0099] In an embodiment, a functional group (e.g.an amino group, a thiol group, a carboxylic acid group) of an amino acid, in the first or second protein, may be modified in order to introduce a specific chemoselective group. These modifications may include the use of thiolation reagents such as the Traut’s reagent (2-iminothiolane), haloacetyls, maleimides, aziridines, acryoloys, arylating agents, vinylsulfones, pyridyl disulfides, TNB-thiols and disulfide reducing agents. Other modifications, such as azides, alkynes, cicloalkynes, etc, may be introduced via coupling reagents, that can also bear or not a linker, bearing the chemoselective group, such as N-succinimidyl-linker-Azide, N- succinimidyl S-acetylthioacetate (SATA), Azido-Linker-Maleimide, N-succinimidyl-linker- DBCO, etc.

[0100] The cargo of interest and the cleavable or degradable linker

[0101] As explained above, a component of the conjugate of the invention is a cargo of interest.

[0102] Said cargo is bound to the first protein of the invention by means of at least a cleavable or degradable linker. Linkers that bind the cargo to the first protein may account for: (i) high plasma stability to minimize untimely cargo release, (ii) preservation of the properties of the first protein and cell-killing capability of the cargo, (iii) high agueous solubility to facilitate conjugation of lipophilic cargos and prevent first protein aggregation, and (iv) cargo release in target environments to maximize the therapeutic effect.

[0103] Cleavable linkers may comprise of two parts: (i) the protein-linker attachment, which defines the bioconjugation methods, the cargo-to-protein ratio and the attachment sites in the first protein, and (ii) the linker-cargo attachment, which controls the cleavage mechanism and the release rate both on- and off-target and defines the chemical nature of the active cargo. Bioconjugation methods for attaching the linker to the peptide are widely known in the art, some of which are common to the strategies to linking the first and second protein and are summarized herein in the section “Linker between the first and the second proteins”. In one embodiment, the linker between the cargo and the first protein is a cleavable linker or comprises a cleavable sequence. In certain embodiments, the linker may be selected or designed to cleave, immolate and / or otherwise specifically degrade in vivo or inside the cell. Cleavable linkers may include chemically or enzymatically sensitive linkages. Cleavable linkers generally rely on processes inside the cell to liberate the cargo, such as reduction in the cytoplasm, exposure to acidic conditions in the lysosome, or cleavage by specific proteases or other enzymes within the cell. Cleavable linkers generally incorporate one or more chemical bonds that are either chemically or enzymatically cleavable while the remainder of the linker may be non-cleavable.

[0104] Preferably, the cleavage or degradable linker is an intracellular cleavable or degradable linker. By “intracellular” is referred herein to a linker that is mainly cleaved or degraded inside the cell, such as inside the lysosomal compartment of the cell. However, intracellular linkers may also be cleaved or degraded in the extracellular media, if the conditions that lead to the cleavage or degradation of the linker are also present in the extracellular media. Preferably, the linker is only cleaved or degraded inside the cell, but not in the extracellular media of the cell.

[0105] In one embodiment, the cleavable linker is a chemically cleavable linker, such as an acid cleavable / pH sensitive linker that is stable (i.e., remains intact) at neutral pH (bloodstream pH 7.3-7.5) but undergoes hydrolysis upon internalization into the mildly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0) of a cancer cell (e.g., a cancer cell) or a reduction-sensitive linker, such as disulfide-bond linkers, which are sensitive to the different reduction potential in the intracellular compartment versus plasma. Chemically labile linkers include, but are not limited to, hydrazone-based linkers, oxime-based linkers, carbonate-based linkers, ester-based linkers, acetal-based linkers, N-ethoxybenzylimidazole linkers, maleic acid-based linkers, disulfide-based linkers, etc. In certain aspects, a linker comprises a chemically labile group such as hydrazone and / or disulfide groups.

[0106] To increase the stability of the hydrazone group of the linker, the linker may be varied by chemical modification, e.g., substitution, allowing tuning to achieve more efficient release in the lysosome with a minimized loss in circulation. Hydrazone-containing linkers may contain additional cleavage sites, such as additional acid-labile cleavage sites, reduction-labile cleavage sites and / or enzymatically labile cleavage sites. Other pH- labile groups that may be incorporated into linkers include maleic acid-derived linkers, such as cis-aconityl groups. Cis-Aconityl chemistry is based on the intramolecular cyclization of maleoyl amide at a pH lower than the pKa of the free carboxylic acid, which is juxtaposed to an amide bond to promote amide hydrolysis under acidic conditions.

[0107] Cleavable linkers may also include a disulfide group. Disulfides are thermodynamically stable at physiological pH and are designed to release the cargo upon cell internalization, wherein the intracellular milieu provides a significantly more reducing environment compared to the extracellular environment. Disulfide bonds are susceptible to nucleophilic attack by thiols, such as (reduced) glutathione (GSH). Disulfide-containing linkers are reasonably stable in circulation, wherein the dominant thiol species in the reduced form of human serum albumin, whose activity toward disulfide bonds is largely hindered due to the steric blockage of the albumin free thiol-containing residue, which is located in a crevice with limited solvent exposure. In contrast, the intracellular milieu contains high levels of (reduced) glutathione, which selectively releases the cargo upon its internalization by the cells. The intracellular enzyme protein disulfide isomerase, or similar enzymes capable of cleaving disulfide bonds, may also contribute to the preferential cleavage of disulfide bonds inside cells. In certain embodiments, the in vivo stability of a disulfide-containing linker may be enhanced by chemical modification of the linker, e.g., use of steric hinderance adjacent to the disulfide bond by the introduction of two methyl groups to the a-carbons bearing the disulfide bonds. The disulfide bond may be internal to the linker or be formed with naturally occurring or engineered first protein cysteine residues.

[0108] Another type of cleavable linker that may be used to connect the cargo with the first protein is a linker that is specifically cleaved by an enzyme. Such linkers are typically peptide-based or include peptidic regions that are preferentially specific substrates for lysosomal proteases.

[0109] Thus, in an embodiment, the cleavable linker is an enzyme-labile linker, such as an enzyme-labile linker that is stable in the bloodstream tumor but undergoes enzymatic cleavage upon internalization into the cancer target cell and / or in the tumor extracellular microenvironment. Enzyme-labile linkers include, but are not limited to, linkers that are cleaved by lysosomal enzymes such as cathepsins, phosphatases, pyrophosphatases, sulfatases, p-galactosidase and p-glucuronidase.

[0110] In an embodiment, the enzyme-labile linker is a lysosomal cleavable peptide that is cleaved in the lysosome of the cancer cell e.g., by a lysosomal protease (such as cathepsin or plasmin). Dipeptide-based linkers such as valine-citrulline (Val-Cit) linkers (maleimidocaproyl-valine-citruline-p-aminobenzyl (e.g. MC-Val-Cit-PAB) linker), , phenylalanine-lysine (Phe-Lys) linkers (e.g. (S)-2-((S)-2-Amino-N-(4-

[0111] (hydroxymethyl)phenyl)-3-phenylpropanamido)-6-(tritylamino)hexanamide (Fmoc-Phe- Lys(Trt)-PAB) linker) and valine-alanine (Val-Ala) linkers (maleimidocaproyl-valyl-alanyl- para-aminobenzyloxy (Mc-Val-Ala-PAB) linker), are linkers designed to be cleaved by lysosomal cathepsins at the amide bond between the P1 residue of the dipeptide (e.g. citrulline) and PT of a self-immolating moiety (e.g, para-aminobenzyl carbamate (PABC)). Cathepsins are overexpressed in tumor cells, and their activity outside the tumor microenvironment is low. The plasma stability of dipeptide linkers can be greatly enhanced by the addition of a polar acidic residue, such as glutamine, at the P3 position in dipeptides and P4 position in tripeptides and so forth (e.g. Glu-Val-Cit vs Val-Cit). In addition, to increase linker cleavage in the tumor environment, peptides can be modified to specifically target Cathepsin B, which is more implicated in tumor progression than other lysosomal proteases, thus increasing the therapeutic window. This can be achieved by removing the P1-P2 amide bond while retaining critical H-bond interactions, for example by replacing the valine amino acid with the cyclobutane- 1 ,1 -dicarboxamide (cBu) moiety.

[0112] In another embodiment, the cleavable linker is a p-glucuronic acid-based linker / Glucoronide linker, such as 2-Amino-4(hydroxymethyl)phenyl methyl 2,3,4-tri-O- acetyl-p-D-glucopyranosiduronate. Glycosidases, such as p-glucuronidases, are a class of hydrolytic lysosomal enzymes that degrade b-glucuronic residues into polysaccharides. The cleavage of the p-glucuronide glycosidic bond by the lysosomal enzyme p-glucuronidase allows easy release of the cargo upon internalization, p- glucuronidases are present abundantly within lysosomes and are overexpressed in several tumor types, while p-glucuronidase activity outside cells and tumor microenvironment is low. p-galactosidase is another class of hydrolytic lysosomal enzyme that degrades p-glycosidic bonds formed between a galactose and another organic moiety which may or may not be another carbohydrate, p-galactosidase- sensitive linkers, such as the maleimidocaproyl-galactoside-p-aminobenzyl, selectively release the cargo upon internalization, in the lysosomal compartment by the action of the p-galactosidase, which its extracellular activity is negligible. Like many other lysosomal enzymes, p-galactosidase is overexpressed in tumor cells. Different cleavable linkages can be incorporated into a single cleavable linker to improve the stability, cargo release, functionality, or to reduce undesirable side effects of the conjugate.

[0113] Enzymatically cleavable linkers may include a self-immolative spacer to spatially separate the cargo from the site of enzymatic cleavage.

[0114] Of course, cleavable linkers may include noncleavable portions or segments, and / or cleavable segments or portions may be included in an otherwise non-cleavable linker to render it cleavable.

[0115] In another embodiment, the linker between the cargo and the first protein is a degradable linker. Degradable linkages include, but are not limited to, ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a biologically active agent, wherein such ester groups generally hydrolyze under physiological conditions to release the biologically active agent. Hydrolytically degradable linkages include, but are not limited to, carbonate linkages; imine linkages resulting from reaction of an amine and an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; acetal linkages that are the reaction product of an aldehyde and an alcohol; orthoester linkages that are the reaction product of a formate and an alcohol; and oligonucleotide linkages formed by a phosphoramidite group, including but not limited to, at the end of a polymer, and a 5'-hydroxyl group of an oligonucleotide.

[0116] In a preferred embodiment, the linker connecting the cargo and the first protein comprises a maleimide Valine-Citruline linker.

[0117] Preferably, the linker connecting the cargo and the first protein comprises or consists of Formula (III) or (IV):

[0118] Formula (III):

[0119]

[0120] VcMMAE Chemical Structure

[0121] Formula (IV):

[0122] MC-Vai-Cit-PAB-MMAF Chemical

[0123] Structure

[0124] As explained above, the cleavable or degradable linker connects or binds the cargo of interest to the first protein of the invention.

[0125] In an embodiment, the cargo of interest is a drug that is toxic for tumor cells. Hence, I a preferred embodiment, the conjugate is represented by the formula (V): (toxic drug - cleavable or degradable linker)n- first protein of the invention- (linker — second protein of the invention)m, where “n” represents the number of linker-cargo units linked to the first protein and “m” represents the number of second protein-linker units linked to the first protein. Preferably, n is 1 , 2, 3, 4, 5, 6, or more, more preferably 1. Preferably, m is 1 , 2, 3, 4, 5, 6, or more, more preferably 1. In an embodiment, the first protein and the second protein are part of a single fusion protein.

[0126] The toxic drug can be any compound, such as chemical compounds, small molecules, proteins, nucleic acids, etc., as long as it is toxic to a tumor cell. Preferably, the toxic drug is toxic only for tumor cells and not for healthy cells. By “toxic drug” is referred herein to a drug or a molecule that reduces or inhibits tumor cell’s survivability, growth, and / or replication. Preferably, a toxic drug is characterized by being capable of killing the tumor cell upon exposure to said drug. In an embodiment, tumor cell’s survivability, growth, and / or replication in reduced in at least 10%, 20, 30, 40, 50, 60, 70, 80, 90, or 100% in the presence of the toxic drug as compared to the same tumor cell type that has not been exposed or treated with said toxic drug. Methods to measure tumor cell’s survivability, growth, and / or replication are known in the art. Preferably, the method to measure tumor cell’s survivability, growth, and / or replication are for in vivo models the measurement of tumor volume and for in vitro models cell death, proliferation and viability assays. For instance, the cell’s survivability can be measured by flow cytometry to distinguish live and dead cell populations.

[0127] Preferably, the toxic drug is a cytotoxic and / or cytostatic agent. The cytotoxic and / or cytostatic agents may be any agents known to inhibit the growth and / or replication of and / or kill cells, and in particular cancer and / or tumor cells.

[0128] In a more preferred embodiment, the cytotoxic and / or cytostatic agent are selected from, monomethylauristatin E (MMAE), monomethyauristatin F (MMAF), N-acetyl g calicheamicin, DM1 , DM4, deruxtecan, pyrrolobenzodiazepine (PBD) dimer.

[0129] In another embodiment, the cargo of interest is a radiopharmaceutical that has utility in theragnostic methods. In another embodiment, the cargo of interest is a compound that has utility in theragnostic, imaging, and / or diagnostics methods. In an embodiment, the cargo of interest is selected from the group consisting of89Zr,18F, and "Tc. Preferred of the invention are:

[0130] Conjugate 1. In an embodiment, the conjugate of the invention comprises: i. a first protein that is capable of binding to, and preferably specifically binds to, IL-1B, ii. a second protein that is bound to the first protein and that comprises a peptide comprising or consisting of SEQ ID NO: 1 , and iii. a cargo, preferably a drug that is toxic for tumor cells, wherein the cargo is bound to the first protein via an intracellular cleavable or degradable linker, wherein the binding of the endocytosis-triggering peptide to the endocytic receptor of the tumor cell causes the internalization of the conjugate and IL-1 B inside the tumor cell.

[0131] Conjugate 2: In an embodiment, the conjugate of the invention comprises: i. a first protein that is capable of binding to, and preferably specifically binds to, IL-1B, ii. a second protein that is bound to the first protein and that comprises a peptide comprising or consisting of SEQ ID NO: 2, and iii. a cargo, preferably a drug that is toxic for tumor cells, wherein the cargo is bound to the first protein via an intracellular cleavable or degradable linker, wherein the binding of the endocytosis-triggering peptide to the endocytic receptor of the tumor cell causes the internalization of the conjugate and IL-1 B inside the tumor cell.

[0132] Conjugate 3: In an embodiment, the conjugate of the invention comprises: i. a first protein that is capable of binding to, and preferably specifically binds to, IL-1 B, ii. a second protein that is bound to the first protein and that comprises a peptide comprising or consisting of SEQ ID NO: 13, and iii. a cargo, preferably a drug that is toxic for tumor cells, wherein the cargo is bound to the first protein via an intracellular cleavable or degradable linker, wherein the binding of the endocytosis-triggering peptide to the endocytic receptor of the tumor cell causes the internalization of the conjugate and IL-1 B inside the tumor cell.

[0133] Conjugate 4: In an embodiment, the conjugate of the invention comprises: i. a first protein that is capable of binding to, and preferably specifically binds to, PD-L1 , ii. a second protein that is bound to the first protein and that comprises a peptide comprising or consisting of SEQ ID NO: 13, and iii. a cargo, preferably a drug that is toxic for tumor cells, wherein the cargo is bound to the first protein via an intracellular cleavable or degradable linker, wherein the binding of the endocytosis-triggering peptide to the endocytic receptor of the tumor cell causes the internalization of the conjugate and PD-L1 inside the tumor cell.

[0134] Conjugate 5: In an embodiment, the conjugate of the invention comprises: i. a first protein that is capable of binding to, and preferably specifically binds to, PD-L1 , ii. a second protein that is bound to the first protein and that comprises a peptide comprising or consisting of SEQ ID NO: 1 , and iii. a cargo, preferably a drug that is toxic for tumor cells, wherein the cargo is bound to the first protein via an intracellular cleavable or degradable linker, wherein the binding of the endocytosis-triggering peptide to the endocytic receptor of the tumor cell causes the internalization of the conjugate and of PD-L1 inside the tumor cell.

[0135] Conjugate 6: In an embodiment, the conjugate of the invention comprises: i. a first protein that is capable of binding to, and preferably specifically binds to, PD-L1 , ii. a second protein that is bound to the first protein and that comprises a peptide comprising or consisting of SEQ ID NO: 2, and iii. a cargo, preferably a drug that is toxic for tumor cells, wherein the cargo is bound to the first protein via an intracellular cleavable or degradable linker, wherein the binding of the endocytosis-triggering peptide to the endocytic receptor of the tumor cell causes the internalization of the conjugate and PD-L1 inside the tumor cell.

[0136] Conjugate 7: In an embodiment, the conjugate of the invention comprises: i. a first protein that is capable of binding to, and preferably specifically binds to, IL-6, ii. a second protein that is bound to the first protein and that comprises a peptide comprising or consisting of SEQ ID NO: 13, and iii. a cargo, preferably a drug that is toxic for tumor cells, wherein the cargo is bound to the first protein via an intracellular cleavable or degradable linker, wherein the binding of the endocytosis-triggering peptide to the endocytic receptor of the tumor cell causes the internalization of the conjugate and IL-6 inside the tumor cell.

[0137] Conjugate 8: In an embodiment, the conjugate of the invention comprises: i. a first protein that is capable of binding to, and preferably specifically binds to, IL-6, ii. a second protein that is bound to the first protein and that comprises a peptide comprising or consisting of SEQ ID NO: 1 , and iii. a cargo, preferably a drug that is toxic for tumor cells, wherein the cargo is bound to the first protein via an intracellular cleavable or degradable linker, wherein the binding of the endocytosis-triggering peptide to the endocytic receptor of the tumor cell causes the internalization of the conjugate and IL-6 inside the tumor cell.

[0138] Conjugate 9: In an embodiment, the conjugate of the invention comprises: i. a first protein that is capable of binding to, and preferably specifically binds to, IL-6, ii. a second protein that is bound to the first protein and that comprises a peptide comprising or consisting of SEQ ID NO: 2, and iii. a cargo, preferably a drug that is toxic for tumor cells, wherein the cargo is bound to the first protein via an intracellular cleavable or degradable linker, wherein the binding of the endocytosis-triggering peptide to the endocytic receptor of the tumor cell causes the internalization of the conjugate and IL-6 inside the tumor cell.

[0139] Conjugate 10: In an embodiment, the conjugate of the invention comprises: i. a first protein that is capable of binding to, and preferably specifically binds to, VEGF-A, ii. a second protein that is bound to the first protein and that comprises a peptide comprising or consisting of SEQ ID NO: 13, and iii. a cargo, preferably a drug that is toxic for tumor cells, wherein the cargo is bound to the first protein via an intracellular cleavable or degradable linker, wherein the binding of the endocytosis-triggering peptide to the endocytic receptor of the tumor cell causes the internalization of the conjugate and VEGF-A inside the tumor cell.

[0140] Conjugate 11 : In an embodiment, the conjugate of the invention comprises: i. a first protein that is capable of binding to, and preferably specifically binds to, VEGF-A, ii. a second protein that is bound to the first protein and that comprises a peptide comprising or consisting of SEQ ID NO: 1 , and iii. a cargo, preferably a drug that is toxic for tumor cells, wherein the cargo is bound to the first protein via an intracellular cleavable or degradable linker, wherein the binding of the endocytosis-triggering peptide to the endocytic receptor of the tumor cell causes the internalization of the conjugate and VEGF-A inside the tumor cell.

[0141] Conjugate 12: In an embodiment, the conjugate of the invention comprises: i. a first protein that is capable of binding to, and preferably specifically binds to, VEGF-A, ii. a second protein that is bound to the first protein and that comprises a peptide comprising or consisting of SEQ ID NO: 2, and iii. a cargo, preferably a drug that is toxic for tumor cells, wherein the cargo is bound to the first protein via an intracellular cleavable or degradable linker, wherein the binding of the endocytosis-triggering peptide to the endocytic receptor of the tumor cell causes the internalization of the conjugate and VEGF-A rest inside the tumor cell.

[0142] Conjugate 13: In an embodiment, the conjugate of the invention comprises: i. a first protein that is capable of binding to, and preferably specifically binds to, EGFR, ii. a second protein that is bound to the first protein and that comprises a peptide comprising or consisting of SEQ ID NO: 13, iii. a cargo, preferably a drug that is toxic for tumor cells, wherein the cargo is bound to the first protein via an intracellular cleavable or degradable linker, wherein the binding of the endocytosis-triggering peptide to the endocytic receptor of the tumor cell causes the internalization of the conjugate and EGFR inside the tumor cell.

[0143] Conjugate 14: In an embodiment, the conjugate of the invention comprises: i. a first protein that is capable of binding to, and preferably specifically binds to, EGFR, ii. a second protein that is bound to the first protein and that comprises a peptide comprising or consisting of SEQ ID NO: 1 , and iii. a cargo, preferably a drug that is toxic for tumor cells, wherein the cargo is bound to the first protein via an intracellular cleavable or degradable linker, wherein the binding of the endocytosis-triggering peptide to the endocytic receptor of the tumor cell causes the internalization of the conjugate and EGFR inside the tumor cell.

[0144] Conjugate 15: In an embodiment, the conjugate of the invention comprises: i. a first protein that is capable of binding to, and preferably specifically binds to, EGFR, ii. a second protein that is bound to the first protein and that comprises a peptide comprising or consisting of SEQ ID NO: 2, and iii. a cargo, preferably a drug that is toxic for tumor cells, wherein the cargo is bound to the first protein via an intracellular cleavable or degradable linker, wherein the binding of the endocytosis-triggering peptide to the endocytic receptor of the tumor cell causes the internalization of the conjugate and EGFR inside the tumor cell.

[0145] Conjugate 16: In an embodiment, the conjugate of the invention comprises: i. a first protein that is capable of binding to, and preferably specifically binds to, EGFR, ii. a second protein that is bound to the first protein and that comprises a peptide comprising or consisting of SEQ ID NO: 14, and iii. a cargo, preferably a drug that is toxic for tumor cells, wherein the cargo is bound to the first protein via an intracellular cleavable or degradable linker, wherein the binding of the endocytosis-triggering peptide to the endocytic receptor of the tumor cell causes the internalization of the conjugate and EGFR inside the tumor cell.

[0146] Conjugate 17: In an embodiment, the conjugate of the invention comprises: i. a first protein that is capable of binding to, and preferably specifically binds to, VEGF-A, ii. a second protein that is bound to the first protein and that comprises a peptide comprising or consisting of SEQ ID NO: 14, and iii. a cargo, preferably a drug that is toxic for tumor cells, wherein the cargo is bound to the first protein via an intracellular cleavable or degradable linker, wherein the binding of the endocytosis-triggering peptide to the endocytic receptor of the tumor cell causes the internalization of the conjugate and VEGF-A inside the tumor cell.

[0147] Conjugate 18: In an embodiment, the conjugate of the invention comprises: i. a first protein that is capable of binding to, and preferably specifically binds to, PD-L1 , ii. a second protein that is bound to the first protein and that comprises a peptide comprising or consisting of SEQ ID NO: 14, and iii. a cargo, preferably a drug that is toxic for tumor cells, wherein the cargo is bound to the first protein via an intracellular cleavable or degradable linker, wherein the binding of the endocytosis-triggering peptide to the endocytic receptor of the tumor cell causes the internalization of the conjugate and PD-L1 inside the tumor cell.

[0148] Conjugate 19: In an embodiment, the conjugate of the invention comprises: i. a first protein that is capable of binding to, and preferably specifically binds to, IL-6, ii. a second protein that is bound to the first protein and that comprises a peptide comprising or consisting of SEQ ID NO: 14, and iii. a cargo, preferably a drug that is toxic for tumor cells, wherein the cargo is bound to the first protein via an intracellular cleavable or degradable linker, wherein the binding of the endocytosis-triggering peptide to the endocytic receptor of the tumor cell causes the internalization of the conjugate and IL-6 inside the tumor cell.

[0149] Conjugate 20: In an embodiment, the conjugate of the invention comprises: i. a first protein that is capable of binding to, and preferably specifically binds to, IL-1B, ii. a second protein that is bound to the first protein and that comprises a peptide comprising or consisting of SEQ ID NO: 14, and iii. a cargo, preferably a drug that is toxic for tumor cells, wherein the cargo is bound to the first protein via an intracellular cleavable or degradable linker, wherein the binding of the endocytosis-triggering peptide to the endocytic receptor of the tumor cell causes the internalization of the conjugate and IL-1 B inside the tumor cell.

[0150] Preferably, the linker between the first and the second protein of conjugates 1-20 comprises or consists of maleimide-PEGe-N-hidroxysuccinimide, wherein “n” represents a natural number of between 1-100, preferably 1-50, more preferably 5-25. Preferably, the linker between the first and the second protein of conjugates 1-20 comprises or consists of maleimide-PEG6-N-hidroxysuccinimide or a derivative thereof. Preferably, the cleavable or degradable linker comprised in conjugates 1-20 comprises or consists of maleimide Valine-Citruline or a derivative thereof. Preferably, in conjugates 1-20, the intracellular cleavable or degradable linker comprises maleimide Valine-Citruline or derivatives thereof, and the linker between the first and the second protein comprises maleimide-PEGn-N-hidroxysuccinimide, wherein “n” is a natural number, preferably 1 , 2, 3 4, 5, 6, 7, 8, 9, 10 or more than 10. Preferably, “n” is any number between 1-50, preferably 1-25, more preferably 5-20. Also preferably, “n” is 4, 8, 12, or 24. Most preferably, “n” is 6. Preferably, the linker between the first and the second protein of conjugates 1-20 comprises maleimide-PEG6-N-hidroxysuccinimide and the intracellular cleavable or degradable linker comprises maleimide Valine-Citruline or derivatives thereof. Preferably, the linker between the first and the second protein of conjugates 1- 20 comprises maleimide-PEG6-N-Hydroxysuccinimidyl ester and the intracellular cleavable or degradable linker comprises maleimide Valine-Citruline or derivatives thereof. Preferably, the linker between the first and the second protein of conjugates 1- 20 consists of maleimide-PEG6-N-Hydroxysuccinimidyl ester and the intracellular cleavable or degradable linker consists of maleimide Valine-Citruline or derivatives thereof.

[0151] Preferably, the targeting moiety of conjugates 1 to 3 and 20 is an antibody comprising a VH and VL domain, wherein the VL domain comprises or consists of SEQ ID NO: 8, and the VH domain comprises or consists of SEQ ID NO: 7 (Canakinumab).

[0152] Preferably, the targeting moiety of conjugates 4 to 6 and 18 is an antibody comprising a VH and VL domain, wherein the VL domain comprises or consists of SEQ ID NO: 4, and the VH domain comprises or consists of SEQ ID NO: 3 (Atezolizumab).

[0153] Preferably, the targeting moiety of conjugates 7 to 9 and 19 is an antibody comprising a VH and VL domain, wherein the VL domain comprises or consists of SEQ ID NO: 12, and the VH domain comprises or consists of SEQ ID NO: 11 (Siltuximab).

[0154] Preferably, the targeting moiety of conjugates 10 to 12 and 17 is an antibody comprising a VH and VL domain, wherein the VL domain comprises or consists of SEQ ID NO: 6, and the VH domain comprises or consists of SEQ ID NO: 5 (Bevacizumab).

[0155] Preferably, the targeting moiety of conjugates 13 to 16 is an antibody comprising a VH and VL domain, wherein the VL domain comprises or consists of SEQ ID NO: 10, and the VH domain comprises or consists of SEQ ID NO: 9 (Cetuximab).

[0156] Preferably, the drug that is toxic and that is included in element iii) of the conjugates of the invention 1-15 is selected from the group consisting of monomethylauristatin E (MMAE), monomethyauristatin F (MMAF), N-acetyl g calicheamicin, DM1 , DM4, deruxtecan, and pyrrolobenzodiazepine (PBD) dimer. Most preferably, the toxic drug is MMAE or MMAF, and the first protein binds to, preferably specifically binds to, IL-1 b. Most preferably, the toxic drug is MMAE or MMAF, and the first protein binds to, preferably specifically binds to, VEGF-A. Most preferably, the toxic drug is MMAE or MMAF, and the first protein binds to, preferably specifically binds to, PD-L1. Most preferably, the toxic drug is MMAE or MMAF, and the first protein binds to, preferably specifically binds to, EGFR.

[0157] Compositions

[0158] A second aspect of the present invention refers to a composition (also called herein “the composition of the invention”), preferably a pharmaceutical composition, comprising the conjugate of the invention as defined in the first aspect or in any of its embodiments. The conjugates described herein may be in the form of compositions comprising the conjugate and one or more carriers, excipients and / or diluents. The compositions may be formulated for specific uses, such as for veterinary uses or pharmaceutical uses in humans.

[0159] The composition can be in any suitable form (depending upon the desired method of administering it to a patient). The pharmaceutical composition can be administered to a patient by a variety of routes such as orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intratumorally, intrathecally, topically or locally. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

[0160] Pharmaceutical compositions may be prepared for storage as lyophilized formulations or aqueous solutions by mixing the conjugate of the invention having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers typically employed in the art (all of which are referred to herein as "carriers"), i.e., buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants, and other miscellaneous additives.

[0161] Methods and Uses

[0162] In a third aspect, the present invention refers to the conjugate of the invention as defined in the first aspect or any of its embodiments, or the composition as defined in the second aspect or any of its embodiments, for use in therapy or in the manufacture of a medicament. Preferably, the conjugate and the composition of the invention are used in a method of treating or preventing cancer.

[0163] Also provided herein are methods of degrading a membrane or extracellular protein of interest. Such methods include contacting the membrane or extracellular protein with the conjugate of the present invention, under conditions in which the first protein will bind to the POI, and the second protein will bind an endocytic receptor of a tumor cell, causing the internalization of the conjugate and the POI, and preferably the degradation of the POI and of the cleavable linker that binds the first protein to the cargo, thereby releasing the cargo.

[0164] Also provided are methods that include administering to an individual in need thereof a therapeutically effective amount of the conjugate of the invention as defined in the first aspect or any of its embodiment, or the composition of the second aspect or any of its embodiment. A variety of individuals are treatable according to the subject methods. Generally, such subjects are “mammals” or “mammalian,” where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some embodiments, the individual is a human.

[0165] An effective amount of the conjugate of the invention (or pharmaceutical composition including same) is an amount that, when administered alone (e.g., in monotherapy) or in combination (e.g., in combination therapy) with one or more additional therapeutic agents, in one or more doses, is effective to reduce the symptoms of a medical condition of the individual (e.g., cancer) by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the symptoms in the individual in the absence of treatment with the bifunctional degrader or pharmaceutical composition.

[0166] The methods and uses defined herein include administering to an individual having cancer a therapeutically effective amount of the conjugate of the invention (or pharmaceutical composition including same. According to such uses, the conjugate is capable of binding and degrading a membrane or extracellular protein of interest that at least contributes to the individual’s cancer, and at the same time the conjugate introduces a cargo into the tumor cell. In an embodiment, the endocytosis-triggering peptide is specific for tumor lung cells, so that the cancer treated is lung cancer. In an embodiment, the endocytosis-triggering peptide is MGS4 (SEQ ID NO: 2), and the conjugate is used in the treatment and / or prevention of lunch cancer.

[0167] In an embodiment, the first protein binds to, preferably specifically binds to, IL-1 B, and the use is in a method of treating and / or preventing a IL-1 B positive cancer. In an embodiment, the first protein binds to, preferably specifically binds to, EGFR, and the use is in a method of treating and / or preventing a EGFR positive cancer. In an embodiment, the first protein binds to, preferably specifically binds to, PD-L1 , and the use is in a method of treating and / or preventing a PD-L1 positive cancer. In an embodiment, the first protein binds to, preferably specifically binds to, IL-6, and the use is in a method of treating and / or preventing a IL-6 positive cancer. In an embodiment, the first protein binds to, preferably specifically binds to, VEGF-A, and the use is in a method of treating and / or preventing a VEGF-A positive cancer. In an embodiment, the conjugate is any of conjugates 1 to 3 and 20, and the use is in a method of treating and / or preventing a IL-1 B positive cancer. In an embodiment, the conjugate is any of conjugates 4 to 6 and 18, and the use is in a method of treating and / or preventing a PDL-1 positive cancer. In an embodiment, the conjugate is any of conjugates 7 to 9 and 19, and the use is in a method of treating and / or preventing a IL-6 positive cancer. In an embodiment, the conjugate is any of conjugates 10 to 12 and 17, and the use is in a method of treating and / or preventing a VEGF-A positive cancer. In an embodiment, the conjugate is any of conjugates 13 to 16, and the use is in a method of treating and / or preventing a EGFR positive cancer.

[0168] By “treat”, “treating” or “treatment” is meant at least an amelioration of the symptoms associated with the medical condition (e.g., cell proliferative disorder, e.g., cancer) of the individual, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the medical condition being treated. As such, treatment also includes situations where the medical condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the individual no longer suffers from the medical condition, or at least the symptoms that characterize the medical condition.

[0169] The uses described herein include administering the conjugate or the composition of the invention to the individual using any available method and route suitable for delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration. Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intra-tracheal, subcutaneous, intradermal, topical application, ocular, intravenous, intratumoral, intraarterial, nasal, oral, and other enteral and parenteral routes of administration. In some embodiments, the administering is by parenteral administration. Routes of administration may be combined, if desired, or adjusted depending upon the bifunctional degrader and / or the desired effect. The conjugates or the compositions of the invention may be administered in a single dose or in multiple doses. In some embodiments, conjugates or the compositions of the invention are administered intravenously. In some embodiments, the conjugates or the compositions of the invention are administered by injection, e.g., for systemic delivery (e.g. intravenous infusion) or to a local site.

[0170] In another embodiment of the third aspect, the conjugate of the invention as defined in the first aspect or any of its embodiment, or the composition as defined in the second aspect or any of its embodiments, is used in diagnostic and / or theragnostic methods. Said use may comprise using a first protein that has utility in the field of diagnostics method (e.g., a labelled protein) or theragnostic (i.e., a radioisotope).

[0171] Kits and Uses

[0172] In a fourth aspect, the present invention provides a kit comprising the conjugate of the first aspect, or the composition of the second aspect, or any of their embodiments, together with instructions on how to use said conjugate or composition. Hence, any of the conjugates and / or compositions described herein may be included in a kit. Such kits may also include components that preserve the conjugates or that protect against their degradation.

[0173] Such kits generally will comprise, in suitable means, distinct containers for each reagent or solution. The kit may comprise one or more containers holding the conjugate or the composition of the invention. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic. A container may have a sterile access port (for example, the container may be a vial having a stopper pierceable by a hypodermic injection needle).

[0174] The kit can further comprise a container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including other pharmaceutically acceptable formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery devices. The delivery device may be pre-filled with the compositions. The kit can also comprise a package insert containing written instructions for methods of treating cancer with the conjugate or the composition of the invention.

[0175] In this aspect, the present invention also refers to medical uses of the kit. The uses of the kit are the same uses as defined in the third aspect of the invention or any of its embodiments. Thus, preferably, the kit is used in a method of treating and / or preventing cancer.

[0176] Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention.

[0177] The following items are also included in the present invention: A conjugate comprising: i. a first protein that is capable of binding to a protein of interest, ii. a second protein that is bound to the first protein and that comprises an endocytosis-triggering peptide that is capable of binding to an endocytic receptor of a tumor cell, and iii. a cargo, preferably a drug that is toxic for tumor cells, wherein the cargo is bound to the first protein via an intracellular cleavable or degradable linker, wherein the binding of the endocytosis-triggering peptide to the endocytic receptor of the tumor cell causes the internalization of the conjugate and of the protein of interest inside the tumor cell, and wherein the protein of interest is a protein present in the extracellular media of a tumor microenvironment or the extracellular domain of a protein present in the surface of a tumor cell, and wherein said protein of interest promotes tumor progression, immunoescape and / or survivability. The conjugate according to item 1 , wherein the intracellular cleavable or degradable linker is a lysosomal cleavable peptide, and wherein the internalization of the conjugate and the protein of interest inside the tumor cell leads the conjugate and the protein of interest to the intracellular lysosomal compartment of the tumor cell. The conjugate according to any one of items 1 or 2, wherein the endocytosis- triggering peptide comprised in the second protein is selected from the list consisting of SEQ ID NO: 1 , 2, and 13. The conjugate according to any one of items 1 to 3, wherein the first protein is an antibody, an aptamer, or a small molecule. The conjugate according to any one of items 1 to 4, wherein the first protein comprises a targeting moiety capable of binding to IL-1 b, IL-6, EGFR, PD-L1 or VEGF-A. The conjugate according to any one of items 1 to 5, wherein the second protein further comprises a flexible linker that binds the second protein with the first protein, preferably wherein the linker comprises maleimide-PEG6-N-Hydroxysuccin- imidyl ester. 7. The conjugate according to any one of items 1 to 6, wherein the intracellular cleavable peptide is selected from the list consisting of cathepsin, phosphatases, pyrophosphatases, sulfatases, p-galactosidase and p-glucuronidase sensitive linker.

[0178] 8. The conjugate according to any one of items 1 to 7, wherein the intracellular cleavable peptide comprises or consists of a maleimide valine citrulline linker.

[0179] 9. The conjugate according to any one of items 1 to 8, wherein the cargo is a drug that is toxic for the tumor cell and is selected from the group consisting of monomethylauristatin E (MMAE), monomethyauristatin F (MMAF), N-acetyl g ca- licheamicin, DM1 , DM4, deruxtecan, and pyrrolobenzodiazepine (PBD) dimer.

[0180] 10. The conjugate according to any one of items 1 to 9, wherein: i. the first protein is capable of binding to IL-1 b, IL-6, EGFR, PD-L1 or VEGF-A, and ii. the second protein comprises a endocytosis-triggering peptide comprising or consisting of any one of SEQ ID NO: 1 , 2, or 13, and wherein the intracellular cleavable linker is maleimide Valine-Citruline.

[0181] 11 . The conjugate according to any one of items 1 to 9, wherein: i. the first protein is capable of binding to I L-1 b, preferably comprising a VH and VL domain, wherein the VL domain comprises or consists of SEQ ID NO: 8, and the VH domain comprises or consists of SEQ ID NO: 7 (Can- akinumab), ii. the second protein comprises an endocytosis-triggering peptide comprising or consisting of SEQ ID NO: 2, wherein the intracellular cleavable linker is maleimide Valine-Citruline, and wherein the linker connecting the first protein and the second protein is malei- mide-PEG6-N-Hydroxysuccinimidyl ester.

[0182] 12. A pharmaceutical composition comprising the conjugate as defined in any one of items 1 to 11 , further comprising a pharmaceutically acceptable carrier. 13. The conjugate according to any one of items 1 to 11 , or the pharmaceutical composition as defined in item 12, for use in medicine.

[0183] 14. The conjugate according to any one of items 1 to 11 , or the pharmaceutical composition as defined in item 12, for use in a method of treating and / or preventing cancer.

[0184] 15. The conjugate as defined in item 11 , for use in cancer, wherein the cancer is lung cancer.

[0185] SEQUENCE LISTING

[0186] R12 peptide: SEQ ID NO: 1 RRRRRRRRRRRR

[0187] MGS4 peptide SEQ ID NO: 2 FHAVPQSFYTAP.

[0188] Atezolizumab Heavy Chain SEQ ID NO: 3

[0189] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYG

[0190] GSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQG

[0191] TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV

[0192] HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT

[0193] CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV

[0194] EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0195] GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV

[0196] LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0197] Atezolizumab Light Chain SEQ ID NO: 4

[0198] DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGV

[0199] PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYH PATFGQGTKVEI KRTVAAPSVFI

[0200] FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY

[0201] SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0202] Bevacizumab Heavy Chain SEQ ID NO: 5

[0203] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYT

[0204] GEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDV

[0205] WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD

[0206] KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYV

[0207] DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS

[0208] KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT

[0209] PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0210] Bevacizumab Light Chain SEQ ID NO: 6

[0211] DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGV

[0212] PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVF

[0213] IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0214] Canakinumab Heavy Chain SEQ ID NO: 7

[0215] QVQLVESGGGVVQPGRSLRLSCAASGFTFSVYGMNWVRQAPGKGLEWVAIIWYDG

[0216] DNQYYADSVKGRFTISRDNSKNTLYLQMNGLRAEDTAVYYCARDLRTGPFDYWGQG

[0217] TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV

[0218] HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT

[0219] CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV

[0220] EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0221] GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV

[0222] LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0223] Canakinumab Light Chain SEQ ID NO: 8

[0224] EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSLHWYQQKPDQSPKLLIKYASQSFSGVP

[0225] SRFSGSGSGTDFTLTINSLEAEDAAAYYCHQSSSLPFTFGPGTKVDIKRTVAAPSVFIF

[0226] PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS

[0227] LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0228] Cetuximab Heavy Chain SEQ ID NO:9

[0229] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGN

[0230] TDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTL

[0231] VTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT

[0232] FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP

[0233] PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV

[0234] HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ

[0235] PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

[0236] SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Cetuximab Light Chain SEQ ID NO: 10

[0237] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPS RFSGSGSGTDFTLSI NSVESEDI ADYYCQQN N N WPTTFGAGTKLELKRTVAAPSVFI F PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0238] Siltuximab Heavy Chain SEQ ID NO:11

[0239] EVQLVESGGKLLKPGGSLKLSCAASGFTFSSFAMSWFRQSPEKRLEWVAEISSGGSY

[0240] TYYPDTVTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYCARGLWGYYALDYWGQGT

[0241] SVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH

[0242] TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC

[0243] PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE

[0244] VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG

[0245] QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL

[0246] DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0247] Siltuximab Light Chain SEQ ID NO:12

[0248] QIVLIQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVP

[0249] VRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSGYPYTFGGGTKLEIKRTVAAPSVFI

[0250] FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY

[0251] SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC iRGD peptide SEQ ID NO: 13: CRGDKGPDC

[0252] R12 modified peptide SEQ ID NO: 14: VSGWRLFKKISGGGGSRRRRRRRRRRRR

[0253] The invention is described below by the following examples, which must be considered as merely illustrative and in no case limiting of the scope of the present invention.

[0254] EXAMPLES

[0255] EXAMPLE 1

[0256] Material and Methods

[0257] AKS Synthesis Synthesis of Antibody Killer Scavengers (AKS) were performed in sterile conditions using endotoxin free reagents and materials. Reagents were purchased in reagent grade from commercial suppliers and used as received. Synthesis was carried away as a two-stage reaction process, firstly the antibody was partially reduced to generate / expose thiol groups from cysteine amino acids and conjugated to the cytotoxic drug. Secondly, the afforded drug conjugated antibody (ADC) was conjugate to an endocytosis-triggering peptide via orthogonal bioconjugation.

[0258] Anti-IL1 b AKS Synthesis

[0259] Briefly, 1 equivalent of AntilLIb antibody (BioXCell, BE0246) was mixed with 2 equivalents of Tris(2-carboxyethyl) phosphine (TCEP)(Sigma-Aldrich, C4706) in 25 mM phosphate buffer at pH 7,5 and left to react protected from light for 30 min at 25°C with shaking (1000 rpm) in a TS-100C BioSan Thermo-Shaker. Reduced antibody was mixed with 2 equivalents of a maleimide valine-citrulline modified cytotoxic drug, either MC-VC-PAB- MMAE (MedChemExpress, HY-15575) or MC-VC-PAB-MMAF (MedChemExpress, HY- 112786). Drugs were previously dissolved at 1 mg / ml in dimethylformamide (DMF). Mixture was left to react protected from light overnight (minimum of 12 hours) at 25°C with shaking (1000 rpm) to afford either Anti-IL1 b MMAE ADC orAnti-IL1 b MMAF ADC.

[0260] The afforded ADC was purified by centrifugal diafiltration (Amicon Ultra-4 Centrifugal Filter 10 kDa mwco) in 25mM MES (2-(N-morpholino)ethanesulfonic acid) buffer at pH 7,0. 1 equivalent of purified ADC was diluted to approximately 5 mg / ml with 25mM MES buffer at pH 7,0 and mixed with 4 equivalents of maleimide-PEG6-N-Hydroxysuccin- imidyl ester (746193, Sigma-Aldrich), a heterobifunctional linker. The mixture was left to react protected from light for 1 h at 25°C with shaking (1000 rpm).

[0261] The afforded maleimide-PEG6-ADC was purified by centrifugal diafiltration in 25mM phosphate buffer at pH7.5 and its concentration adjusted to approximately 1.5 mg / ml. Finally, 1 equivalent of the maleimide-PEG6-ADC was mixed with 4 equivalents of an endocytosis-triggering peptide, either R12C (SEQ ID NO 1 : RRRRRRRRRRR) (Gene- Script) or MGS4C (SEQ ID NO 2: FHAVPQSFYTAP)(GeneScript). A cysteine amino acid was added at the C-terminus of SEQ ID NO: 1 and 2 in order to chemoselectively conjugate it to the antibody. The mixture was left to react protected from light overnight (minimum of 12 hours) at 25°C with shaking to afford either Anti-111 b MMAE R12 AKS, Anti- 111b MMAF R12 AKS, Anti-ll1b MMAE MGS4 AKS orAnti-H1b MMAF R12 AKS.

[0262] AKSs were characterized using an ACQUITY UPLC system (Waters) equipped with a quaternary pumping system, a fixed loop injector, and coupled to a fluorescence detector. Column used was a Xbridge SEC 250A 2.5 pm column (7,8x300 mm). Mobile phase consisted of 100mM phosphate buffer at pH 6,8, column temperature was 25°C and injection volume was 10pl. Flow rate was set to 1ml / min.

[0263] Synthesis of AKS Against Proteins of Interest

[0264] Synthesis of AKS against POI; Epithelial Growth Factor Receptor (EGFR), Interleukin 6 (IL6), Vascular Endothelial Growth Factor (VEGF), Interleukin ip (IL1 p), Programmed Death-Ligand 1 (PD-L1) were carried out as described in the previous paragraph. Briefly, 1 equivalent of the anti-POl antibody (EGFR: Cetuximab Biosimilar (IchorBio, ICH4004), VEGF: Bevacizumab Biosimilar (IchorBio, ICH4003), IL6 Siltuximab Biosimilar (Ichorbio, ICH5104), IL1b: Canakinumab Biosimilar (IchorBio, ICH5115), Atezolizumab Biosimilar (IchorBio, ICH4018)), was partially reduced with 2 equivalents of TCEP for 30 min at room temperature with shaking (1000 rpm). Reduced antibody was mixed with 2 equivalents of a maleimide valine-citrulline modified cytotoxic drug, either MC-VC-PAB-MMAE or MC-VC-PAB-MMAF. Drugs were previously dissolved at 1 mg / ml in dimethylformamide (DMF). Mixture was left to react protected from light overnight (minimum of 12 hours) at 25°C with shaking (1000 rpm) to afford either Anti-POl MMAE ADC or Anti-POl MMAF ADC.

[0265] The afforded ADC was purified by centrifugal diafiltration (Amicon Ultra-4 Centrifugal Filter 10 kDa mwco) in 25mM MES buffer at pH 7,0. 1 equivalent of purified ADC was diluted to approximately 5 mg / ml with 25mM MES buffer at pH 7,0 and mixed with 4 equivalents of maleimide-PEG6-N-Hydroxysuccinimidyl ester, a heterobifunctional linker. The mixture was left to react protected from light for 1 h at 25°C with shaking (1000 rpm).

[0266] The afforded maleimide-PEG6-ADC was purified by centrifugal diafiltration in 25mM phosphate buffer at pH7.5 and its concentration adjusted to approximately 1.5 mg / ml. Finally, 1 equivalent of the maleimide-PEG6-ADC was mixed with 4 equivalents of an endocytosis-triggering peptide, either R12C (SEQ ID NO: 1 - RRRRRRRRRRRR)(Gene- Script) or MGS4C (SEQ ID NO 2: FHAVPQSFYTAP) (GeneScript). The mixture was left to react protected from light overnight (minimum of 12 hours) at 25°C with shaking to afford either Anti-POl MMAE R12 AKS, Anti-POl MMAF R12 AKS, Anti-POl MMAE MGS4 AKS or Anti-POl MMAF R12 AKS.

[0267] Cell Culture

[0268] A549 (CCL-185, ATCC) and HeLa (CRM-CCL-2, ATCC) cells were routinely cultured in Dulbecco’s Modified Eagle’s High Glucose (4,5 g / L) Medium (DMEM) supplemented with 10% (v / v) FBS, 1% (v / v) Penicillin / Streptavidin and 1 % (v / v) Glutamax. NCI-H1299 (CRL- 5803 , ATCC) were incubated in Roswell Park Memorial Institute (RPMI) 1460 Medium supplemented with 10%(v / v) FBS, 1 % (v / v) Penicillin / Streptavidin and 1 % (v / v) Gluta- max. All cell lines were incubated in a 37°C humidified (95%) CO2 (5%) incubator.

[0269] Toxicity Assays

[0270] HeLa, A549 NCI-H1299 cells were seeded in 96-Well culture-treated flat-bottom microplates and left to attach overnight (minimum 12h). Cells were treated with 100nM of either anti-target antibody, anti-target MMAE ADC, anti-target MMAF ADC, anti-target MMAE R12 AKS, anti-target MMAE MGS4 AKS, anti-target MMAF R12 AKS, anti-target MMAF MGS4 AKS. for 24h or 48h. Cell viability was assessed using the alamarBlue assay (DAL1100, Thermo Fisher Scientific) following manufacturer’s instructions. Absorbance was measured in a Synergy HTX multimode reader (Biotek).

[0271] Phase Contrast Microscopy

[0272] After treatment, cells were also inspected by phase contrast microscopy and images were taken to document cell morphology and density in each condition. An Oxion Inverso Range (Euromex) microscope was used.

[0273] AKS / Target Internalization Assays

[0274] A549, NCI-H1299 and HeLa cells were seeded in 12-Well Tissue Culture Plates and left to attach overnight (minimum 12h). Cells were treated with one of the following drugs: 100nM of target protein (IL1 b), 100nM of Anti-target antibody, Anti-target MMAE ADC, Anti-target MMAF ADC, Anti-target MMAE R12 AKS or Anti-target MMAF R12 AKS for 24h in either presence of absence of 100nM of the Lysosomal inhibitor Bafilomicyn A1 (SC-201550A, Santa Cruz Biotechnology).

[0275] After treatment, cells were washed with sterile PBS and harvested using Trypsin 0.025% . Cell pellets were washed twice with PBS and cells were lysed using RIPA Lysis Buffer (89900, Thermo Fisher Scientific) supplemented with Halt™ Protease and Phosphatase Inhibitor (78442, Thermo Fisher Scientific) on ice for 30 min. The lysates were centrifuged at 21000 g for 15 min at 4°C and protein concentration was measured by Pierce BCA assay (23225, Thermo Fisher Scientific). Equal amounts of cell lysates were separated by SDS-PAGE (10% Bis-Tris Gel) and transfer to a nitrocellulose membrane. Then, membranes were blocked for 1h at room temperature in PBS-T with 5% non-fat dried milk. Blocked membranes were incubated with primary antibody, either Goat anti-IgG antibody HRP (1 :5000)( PA1-32045, Thermo Fisher Scientific) or rabbit multiclonal AntilLI b antibody (1 :5000) (ab283822, Abeam), overnight at 4°C, then washed thrice with PBS-T and incubated for 1 h at room temperature with either, HRP-conjugated anti- rabbit IgG antibody (1 : 10000) (32460, Thermo Fisher Scientific). Protein bands were developed with Supersignal West Atto Chemiluminiscent Substrate (A38555, Thermo Fisher Scientific) and imaged using a C-Digit Blot Scanner (Lycor). Vinculin and Tubulin were used as loading control for Western Blot for normalization purposes, anti-vinculin antibody (SC-73614, Santa Cruz Biotechnology) was used as primary antibody

[0276] (1 :10000), or anti-Tubulin antibody (SC-166729, Santa Cruz Biotechnology) was used as primary antibody (1 :10000) and HRP-Conjugated Goat anti IgG (P0447, Dako) as secondary antibody (1 :10000).

[0277] Tumor Selective Peptides Internalization Assays A549, NCI-H1299 and HeLa cells were seeded in 12-Well Tissue Culture Plates as described before. Cells were treated with either 100nM of mouse IgG B6 antibody or mouse IgG B6 MGS4 LYTAC for24h in presence of absence of 100nM of the Lysosomal inhibitor Bafilomicyn A1 . Cells were collected, lysates processed and proteins Western blotted as described in the previous section. AKS construct used in Example 1

[0278] RESULTS

[0279] AKS ELICIT CYTOTOXIC EFFECTS COUPLED TO TARGETED PROTEIN DEGRADATION OF EXTRACELULAR IL1b.

[0280] Lysosomal Targeting Chimeras (LYTACs) are bifunctional molecules that engage simultaneously an extracellular target protein and an endocytic receptor, triggering the internalization of the whole complex and directed it to its degradation through the endo-lyso- somal system. Described lysosomal degraders pair in one single structure a molecule that specifically recognize and bind the extracellular protein targeted for its degradation and, an endocytic triggering molecule that will drive the internalization of the complex and its sorting to the cell lysosomes. On the other hand, Antibody Drug Conjugates (ADCs) are monoclonal antibodies conjugates to a drug payload via a linker that will release the drug when the ADC is internalized into the intracellular space. ADC’s monoclonal antibodies are commonly selected against tumor specific antigens, usually a membrane receptor, to increase tumor selectivity. Linkers selected to attach the payload are usually sensible to the lysosomal conditions (pH, cathepsins etc.) leading to the release of the cytotoxic drug after the internalization of the ADC by the cells.

[0281] We hypothesized that the combination of these two concepts could lead to a novel mechanism action or a synergetic improvement on the already established LYTACs and ADCs functionality. These novel compounds, christened as Artificial Killer Scavengers (AKS), could overcome limitations of the ADCs on payload delivery to tumor cells while impacting the tumor microenvironment targeting not only tumor specific antigens but also cytokines and other modulators that are not intrinsically related to the tumor cells themselves but promote tumor progression, immunoescape and survivability.

[0282] As proof of concept, we developed an AKS based on a monoclonal antibody against IL- 1 b. We chose I L-1 b as it is a soluble cytokine that has pleiotropic effects in cancer, promoting angiogenesis, cancer cell proliferation, migration and metastasis among others. To avoid any biological effects of IL-1 b on human tumor cells that could interfere with the AKS toxicity / targeted protein degradation functionality we chose to use mouse I L-1 b, as it has less than 60% of homology to the human I L-1 b. The mouse-IL-1 b AKS was synthesized following a two-step strategy, firstly the antibody was partially reduced and conjugated to a cytotoxic drug, either MMAE or MMAF, through a cathepsin sensitive linker. Secondly, the afforded Anti I L1 b ADC was orthogonally conjugated to the cell-penetrating peptide (CPP) R12 to afford the Anti IL1 B AKS. LIPLC SEC chromatographic analysis showed no antibody fragmentation or aggregation after each synthetic step.

[0283] To test if AKS internalization and toxicity was specifically driven by the R12 CPP, we tested the Anti IL-1 b MMAE R12 and MMAF R12 AKSs and the Anti IL-1 b MMAE and MMAF ADCs toxicity on HeLa cells. Effect on cell viability was only observed in cells treated with AKSs, a reduction in cell viability compared to controls of 42,4% for Anti IL- 1 b MMAE R12 and 52,8% for Anti IL-1b MMAF R12. Comparatively, cells treated with Anti IL-1 b MMAE or Anti I L-1 b MMAF ADCs showed no significant reduction in cell viability compared to controls (figure 1). This result suggested that the toxicity was related to the presence in one single structure of a cytotoxic payload (MMAE or MMAF) and a CCP (R12). Moreover, since cytotoxic payloads can only be released from the antibody after the cleavage of the linker by lysosomal cathepsins, the diminution of cell viability only of AKS treated cells indicates that not only AKSs are differentially internalized compared to ADCs by HeLa cells, but that AKS are also sorted to the lysosomes.

[0284] One key aspect of AKSs is their selectivity towards tumor cell lines. To test that selectivity, we synthesized anti-IL-1 b AKS bearing the MGS4 peptide as internalizing moiety. MGS4 has been described as an internalizing peptide with selectivity towards certain lineages of lung cancer such as NCI-H1299 cell line. HeLa an NCI-H1299 cell lines were treated with one of the following drugs: Anti I L-1 b MMAE R12, MMAF R12, MMAE MGS4 or MMAF MGS4 AKS and the Anti IL-1 b MMAE and MMAF ADCs. A significant reduction in cell viability was only observed in HeLa cells treated with the Anti I L-1 b MMAE R12 and Anti I L-1 b MMAF R12 AKS (65,4 % and 62,1 % respectively)(Figure 2 a)), no significant toxicity was observed in those cells treated with Anti I L-1 b MMAE or MMAF ADCs or Anti IL-1 b MMAE MGS4 o MMAF MGS4AKS. Notably, NCI-H1299 cells showed a significant reduction in cell viability when treated with either set of AKSs (Figure 2 b)), the ones that bore the CPP R12 (anti-H1b MMAE R12 53,7%, anti-H1b MMAF R12 54,5%) and the ones that bore the lung cancer specific peptide MGS4 (anti-IL-1 b MMAE MGS4 12 % 46%, anti-l L-1 b MMAF 12 55,2%), while no reduction in viability was observed in those cells treated with the MMAE or MMAF ADCs. All this evidence supports a selective internalizing mechanism of AKSs driven by tumor specific peptides, which could improve both safety and efficiency in delivering cytotoxic drugs to specific cancer cell lines. Next, we investigated the targeted protein degradation of extracellular targets mediated by AKSs. Functionally, LYTACs capture an extracellular target and drive its degradation through the lysosomal pathway, Anti IL-1 b AKSs should perform in the exact way, capturing mouse-IL-1 b in the extracellular space, inducing its endocytosis and sorting to the lysosomes. HeLa cells were treated with equimolar amounts (100nM) of I L-1 b and one of the following drugs: Anti IL-1 b MMAE R12 AKS, MMAF R12 AKS, MMAE ADC or MMAF ADC in presence or absence of the lysosomal inhibitor bafilomycin A1 for 24 hours. After treatment cells were harvested and cell lysates were analyzed by western blot to determine the presence of IL1 b and the anti-IL1 b antibody used in the AKSs and ADCs synthesis. Basal internalization of IL-1 b was observed in all conditions, suggesting that IL-1 b is internalized interspecifically. However, when cells are treated with AKSs, either MMAE R12 or MMAF R12, in presence of bafilomycin A1 there was a significative increase of intracellular levels of IL-1 b. Thus indicate an active internalization of the IL- 1 b mediated by the AKSs and intracellular accumulation when the lysosomal degradation mechanisms are inhibited (Figure 3 a)). Accordingly, levels of intracellular Anti IL-1 b antibody were only detected in those cells treated with AKSs with a significant accumulation in cells treated with bafilomycin A1 (Figure 3 b)). All this evidence supports an active internalization and degradation of I L-1 b mediated by AKSs in a LYTAC-like fashion coupled to the delivery of cytotoxic payloads to tumoral cell in an ADC-like fashion.

[0285] This observation correlated with the changes in cellular morphology and cell density observed by phase contrast microscopy, cells treated with Anti I L-1 b ADCs (Figure 4 b)) were morphologically indistinguishable from controls (Figure 4 a)), cells treated with anti- IL-1 b AKSs (Figure 4 c)) showed evident signs of distress and cell density plummeted compared with controls. However, when bafilomycin A1 was present cells treated with AKSs were morphologically close to controls with no visual difference in cell density either (Figure 4 d)).

[0286] TUMOR SELECTIVE PEPTIDE MGS4 DRIVES AKS INTERNALIZATION DIFFERENTIALLY IN LUNG CANCER CELLS

[0287] One key feature of AKSs is their selectivity towards cancer cells, specifically in this application towards lung cancer cells. This selectivity increases the chances that cytotoxic payloads are preferentially delivered to cancer cells, thus potentially minimizing off-target toxicity. This specificity can be achieved using tumor-homing peptides, short peptides that specifically engage with tumoral cells or cells in the tumor microenvironment. MGS4 is a tumor-homing peptide used to deliver cytotoxic payloads to a wide subset of NonSmall Cell Lung Cancer cell lines. First, we studied the selective internalization of AKSs developed using MGS4 or R12 as internalizing molecules. The MGS4 peptide is selective towards non-small cell lung cancer cell lines, and it is positively internalized in A549 and NCI-H 1299 cell lines, with higher selectivity towards the NCI-H 1299 cells. HeLa (cervical adenocarcinoma), A549 (lung adenocarcinoma) and NCI-H 1299 (lung adenocarcinoma) cell lines were treated for 24h in the presence of absence of Bafilomycin A1 (100nM) with 100nM of one of the following drugs: anti-IL-1 b MMAE R12 , MMAF R12, MMAE MGS4 or MMAF MGS4 AKSs, anti- IL-1 b MMAE or MMAF ADCs. Intracellular levels of the IgG antibody used to synthesize the drugs were positively detected in those cells treated with R12 AKS, independently of their lineage (HeLa Figure 5 a), A549 Figure 5 b) and NCI-H 1299 Figure 5 c)) which correlates with the unspecific mechanism of internalization of the CPP. Moreover, the inhibition of the lysosomal degradation pathways led to the detection of higher intracellular levels of the anti I L-1 b antibody, supporting the thesis of R12 AKSs lysosomal shuttling and degradation. However, for those AKSs conjugated to MGS4, the intracellular levels of the anti IL-1 b antibody were only detected faintly in cell lysates from A549 (Figure 5 b)) and more clearly in lysates of H1299 (Figure 5 c)) in those cells treated with both the MGS4 AKS and bafilomycin (Figure 5), which indicates the selectivity of MGS4 AKSs towards the lung adenocarcinoma cell lines. Moreover, the lack of detection of internalized IgG antibody in A549 and NCI-H1299 cells treated with MGS4 AKSs when the lysosomal maturation was not inhibited indicates that the MGS4 AKSs are driven to the lysosomes for its degradation.

[0288] Next, we analyzed the targeted degradation of I L-1 b by the MGS4 and R12 AKSs in the NCI-H1299 cell line, which showed significant internalization rates of the AKSs for both peptides. NCI-H 1299 cells were treated for 24h either in the presence or absence of bafilomycin A1 (100nM) with 100 nM of IL1 b and 100nM of one of the following drugs: Anti IL-1b MMAE ADC, MMAF ADC, MMAE R12 ADK, MMAF R12 ADK, MMAE MGS4 ADK or MMAF MGS4 ADK. Intracellular levels of the Anti IL-1 b were positively detected in those cells treated with R12 and MGS4 AKSs (Figure 6) only when the lysosomal degradation pathways were inhibited. This supports the thesis of R12 and MGS4 AKSs lysosomal shuttling and degradation of a targeted extracellular protein (I L1 b). Noteworthy, intracellular levels of IL-1b in NCI-H1299 cells were significantly higher in those treated with R12 AKSs compared to MGS4 AKSs. This could be explained due to the existence of multiple internalization pathways for the polyarginine CPP, endocytic internalization of R12 has been related to unspecific binding to negatively charged cell surface glycoproteins, whereas MGS4 selectivity towards specific subsets of NSCLC cells lines points to a receptor mediated internalization and therefore a more selective internalization pathway.

[0289] EXAMPLE 2

[0290] METHODOLOGY

[0291] Crystal violet staining

[0292] The cell viability in the presence of AKS and related compounds was evaluated by using crystal violet staining. Cells were seeded in 96-well plates at a density of fifteen thousands cells per well in 100 pL of complete culture medium and incubated under standard conditions (37 °C, 5% CO2, humidified atmosphere) for 24 hours to allow adhesion and recovery. On the next day, the culture medium was replaced with fresh medium containing the treatments to be tested. Treatments included AKS at a final concentration of 100 nM, as well as respective controls, including the cytotoxic payloads MMAF or MMAE, an antibody-drug conjugate (ADC), and AKS conjugated to either the R12 peptide or the MGS4 peptide. A vehicle-only control was included to serve as a negative control. Cells were incubated with the respective treatments for 24 or 48 hours under standard culture conditions.

[0293] After the treatment period, the medium was removed, and cells were gently washed with phosphate-buffered saline (PBS). Cells were subsequently fixed by adding 100 pL of PFA per well for 10 minutes at room temperature, followed by a PBS wash Fixed cells were stained with 50 pL of 0.5% (w / v) crystal violet solution per well for 10 minutes at room temperature. Excess dye was removed by careful washing with distilled water until the wash solution ran clear, and the plates were allowed to dry completely. To quantify the stained cells, 100 pL of absolute methanol was added to each well to solubilize the bound dye. The optical density of each well was measured at 570 nm using a microplate reader.

[0294] The absorbance values were used as a measure of relative cell viability, with values normalized to the vehicle control to determine the percentage of viable cells. Comparisons were performed to assess the effects of AKS and the control treatments (MMAF, MMAE, ADC, AKS-R12, and AKS-MGS4) at both 24-hour and 48-hour time points.

[0295] Quantification of protein internalization / degradation by western blot The amount of target protein was analyzed by Western Blot. In short, cells were treated with either 100nM AKS, as well as respective controls, including the cytotoxic payloads MMAF or MMAE, an antibody-drug conjugate (ADC), and AKS conjugated to the R12 peptide and / or MGS4 peptide, in presence of proteases or lysosome inhibitors. After 24h cells were washed with PBS, lifted with trypsin, spun at 300g for 5min, washed with PBS and spun again at 300g for 5min to remove traces of treatments. Cell pellet were solubilized with RIPA lysis buffer supplemented with protease / phosphatase inhibitors (Halt™ Protease Inhibitor Cocktail, ThermoFisher) and lysates were spun at 16000g for 10min. The protein concentration of the supernatant was quantified by the BCA reagent.

[0296] Samples were boiled with Laemmli buffer 1x and equal amount of protein per lane was loaded in a polycrilamide gel for electrophoresis. After semi-dry transfer to a PVDF membrane, blots were blocked with 5%milk, incubated with the respective primary and secondary antibodies and developed with ECL substrate.

[0297] AKS construct used in Example 2

[0298] RESULTS

[0299] AKS based on atezolizumab (anti PD-L1 antibody)

[0300] The effect of AKS based on Atezolizumab biosimilar, named as Atezolizumab-R12(AE), on cell viability was evaluated in two human cell lines, PC9 and HCC827, using a crystal violet staining assay as previously described. Control treatments included the cytotoxic payloads MMAE and antibody-drug conjugate (ADC), and AKS conjugated to the R12 peptide. Vehicle-treated wells were included as negative controls.

[0301] In both PC9 and HCC827 cell lines, treatment with the AKS conjugate Atezolizumab- R12(AE) resulted in a significative decrease in cell viability compared to vehicle and other control treatments. Specifically, cells treated with MMAE, or the ADC showed minimal reduction in viability at both 24 and 48 hours, indicating limited cytotoxic effects under these conditions. In contrast, the Atezolizumab-R12(AE) conjugate reduced cell viability in a 60-80% compared to control levels, demonstrating a potent cytotoxic effect in both cell lines. No significant differences in viability were observed between the 24-hour and 48-hour treatment periods, suggesting that the effect of AKS on cell viability was largely established within the first 24 hours (Fig. 7).

[0302] On another hand, after the treatment of HCC827 cells with atezolizumab alone, atezolizumab-R12, atezolizumab-MMAE and atezolizumab-MMAE -R12, the total levels of PD-L1 significantly degreased in cells treated with Atezolizumab-R12 conjugate (an effect, which was reverted by co-treatment with bafilomycin 100nM) and with Atezolizumab-R12-MMAE conjugate (Fig. 8)

[0303] These results indicate that the AKS conjugate Atezolizumab-R12(AE) significantly reduces the viability of PC9 and HCC827 cells, and the PD-L1 expression in HCC827 cells, whereas unconjugated cytotoxic payloads and ADC controls exhibit minimal activity under the same experimental conditions.

[0304] AKS based on bevacizumab (anti VEGF-A antibody)

[0305] The effect of AKS based on bevacizcumab biosimilar on the viability was evaluated in two cell lines H1299 and HeLa after 24 hours of treatment. Cell viability was measured relative to untreated control cells (C). The treatments included the cytotoxic payload alone (MMAF), a conventional Bevacizumab antibody-drug conjugate (Beva ADC(AF)), and two Bevacizumab-based AKS constructs, Beva-R12(AF) and Beva-MGS4(AF) (Figure 9).

[0306] In H1299 cells (Fig.9, upper panel), treatment with the cytotoxic payload alone (MMAF) reduced viability moderately compared to control, indicating intrinsic sensitivity to the payload. The conventional Bevacizumab ADC(AF) further decreased cell viability, showing the added effect of targeted delivery. Notably, the Beva-R12(AF) and Beva- MGS4(AF) AKS constructs produced a more pronounced reduction in cell viability, with Beva-R12(AF) showing the strongest cytotoxic effect, indicating enhanced cell killing mediated by the AKS constructs compared to both the cytotoxic payload alone and the conventional ADC.

[0307] In HeLa cells (Fig. 9, bottom panel), a similar trend was observed. The cytotoxic payload (MMAF) reduced cell viability modestly, while the Beva ADC(AF) showed a slightly greater effect. Both Beva-R12(AF) and Beva-MGS4(AF) further decreased cell viability, with Beva-R12(AF) again demonstrating the most significant cytotoxicity. These results suggest that the R12 and MGS4 AKS constructs enhance the potency of the cytotoxic payload in different cell types, outperforming both the free payload and conventional ADC formats.

[0308] Additionally, we evaluated the capacity of a bevacizumab conjugated to MGS4 to provoke the internalization of the soluble target of the antibody - VEGF-A by western blot (Fig.10). When we blotted cell lysates of H1299 treated with 100nM VEGF , bevacizumab, bevacizumab-MGS4 conjugates ± E64 protease inhibitor for 24h, we observed that there was a basal internalization of VEGF upon treatment with bevacizumab (lines 4 and 5), but the internalization of VEGF was significantly increased upon treatment with bevacizumab-MGS4 conjugates (lines 6 and 7), which was increased even further upon treatment with E64, demonstrating the increased VEGF internalization and its degradation in cells treated with VEGF and bevacizumab-MGS4 conjugates.

[0309] Taken together, the data demonstrates that Bevacizumab-based AKS constructs, particularly Beva-R12(AF), exhibit superior cytotoxic activity compared to standard ADCs and the cytotoxic payload alone. This, combined with the increased capacity of internalization of the soluble target VEGF-A by the bevacizumab-MGS4 conjugate, supports their potential use in targeted therapeutic applications.

[0310] AKS based on cetuximab (anti EGFR antibody)

[0311] The effect of AKS based on cetuximab biosimilar on the viability was evaluated in two cell lines A549 and H1975 after 24 hours of treatment. The effects of Cetuximab-based AKS constructs on A549 and H1975 on cell viability were evaluated after 24 hours of treatment by crystal violet and bright field microscopy. For crystal violet, cell viability was measured relative to untreated control cells (C). The treatments included the cytotoxic payload alone (MMAE), cetuximab-R12 (Cetuximab-LYTAC) a conventional Cetuximab antibody-drug conjugate (Cetuximab ADC) and the Cetuximab-R12-based AKS construct (Cetuximab AKS).

[0312] In A549 cells (Fig.11 , left top), the conventional Cetuximab ADC(AE) showed decreased viability, however, the Cetuxuximab AKS construct led to even further reduction in cell viability, demonstrating enhanced cytotoxic potency relative to MMAE alone and Cetuximab ADC.

[0313] In H1975 cells (Fig.11 , left bottom), a similar pattern was observed. Although cetuximab ADC(AE) induced a moderate reduction in viability, cetuximab AKS construct achieved a markedly greater cytotoxic effect, underscoring the superior activity of the AKS in comparison with standard ADC approaches. Phenotypic changes related to AKS treatment are observed in microscopy bright field images (Fig. 11 , right) where in both cases, A549 and H1975, treatment with AKS induces a remarked apoptotic phenotype compared to control and ADC treatments. Additionally, we evaluated the capacity of a Cetuximab alone (Ctx), Ctx with toxic load MMAE (Ctx-AE), Ctx-MGS4 or Ctx-R12 conjugates, Ctx-MGS4 or Ctx-R12 conjugates with toxic load (Ctx-R12-AE or Ctx-MGS4-AE), to reduce the total EGFR in H1299 cells upon 24h treatment ± lysosome inhibitor bafilomycin (Fig. 12). It is well known that treatment with Ctx alone reduces EGFR, and this was confirmed here (lane 1 vs lane 3). While we observed that the reduction of EGFR by Ctx-AE, Ctx-R12 and Ctx-MGS4 was similar to the one induced by Ctx alone (lanes 5,11 and 13 vs. lane 3), we saw that the AKS format further reduced the total EGFR, especially in the case of MGS4 (lane 7 vs. lane 5 and lane 13 vs. lane 9). Bafilomycin treatment partially reverted this degradation especially in the Ctx-MGS4-AE treatment , confirming that it is lysosome-dependent (lane 10 vs lane 9) .

[0314] Overall, these data demonstrate that Cetuximab-based AKS constructs, particularly Cetux-R12-AE, display enhanced cytotoxicity in both A549 and H1975 cells, outperforming both the unconjugated payload and conventional ADCs. Additionally, the AKS constructs, particularly Cetux-MGS4-AE, outperformed cetuximab alone, the ADC format and the antibody-peptide format in reducing total EGFR protein. Together, results highlight the inventive advantage of the AKS format for targeted cancer therapy.

[0315] EXAMPLE 3

[0316] AKS construct used in Example 3 To evaluate the effect of A) atezolizumab B) atezolizumab, conjugated with MMAE and C) atezolizumab conjugated with MMAE and either R12 peptide (SEQ ID NO 1 : RRRRRRRRRRRR) or modified R12 peptide (SEQ ID NO 14: VSGWRLFKKIS- GGGGS-RRRRRRRRRRRR) on cell viability, 40 000 PC9 cells were seeded in 96 well plate in 100ul media.

[0317] On the next day, when cell have already attached with about 60% confluence, old media was removed and fresh media containing the abovementioned treatments at 100nM final concentration was applied for further 24h. On the third day, media with treatments was removed, cells were gently washed with PBS twice and fixed with paraformaldehyde for 5min. After an additional PBS to remove paraformaldehyde, cells were stained with 0.1 % Crystal violet in 20% methanol solution for 10min. The plate was the extensively washed with water until no dye was further eluting. Then the plate was dried and the dye was eluted with 100% methanol. The absorbance at 590nM was read and results were rep- resented as % of the absorbance of the untreated cells (Figure 13).

Claims

1. CLAIMS1 . A conjugate comprising: i. a first protein comprising a targeting moiety that specifically binds to IL- 1 b, EGFR, PD-L1 or VEGF-A, ii. a second protein that is bound to the first protein and that comprises an endocytosis-triggering peptide that is capable of binding to an endocytic receptor of a tumor cell, wherein the endocytosis-triggering peptide comprises SEQ ID NO: 1 , 2, or 14 and iii. a cargo that is toxic for tumor cells, wherein the cargo is bound to the first protein via a linker comprising maleimide Valine-Citruline, and wherein the binding of the endocytosis-triggering peptide to the endocytic receptor of the tumor cell causes the internalization of the conjugate and of the protein of interest inside the tumor cell, and wherein the second protein further comprises a flexible linker that binds the second protein with the first protein, wherein the flexible linker comprises maleimide-PEGe-N-Hydroxysuccinimidyl ester .

2. The conjugate according to claim 1 , wherein: i. the first protein comprises a targeting moiety that specifically binds to IL- 1 b, preferably comprising a VH and VL domain, wherein the VL domain comprises or consists of SEQ ID NO: 8, and the VH domain comprises or consists of SEQ ID NO: 7 (Canakinumab), and ii. the second protein comprises an endocytosis-triggering peptide comprising or consisting of SEQ ID NO: 1 or 2.

3. The conjugate according to claim 1 , wherein: i. the first protein comprises a targeting moiety that specifically binds to EGFR, preferably comprising a VH and VL domain, wherein the VL domain comprises or consists of SEQ ID NO: 10, and the VH domain comprises or consists of SEQ ID NO: 9 (Cetuximab), and ii. the second protein comprises an endocytosis-triggering peptide comprising or consisting of SEQ ID NO: 1 or 2.

4. The conjugate according to claim 1 , wherein: i. the first protein comprises a targeting moiety that specifically binds to POLI , preferably comprising a VH and VL domain, wherein the VL domain comprises or consists of SEQ ID NO: 4, and the VH domain comprises or consists of SEQ ID NO: 3 (Atezolizumab), andii. the second protein comprises an endocytosis-triggering peptide comprising or consisting of SEQ ID NO: 1 or 14.

5. The conjugate according to claim 1 , wherein: i. the first protein comprises a targeting moiety that specifically binds to VEGF-A, preferably comprising a VH and VL domain, wherein the VL domain comprises or consists of SEQ ID NO: 6, and the VH domain comprises or consists of SEQ ID NO: 5 (Bevacizumab), and ii. the second protein comprises an endocytosis-triggering peptide comprising or consisting of SEQ ID NO: 1 or 2.

6. The conjugate according to any one of claims 1 to 5, wherein the cargo is selected from the group consisting of monomethylauristatin E (MMAE), monomethyauri- statin F (MMAF), N-acetyl g calicheamicin, DM1 , DM4, deruxtecan, and pyrrolo- benzodiazepine (PBD) dimer.

7. A pharmaceutical composition comprising the conjugate as defined in any one of claims 1 to 6, further comprising a pharmaceutically acceptable carrier.

8. The conjugate according to any one of claims 1 to 6, or the pharmaceutical composition as defined in claim 7, for use in medicine.

9. The conjugate according to any one of claims 1 to 6, or the pharmaceutical composition as defined in claim 7, for use in a method of treating and / or preventing cancer.

10. The conjugate as defined in claim 2, for use in a method of treating and / or preventing a IL-1 b positive cancer.11 . The conjugate as defined in claim 3, for use in a method of treating and / or preventing a EGFR positive cancer.

12. The conjugate as defined in claim 4, for use in a method of treating and / or preventing a PD-L1 cancer.

13. The conjugate as defined in claim 5, for use in a method of treating and / or preventing a VEGF-A cancer.

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