Detection methods and combination therapies of cells with extracellular ras expression

Abstract

The present invention relates to a combination of an antigen binding protein (ABP) or an isolated nucleic acid comprising a sequence encoding the ABP and a modulator compound for use as medicament. The ABP comprises at least a first antigen binding domain capable of binding to an extracellular Ras antigen. The invention also relates to a pharmaceutical composition comprising the combination and a composition or kit comprising an antigen binding protein (ABP) or an isolated nucleic acid comprising a sequence encoding the ABP and a modulator compound. The invention also relates to an in vitro 3D cell culture model and an in vitro method for detecting the expression of predominantly extracellular Ras proteins on the surface of predominantly living cells. In addition, the present invention relates to novel cytoplasmic antigens of human, or animal, or insect, or plant or algae origin relocated and expressed on the extracellular side of the plasma membrane of viable cells which can be used for diagnostics and therapy in human and veterinarian medicine or for agricultural purpose. The invention also relates to novel antigen binding compounds (ABC) for use as medicament, or supplement or fertilizer on cells of human, or animal, or insect, or plant or algae origin.

Description

[0001]

[0002] December 8, 2025 066094 K

[0003] Dr. Thomas Bumm

[0004] Josef Wiesner Stra&e 1

[0005] 91443 Scheinfeld

[0006] GERMANY

[0007] Bumm UG

[0008] Rheinpromenade 4a

[0009] 40789 Monheim

[0010] GERMANY

[0011] “Detection methods and combination therapies of cells with extracellular Ras expression”

[0012] FIELD OF THE INVENTION

[0013] The present invention pertains to targeting extracellular Ras antigen by a combination of an antigen-binding protein and a modulator compound for use as medicament, in particular for use in a method of diagnosis, prevention and / or treatment of a proliferative disorder, such as cancer, and / or for use in adoptive, target-cell specific immunotherapy and / or for use in drug development and / or for use in a method of diagnosis, prevention and / or treatment of a non- malignant disease. The invention also relates to a pharmaceutical composition, a composition and a kit. Further, an in vitro 3D cell culture model is provided and an in vitro method for detecting the expression of predominantly extracellular Ras proteins on the surface of predominantly living cells. BACKGROUND OF THE INVENTION

[0014] The superfamily of Ras (" Rat sarcoma virus") proteins belong to a class of cytosolic proteins called small GTPase. The Ras superfamily comprise over 150 members and is divided into five major families: Ras, Rho, Arf / Sar, Ran, and Rab. Ras proteins are binary molecular switches that cycle between active guanosine triphosphate (GTP)-bound and inactive guanosine diphosphate (GDP)-bound states, transmitting signals within cells (cellular signal transduction), and are important for regulating cell growth, cell proliferation, and cell differentiation.

[0015] The RAS family is comprised of 36 genes, with more than 40 different proteins reported. There are three RAS genes in the human genome, often mutated in human cancer, namely Kirsten rat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral (v-RAS) oncogene homolog (NRAS), and Harvey rat sarcoma viral oncogene homolog (HRAS). For KRAS, two isoforms arise from alternative RNA splicing, namely KRAS4A and KRAS4B. Thus, due to alternative splicing of the mRNAand post-translational modifications of the proteins, there are different post-translational modified Ras Proteins expressed in the cytosol of cells.

[0016] The amino-terminal catalytic domains of H-Ras, N-Ras and K-Ras are highly conserved (90-100% identical), but the carboxy terminal sequences diverge significantly, carrying the hypervariable region (HVR). The HVR comprises the anchor sequences, important for attachment to the cytoplasmic part of the membrane and to sort, or target, to different membrane microdomains. The interaction of Ras with the plasma membrane is highly dynamic and Ras is present on endosomes and other intracellular membranes, such as the endoplasmic reticulum (ER) and Golgi [1],

[0017] There are many different proteins in the cell with high binding affinity and / or specificity for Ras, important for regulating the Ras signaling cascade. The Ras GTPases are continuously cycling between inactive (RAS’GDP) and active (RAS’GTP) conformations in a process modulated by negative (GTPase Activating Proteins, GAPs) and positive (Guanine nucleotide Exchange Factors, GEFs) regulators.

[0018] Owing to the Ras family proteins’ essential role in modulating a wide range of cellular processes, several human diseases are caused by the dysregulation or dysfunction of Ras related signaling pathways. These include cancer, developmental-, neurocognitive- and neurodegenerative disorders, as well as metabolic and cardiovascular diseases [2],

[0019] Mutations in Ras genes can lead to the production of permanently activated Ras proteins, which can cause overactive signaling inside the cell, even in the absence of incoming signals. Accumulation of constantly active Ras proteins permanently stimulating cell growth plays an important part in cancer cell proliferation and survival. Thus, Ras genes are protooncogenic, i.e. mutations in Ras genes can render them oncogenic, and this is causally linked to the development of certain types of cancer. Importantly, the three Ras genes in humans (HRAS, KRAS, and NRAS) are the most common oncogenes in human cancer, and oncogenic mutations that permanently activate Ras (gain-of-function) are found in 20 to 27% of all human tumors (range 0.8%-90%) (e.g., pancreatic cancer, but also lung or colorectal carcinoma are often characterized by a high percentage of oncogenic Ras mutations).

[0020] Prior art reports small Ras peptides to be expressed on the cell surface via the human major histocompatibility complex (MHC) known as the human leukocyte antigen (HI_A). Therefore, all current cancer immunotherapies against extracellular Ras use T-cell receptors (TCR) or antibody single-chain variable fragments (scFv) targeting Ras mutant peptides expressed via H LA class I or II [3-7],

[0021] It was recently found (PCT / EP2024 / 065774) that extracellular Ras protein is also accessible on cancer cells outside the MHC system. This Ras expression on the cell surface can be used for immunotherapy with different formats of antibodies, chimeric antigen receptor T cells (CART) or small chemical binders. In contrast to prior art, Antigen Binding Proteins (ABPs) against extracellular Ras independent of the MHC system are not restricted to a specific HLA genotype. Twelve different bi-specific T cell recruiting Diabodies had been designed targeting extracellular Ras and targeting the human CD3 receptor complex on T cells being effective in killing tumor cells. The binding sites for these Ras ABPs are in the normally intracellular Ras domains, adapted to the different harsh and densely packed conditions of the cytosol.

[0022] Prior art also reports on combination therapies in tumor therapy, for example the combination of antiangiogenic agents like Bevacizumab® with immune checkpoint inhibitors, for increasing treatment efficiency. However, 3D tumor models, where the tumor microenvironment (TME) is supporting the tumor cells and possibly impede the immune cells, had not been used. In addition, the receptors targeted in prior art immunotherapies are native receptors having transmembrane domains reaching into the cytosol. It is a particular challenge to target extracellular Ras because it only reaches a short distance into the lipid layer and may be redistributed to the cytosol or intracellular membrane.

[0023] Hence, it was an object of the present invention to provide a proper model for testing combination therapies comprising an ABP targeting extracellular Ras antigen not in the context of HLA / MHC and to identify suitable combinations for use as medicament, in particular for use in a method of diagnosis, prevention and / or treatment of a proliferative disorder, such as cancer. In addition, it was aimed at providing an ABP targeting extracellular Ras antigen for use in a method of diagnosis, prevention and / or treatment of a non-malignant disease.

[0024] Prior art reports methods and compositions for detecting the location of Ras proteins in cells, but fail to report their location on the extracellular side of the plasma membrane

[0016] , Therefore, there is also a need for new methods to predominantly detect extracellular Ras expression on viable cells. Hence, it is an object of the present invention to provide such methods involving an ABP targeting extracellular Ras antigen, or involving a detecting compound targeting a specific tag sequence connected to antigens of this invention. In addition, was aimed to provide such methods for use in drug development. Those methods and compositions preferably have a good signal to noise ratio to avoid false positive and false negative results.

[0025] The plasma membrane (PM) of eukaryotes, also known as the cell membrane, is a semipermeable biological membrane that separates and protects the interior of a cell from the outside environment, also known as the extracellular space. The PM is made up of a large number of lipids, predominantly phospholipids, and a variety of proteins that either span the membrane thickness or attach via lipid binding motifs from the cytoplasmic or extracellular side to it. These lipid binding motifs can be specific amino acids sequences, for example the pleckstrin homology (PH) domains, that harbor lipid-binding pockets for recognition of specific PM lipids, or post-translationally modifications (PTMs) of amino acids.

[0026] The PH domains can bind phosphoinositide lipids (PIPs) phosphorylated at different sites on the inositol ring

[0017] , While PH domain PIP binding does not always indicate binding to PM it is generally the case that PIP interactions play an important role in directing PH domain proteins to their appropriate subcellular locations, where they interreact with other PH domain proteins to form signal transduction complexes and cytoskeleton structures. For the post translational modifications (PTMs), over 620 types have been identified to regulate protein activity

[0018] , One of the most common PTMs is protein lipidation, that can attach up to seven different types of lipids, including fatty acids (FA), lipoic acids, isoprenoids, sterols, phospholipids, glycosylphosphatidylinositol anchors, and lipid-derived electrophiles, to proteins. Based on the location of the modified proteins, protein lipidation can be divided into two categories. The “first category” comprises lipid modifications that occur in the cytoplasm or on the cytoplasmic side of membranes, including S-, N- orO-palmitoylation, N-myristoylation and S-prenylation. The “second category” is composed of lipid modifications that occur in the lumen of secretory organelles, such as glycosylphosphatidylinositol (GPI) anchor and cholesterylation. For the “first category”, the 16-carbon fatty acid palmitate covalently linked to cysteine residues of proteins via the labile thioester bond is defined as protein S-palmitoylation. The less frequently occurring O-palmitoylation is characterised by the attachment of palmitate to serine residues. N-palmitoylation is characterised by the attachment of palmitate to the N-terminus of a protein. The attachment of 14-carbon myristic acid to N-terminal glycine residues via an amide bond in a manner of co- or posttranslational modification is referred to as N-myristoylation. S-prenylation refers to the attachment of a 15-carbon farnesyl or a 20-carbon geranylgeranyl isoprenoid lipid onto cysteine residues of proteins via a thioether bond.

[0027] Therefore, it was another object of the present invention to provide a proper model for testing the cellular location of different human and animal cytoplasmic expressed protein-domains, containing a pleckstrin homology (PH) domain and / or one or more PTM lipidation motifs. In addition, it was aimed at providing evidence that cytoplasmic proteins other than the three major Ras oncogenes (KRAS, NRAS and HRAS), containing PH and / or PTM lipidation motifs, can be found on the extracellular plasma membrane for use in a method of diagnosis, prevention and / or treatment of a malignant or non-malignant disease. Also, it was another object of the present invention to provide evidence that drugs or compounds targeting the cell lipid metabolism, the lipid composition of the PM or PTM lipidation of proteins, can be used in a method of drug development, diagnosis, prevention and / or treatment of a malignant or non-malignant disease.

[0028] Plants also use lipid post-translational mechanisms for modification of cytoplasmic proteins

[0019] , In plants there are three types of known intracellular lipid modifications of proteins including prenylation, N-myristoylation and S-acylation. Prenylation involves the addition of a single 15-carbon farnesyl or single or dual 20-carbon geranylgeranyl moieties to one or two cysteines near the C-terminus of target proteins. Three separate heterodimeric enzymes perform prenylation: protein farnesyltransferase (PFT), protein geranylgeranyltransferase-l (PGGT), and Rab geranylgeranyltransferase (Rab-GGT). PFT recognizes a C-terminal CaaX box, where C is the prenylated cysteine, a is usually aliphatic, and X is usually alanine, cysteine, glutamine, methionine, or serine. PGGT recognizes a similar sequence, except the X is almost always leucine. A wide range of critical signaling proteins is prenylated by PFT and PGGT, including members of the Ras superfamily of small GTPases, heterotrimeric G protein y subunits, certain classes of protein kinases, and many others. N-myristoylation involves the addition of a 14 carbon saturated myristate group to the N-terminal glycine of target proteins. It is carried out by a monomeric enzyme, N-myristoyltransferase (NMT). The absolute requirement for enzymatic activity is an N-terminal glycine on the target protein, while amino acid residues two through six are used in substrate recognition, residues 7 through 10 are less restricted, and residues 11 through 17 are hydrophilic. Protein S-acylation is the covalent attachment of a fatty acid to a cysteine residue. Commonly the acyl group involved is palmitate, a 16-carbon saturated fatty acid, but other groups, including shorter or longer or unsaturated groups, can be added. Unlike prenylation or myristoylation, which have specific target sequences at the C- and N-terminus of the protein, the prenylated cysteine can be localized anywhere in the plant protein, though it is common to find acylated cysteines in close proximity to myristoylated glycines on the N-terminus or prenylated cysteins on the C-terminus.

[0029] Therefore, it was another object of the present invention to provide evidence that protein domains of different plant species containing PTM lipidation motifs, can relocalize cytoplasmic proteins or peptides on the extracellular plasma membrane. In addition, it was another object of the present invention to provide evidence that these extracellular expressed proteins of the GTPase family on plant cells, can be used in a method of treatment, fertilization and / or supplementation for agriculture and / or botanical purpose.

[0030] In the last years, lysosomes have emerged as multifaceted intracellular compartments where specific cargo proteins, with or without N-terminal signal sequence, can converge before being released into the extracellular space by lysosomal exocytosis

[0020] , Prior art reports members of different intracellular heat shock protein (HSP) families are exported by cells via the lysoendosomal and exosomal pathway and can be detected on the extracellular side of the plasma membrane

[0021] , HSPs lack classical N-terminus signal sequence required for conventional protein secretory pathway. For human KRAS mutant tumor cells, exosomes are reported to contain many tumor-promoting cytoplasmic proteins, including KRAS, SRC family kinases and integrins

[0022] , Various factors, such as cellular stress and the presence of oncogenes, such as Src, EGFR, and KRAS, can impact the composition of exosomal cargo. The presence of Src in cells increases the release of exosomes containing proinflammatory proteins, while the presence of KRAS leads to an increase in the number of exosomes containing proteins involved in cell growth and survival. Modulating cell signaling with drugs can affect exosome contents, formation and quantity

[0023] , Lipids constitute another crucial class of macromolecules that are packaged into exosomes. These vesicles are particularly rich in various lipids, including cholesterol, phosphatidylcholine, phosphatidylserine, sphingomyelin, and ceramide. These lipids play diverse and essential roles in exosome biogenesis, uptake, and the functional impact of exosomes on recipient cells. Simvastatin, an HMG-CoA reductase inhibitor, prevents the synthesis of cholesterol and affects the intracellular concentrations of exosome-associated proteins. Furthermore, proteins involved in cytoskeletal organization are essential for both exosome release and endocytic processes. A competitive inhibitor of the ROCK family, which includes ROCK1 and ROCK2, is causing in tumor cells a decrease in microvesicles and exosome-sized secretion. Additionally, inhibitors targeting protein kinases, such as imatinib and dasatinib, are effective at preventing the activation of ERK, which is necessary for microvesiculation.

[0031] Therefore, it was another object of the present invention to provide a proper model for testing different drugs and compounds, targeting parts of the lysoendosomal and exosomal pathways, on the relocation of cytoplasmic proteins, containing a PH domain and / or one or more PTM lipidation motifs, on the extracellular PM. In addition, it was aimed at providing evidence for drugs and compounds, targeting parts of the lysoendosomal and exosomal pathways, for use in a method of drug development, diagnosis, prevention and / or treatment of a malignant or non-malignant disease.

[0032] Prior art reports extracellular HSPs can exacerbate cell growth

[0024] . These originally cytoplasmic and nuclear proteins can engage surface receptors and trigger intracellular signaling in an autocrine or paracrine fashion. The localization of HSPs within the lipid bilayer of cellular plasma membranes and their interacting with membrane lipids, possibly stabilizing membranes, can regulate the physical properties and organization of membrane microdomain compositions. Therefore, it was another object to provide proper models for testing binding of ABPs and antigen binding compounds (ABCs) to extracellular Ras, is modulating cell metabolism and cell migration. In addition, it was aimed at providing evidence for ABPs and ABCs of the present invention, stimulating and / or supporting the growth of cells, for use in a method of drug development, diagnosis, prevention and / or treatment of a malignant or non-malignant disease.

[0033] In the last years, click chemistry has made a revolution in the field of chemical biology and especially in the field of cancer therapy

[0025] , Click chemistry is a molecular assembly technique, enabling swift and selective molecule combination to create drug compounds with novel properties. Prior art reports the development of a KRAS G12C mutant specific inhibitor using click chemistry

[0026] , Also click chemistry is reported by prior art for generating albumin nanoparticles for anticancer treatment, or connecting the small molecule ligand Acetazolamide to human serum albumin fused with the chemotherapeutic agent Paclitaxel for targeting tumor hypoxia [27, 28], No prior art reports, connecting a Ras protein targeting small molecule inhibitor (SMI) with click chemistry to a serum protein, for targeting Ras on the extracellular cell membrane for therapy.

[0034] Therefore, it was another object of the present invention to provide evidence for, click chemistry can be used for connecting Ras specific SMIs as antigen binding compounds (ABCs) to human serum albumin for targeting extracellular Ras on malign-cells. In addition, it was aimed at providing evidence for using click chemistry to generate drugs or compounds with novel properties against extracellular antigens of this invention, for use in a method of drug development, diagnosis, prevention and / or treatment of a malignant and / or non-malignant disease.

[0035] SUMMARY OF THE INVENTION

[0036] It has presently found by using a 3D tumor cell model that combinations of an ABP of the present invention with at least one other compound surprisingly have a very good anti-tumor effect which is better than an ABP alone. In addition, it has been shown that the ABP of the present invention bind to extracellular Ras antigen not in the context of or not restricted to, a human leukocyte antigen (HLA)-peptide complex, or specific HLA alleles, presented on the cell surface by the major histocompatibility complex (MHC). Further, a stimulating effect on tumor cells and non-malignant cells using ABPs alone has been demonstrated. In addition to the three Ras antigens (KRAS, NRAS and HRAS), it has been shown for various cytoplasmic proteins of human, animal and plant origin, that plasma membrane binding domains, containing a PH domain and / or one or more PTM lipidation motifs, can redirect proteins for localization on the extracellular side of the plasma membrane. Further, the extracellular Ras antigen expression on primary cells from leukemia patients has been demonstrated.

[0037] Further, different methods have been identified for detecting predominantly extracellular Ras protein on the surface of predominantly living cells. In addition, the importance of optimizing these methods has been demonstrated. Further it has presently found by using a chemical synthesis method that ligates at least one other first compound, the first compound is a chemical compound with high binding affinity to Ras antigen, via connecting linkers to a second compound, the second compound being albumin with no anti-tumor efficacy and no cytoplasmic uptake but with a high extracellular distribution, is generating a novel antigen binding fusion compound surprisingly having a very good anti-tumor efficacy which is similar or even better as the anti-tumor efficacy of the individual Ras inhibitor alone, or what is even more surprising for some antigen binding fusion compounds a positive stimulatory and activating effect on specific cells is reported in contrast if the individual compounds are used alone with no effect or even an inhibitory effect on the target cells.

[0038] In a first aspect of the invention, a combination of an antigen binding protein (ABP) or an isolated nucleic acid comprising a sequence encoding the ABP and a modulator compound for use as medicament is provided, wherein the ABP comprises at least a first antigen binding domain capable of binding to an extracellular Ras antigen and wherein the modulator compound is selected from the group consisting of

[0039] a) an All-trans-retinoic acid (ATRA) based modulator,

[0040] b) a modulator of the cholesterol and triglyceride synthesis or metabolism, preferably Simvastatin, Fluvastatin, Lapaquistat, or Bemfivastatin

[0041] c) an immunomodulatory drug targeting cereblon, preferably Lenalidomide, Iberdomide, HOMO-Protac cereblon degrader 1, or Eragidomide,

[0042] d) a modulator of monomeric GTP-binding proteins, preferably the Ras family, preferably the Ras signaling complex, preferably NSC-70220, SAH-SOS1A TFA, Adagrasib, BI-2852, MRTX-1133, RMC-0331 or SOS1 -activator 1,

[0043] e) a kinase modulator, preferably of the Rho kinase pathway, preferably Narciclasine, f) a tyrosine kinase modulator, preferably of the JAK signaling pathway, preferably Ruxolitinib,

[0044] g) a modulator of esterases, preferably of the phosphodiesterase pathway, preferably Anagrelide,

[0045] h) a lipid, preferably a lipid part of a cell membrane

[0046] i) a fatty acid, preferably a saturated fatty acid or unsaturated fatty acid

[0047] j) a modulator of the gut microbiome, and

[0048] k) a combination of two or more of a) to k).

[0049] In a preferred embodiment, said extracellular Ras antigen is not part of, or is not restricted to, a human leukocyte antigen (HLA)-peptide complex, or specific HLA alleles, presented on the cell surface by the major histocompatibility complex (MHC).

[0050] In another preferred embodiment, said ABP comprises one or more additional antigen binding domain(s) that is capable of binding to antigen(s) present on a mammalian T-cell, preferably a human cluster of differentiation 3 (CD3) antigen or a human T cell receptor (TCR).

[0051] In a further preferred embodiment, the first antigen binding domain comprises an amino acid sequence having a sequence identity of at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% sequence identity, to an amino acid sequence selected from SEQ I D NO: 20 to 31, 66 to 71, 134 to 178 and 290 to 396, or, in each case independently, optionally with no more than ten, nine, eight, seven, six, five, four, preferably no more than three, two or one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.

[0052] The isolated nucleic acid may be comprised in an expression construct, preferably further comprising promoter and / or terminator sequences. The isolated nucleic acid and / or the expression construct may be comprised in a recombinant host cell.

[0053] In a preferred embodiment, the ABP is PCC04D diabody (SEQ ID NOs 23 and 231).

[0054] In another preferred embodiment, the ABP enhances a cell-mediated immune response, such as the immune response mediated by an activated cytotoxic T-cell (CTL) to a mammalian cell expressing said extracellular Ras antigen. Further, the ABP may be selected from the group consisting of an immunoglobulin molecule, such as an IgG, IgE, IgD, IgA, or IgM immunoglobulin, preferably an IgG immunoglobulin, a monoclonal antibody, a chimeric antibody, a bispecifc ABP, such as a bispecific antibody, a CDR-grafted antibody, a humanized antibody, a single domain antibody, such as a VHH single domain antibody, a hemibody antibody, a single-chain KeyLock-antibody, a diabody, a single chain diabody, a variable domain of the antibody heavy chain or antibody light chain, a multispecific antibody, a Chimeric Antigen Receptor (CAR), alternative protein binders including monobodies (derived from fibronectin type III), anticalins (derived from lipocalins), affibodies (derived from immunoglobulin-binding protein A), DARPins (Designed Ankyrin Repeat Proteins), proteins with repeating motifs like leucine-rich repeats (LRRs), ankyrin repeats (ARs), Armadillo repeats (Arms), tetratricopeptide repeats (TPRs), and / or a fragment of an antibody, such as a fragment of a monoclonal antibody, for example a single chain Fv (scFv), (scFv)2, a Fv, a disulfide linked Fv, Fab, Fab', F(ab')2 ora scFv-Fc, preferably wherein said ABP is a bispecific antibody or a diabody.

[0055] In another embodiment, the ABP comprises at least one antigen binding domain capable of binding with one, two, three, four, or preferable more than five amino acids to a Ras antigen, wherein the at least one antigen binding domain is alone or in a bipartite complex with another Ras binding protein, or in a tripartite complex with another Ras binding protein and the membrane, or in a multipartite complex with one or more Ras binding proteins and / or the membrane, and wherein said amino acids in the ABP optionally comprise:

[0056] (i) a domain comprising the RBD-CRD region (amino acids 52 to 188) of the human RAF1 protein as set forth in SEQ ID NO: 23,

[0057] (ii) optionally wherein one or multiple amino acids as depicted in Tables A and B are in contact with KRAS residues, or wherein one or multiple amino acids as depicted in Table C are in contact with the membrane, or wherein one or multiple amino acids as depicted in Table D are in contact with KRAS residues in a tripartite complex comprised of RBD-CRD, KRAS and the membrane, or

[0058] (iii) a domain comprising the CDC25H region (amino acids 780 to 1019) of the human SOS1 protein as set forth in SEQ ID NO: 24, optionally wherein one or multiple amino acids as depicted in Table E are in contact with KRAS residues, or (iv) a domain comprising the CDC25 region (amino acids 1038 to 1270) of the human RASGRF1 protein as set forth in SEQ ID NO: 29, optionally wherein one or multiple amino acids as depicted in Table F are in contact with Ras residues in a tripartite complex with Ras, Sos1 and RasGRF1, or

[0059] (v) a domain comprising the RAS binding region (amino acids 274 to 364) of the human RASSF5 protein (UniProt Q8WWW0-1) as set forth in SEQ ID NO: 377, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0060] (vi) a domain comprising the RAS binding region (amino acids 201 to 363) of a splice variant of the human RASSF5 protein (UniProt Q8WWW0-2) as set forth in SEQ ID NO: 378, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0061] (vii)a domain comprising the RAS binding region (amino acids 201 to 363) of the human RASSF1 protein (UniProt Q9NS23-1) as set forth in SEQ ID NO: 379, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0062] (viii) a domain comprising the RAS binding region (amino acids 176 to 264) of the human RASSF2 protein (UniProt P50749-1) as set forth in SEQ ID NO: 380, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0063] (ix) a domain comprising the RAS binding region (amino acids 6 to 89) of the human RASSF7 protein (UniProt Q02833-1) as set forth in SEQ ID NO: 381, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0064] (x) a domain comprising the RAS binding region (amino acids 19 to 91) of the human ARAF protein (UniProt P10398-1) as set forth in SEQ ID NO: 382, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0065] (xi) a domain comprising the RAS binding region (amino acids 19 to 148) of the human ARAF protein (UniProt P10398-1) as set forth in SEQ ID NO: 383, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0066] (xii)a domain comprising the RAS binding region (amino acids 151 to 232) of the human BRAF protein (UniProt P15056) as set forth in SEQ ID NO: 384, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0067] (xiii) a domain comprising the RAS binding region (amino acids 151 to 320) of the human BRAF protein (UniProt P15056) as set forth in SEQ ID NO: 385, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0068] (xiv) a domain comprising the RAS binding region (amino acids 56 to 131) of the human RAF1 protein (UniProt P04049-1) as set forth in SEQ ID NO: 386, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0069] (xv) a domain comprising the RAS binding region (amino acids 1235 to 1451) of the human NF1 protein (UniProt P21359-1) as set forth in SEQ ID NO: 387, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0070] (xvi) a domain comprising the RAS binding region (amino acids 748 to 942) of the human RASA1 protein (UniProt P20936-1) as set forth in SEQ ID NO: 388, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0071] (xvii) a domain comprising the RAS binding region (amino acids 302 to 512) of the human RASA4 protein (UniProt 043374-1) as set forth in SEQ ID NO: 389, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0072] (xviii) a domain comprising the RAS binding region (amino acids 938 to 1130) of the human RGS12 protein (UniProt 014924-1) as set forth in SEQ ID NO: 390, optionally wherein one or multiple amino acids are in contact with RAS residues.

[0073] In another preferred embodiment, the ABP comprises at least one antigen binding domain capable of binding to an extracellular Ras antigen, preferably provided that said extracellular Ras antigen is not part of, or is not restricted to, a HI_A-peptide complex, or specific HLA alleles presented on the cell surface, and wherein said Ras binding ABP comprises a diabody format as set forth in SEQ ID NO: 228 to 239, wherein in each case independently comprise a sequence having no more than three or two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to SEQ ID NO: 224, and 228 to 239, respectively; or comprising a sequence having at least 75% sequence identity, preferably at least 80% sequence identity, more preferably at least 90% sequence identity, and most preferably at last 95% sequence identity with SEQ ID NO: 224, and 228 to 239.

[0074] In a preferred embodiment, the combination is for use in a method of diagnosis, prevention and / or treatment of a proliferative disorder, such as cancer, and / or for use in adoptive, targetcell specific immunotherapy and / or for use in drug development and / or for use in a method of diagnosis, prevention and / or treatment of a non-malignant disease.

[0075] In a second aspect of the invention, a pharmaceutical composition for use as medicament is provided comprising the combination as disclosed supra and a pharmaceutically acceptable carrier, stabilizer and / or excipient.

[0076] In a third aspect of the invention, a composition comprising an antigen binding protein (ABP) or an isolated nucleic acid comprising a sequence encoding the ABP and a modulator compound is provided, wherein the ABP comprises at least a first antigen binding domain capable of binding to an extracellular Ras antigen and wherein the modulator compound is selected from the group consisting of

[0077] a) an All-trans-retinoic acid (ATRA) based modulator,

[0078] b) a modulator of the cholesterol and triglyceride synthesis or metabolism, preferably Simvastatin, Fluvastatin, Lapaquistat, or Bemfivastatin

[0079] c) an immunomodulatory drug targeting cereblon, preferably Lenalidomide, Iberdomide, HOMO-Protac cereblon degrader 1, or Eragidomide,

[0080] d) a modulator of monomeric GTP-binding proteins, preferably the Ras family, preferably the Ras signaling complex, preferably NSC-70220, SAH-SOS1A TFA, Adagrasib, BI-2852, MRTX-1133, RMC-0331 or SOS1 -activator 1,

[0081] e) a kinase modulator, preferably of the Rho kinase pathway, preferably Narciclasine, f) a tyrosine kinase modulator, preferably of the JAK signaling pathway, preferably Ruxolitinib,

[0082] g) a modulator of esterases, preferably of the phosphodiesterase pathway, preferably Anagrelide,

[0083] h) a lipid, preferably a lipid part of a cell membrane

[0084] i) a fatty acid, preferably a saturated fatty acid or unsaturated fatty acid

[0085] j) a modulator of the gut microbiome, and k) a combination of two or more of a) to k).

[0086] In a preferred embodiment, the composition is a pharmaceutical composition, preferably additionally comprising a pharmaceutically acceptable carrier, stabilizer and / or excipient.

[0087] In a fourth aspect of the invention, a kit comprising an antigen binding protein (ABP) or an isolated nucleic acid comprising a sequence encoding the ABP and a modulator compound is provided, wherein the ABP comprises at least a first antigen binding domain capable of binding to an extracellular Ras antigen and wherein the modulator compound is selected from the group consisting of

[0088] a) an All-trans-retinoic acid (ATRA) based modulator,

[0089] b) a modulator of the cholesterol and triglyceride synthesis or metabolism, preferably Simvastatin, Fluvastatin, Lapaquistat, or Bemfivastatin

[0090] c) an immunomodulatory drug targeting cereblon, preferably Lenalidomide, Iberdomide, HOMO-Protac cereblon degrader 1, or Eragidomide,

[0091] d) a modulator of monomeric GTP-binding proteins, preferably the Ras family, preferably the Ras signaling complex, preferably NSC-70220, SAH-SOS1A TFA, Adagrasib, BI-2852, MRTX-1133, RMC-0331 or SOS1 -activator 1,

[0092] e) a kinase modulator, preferably of the Rho kinase pathway, preferably Narciclasine, f) a tyrosine kinase modulator, preferably of the JAK signaling pathway, preferably Ruxolitinib,

[0093] g) a modulator of esterases, preferably of the phosphodiesterase pathway, preferably Anagrelide,

[0094] h) a lipid, preferably a lipid part of a cell membrane

[0095] i) a fatty acid, preferably a saturated fatty acid or unsaturated fatty acid

[0096] j) a modulator of the gut microbiome, and

[0097] k) a combination of two or more of a) to k).

[0098] In a fifth aspect of the invention, an in vitro 3D cell culture model is provided comprising tumor cells and at least one non-malignant cell selected from the group comprising or consisting of a fibroblast, an endothelial cell of an arterial blood vessel, an endothelial cell of a venous blood vessel, an endothelial cell of a lymphatic vessel, a tissue macrophage, a fat cell, an osteoblast, a chondrocyte, a smooth muscle cell, a preadipocyte, a pericyte, a mesenchymal stem cell, a melanocyte, a keratinocyte, hematopoietic progenitors, a dendritic cell, a skeletal muscle cell, a T cell and a B cell, preferably wherein the at least non-malignant cell is a T cell, a fibroblast and an endothelial cell.

[0099] In a preferred embodiment, the in vitro 3D cell culture model further comprises the combination or the ABP as disclosed supra for testing the influence of the combination on the viability and extracellular Ras expression of the at least one tumor cell.

[0100] In a sixth aspect of the invention, an in vitro method for detecting the expression of predominantly extracellular Ras proteins on the surface of predominantly living cells is provided selected from the group consisting of

[0101] a) a method using a Ras protein labeling compound with substantially no penetration inside the cell and / or predominantly binding to the extracellular Ras protein b) a method using an agent blocking binding to intracellular Ras proteins and / or using a washing step to substantially remove binding to intracellular Ras proteins

[0102] c) a method using a Ras protein labeling compound as of a) and one or more labeling compounds binding to intracellular proteins not belonging to the Ras protein family d) a method using a dual-antigen protein-protein interaction (PPI) reporter

[0103] e) a method using cells expressing an altered Ras protein, preferably a Ras-tag fusion protein, wherein the tag can be detected on the extracellular side of the cell, optionally wherein the tag requires a second tag to be detectable (split-tag PPI)

[0104] f) a method using an ABP as defined supra

[0105] g) a method using 2D, 3D cell culture and / or spheroid or organoid cultures and / or stem cell-based embryo-like structures

[0106] h) a method combining two or more of a) to g).

[0107] In a seventh aspect of the invention an in vitro method for detecting modulation of cell activity induced by binding of ABPs or compounds to extracellular Ras on the surface of predominantly living cells is provided selected from the group consisting of

[0108] a) a method using an agent to detect a cytokine level

[0109] b) a method using at least one electrode to detect a change in cells’ electrical potential c) a method using genetically modified cells with a reporter plasmid to detect and / or measure activation of a signaling cascade

[0110] d) a method combining two or more of a) to c). In an eighth aspect of the invention, an in silica method for detecting modulation of extracellular Ras on the surface of artificial cells is provided selected from the group consisting of

[0111] a) a method using artificial cell membranes

[0112] b) a method using artificial intelligence for calculating the extracellular Ras interface with small molecule compounds, with other proteins, with the lipid cell membrane and / or with the glycocalyx.

[0113] In a ninth aspect of the invention, the extracellular target antigen depicted in the first, second, third, fourth, fifth, sixth, seventh and eight aspects of this invention can belong to a protein outside the three RAS genes (KRAS, NRAS and HRAS), containing no N-terminal secretion signal for the ER-GOLGI pathway, no transmembrane domain for integrating into the cell membrane but instead containing a PH domain and / or one or more PTM lipidation motifs for plasma membrane binding and can be detected on the extracellular surface of predominantly living cells is provided selected from the group consisting of

[0114] a) The Ras super-family

[0115] b) The Rho family

[0116] c) The Arf / Sar family

[0117] d) The Ran family

[0118] e) The Rab family

[0119] f) The protein kinase family (the non-receptor protein kinase family, protein kinase A, G, and C families, calmodulin-dependent protein kinase family, casein kinase 1 family, cyclin-dependent kinase family, mitogen-activated protein kinase family, glycogen synthase kinase (GSK3) family, CDC-like kinase (CLK) family, STE group of kinase families (homologs of yeast Sterile 7, Sterile 11, Sterile 20 kinases), tyrosine kinase family and tyrosine kinase-like family.

[0120] g) GTPase binding proteins

[0121] In a tenth aspect of the invention, the cells expressing the extracellular target antigen depicted in the first, second, third, fourth, fifth, sixth, seventh, eighth and ninth aspects of this invention can be a eukaryote cell of a) human origin

[0122] b) animal origin

[0123] c) insect origin

[0124] d) plant origin

[0125] e) seaweed origin

[0126] f) fungi origin

[0127] g) algae origin

[0128] h) yeast origin

[0129] In an eleventh aspect of the invention, instead of an ABP depicted in the first, second, third, fourth, fifth, sixth and seventh aspects of this invention, an antigen binding compound (ABC) can be used, consisting of at least two different compounds ligated together, with the first compound having a high binding affinity and / or specificity against target antigens depicted in the first, second, third, fourth, fifth, sixth, seventh, eighth and ninth aspects of this invention is provided selected from the group consisting of

[0130] a) a chemical probe using chemical synthesis

[0131] b) a small molecule modulator

[0132] c) a small molecule chemical activator

[0133] d) a small molecule inhibitor

[0134] e) a KRAS inhibitor

[0135] f) a KRAS mutant specific inhibitor

[0136] g) a NRAS inhibitor

[0137] h) a NRAS mutant specific inhibitor

[0138] a HRAS inhibitor j) a HRAS mutant specific inhibitor

[0139] k) a RAS mutant specific inhibitor

[0140] l) a pan RAS inhibitor

[0141] m) a KRAS activator

[0142] n) a NRAS activator

[0143] o) a HRAS activator

[0144] p) a pan RAS activator

[0145] q) a Rho inhibitor

[0146] r) a Arf / Sar inhibitor

[0147] s) a Ran inhibitor

[0148] t) a Rab inhibitor

[0149] u) a kinase inhibitor

[0150] v) a tyrosine kinase inhibitor

[0151] w) a Src kinase inhibitor

[0152] x) a Blk inhibitor

[0153] y) a small GTPase inhibitor

[0154] In a preferred embodiment, the second compound has a significant higher extracellular distribution property under physiological conditions as the first compound, has no significant cell toxicity and / or no significant binding affinity to the target antigen and no significant cytoplasmic uptake is provided selected from the group consisting of

[0155] a) serum proteins

[0156] b) albumin (Albutein, Plasbumin, Buminate, Alburx, Flexbumin, Plasbumin-25, Albuked, Kedbumin) c) isolated albumin domains

[0157] d) immunoglobulins (lgG1, lgG2, lgG3, lgG4, IgE, IgD, IgA, or IgM)

[0158] e) plasma protein fraction systemic (Octaplas)

[0159] f) hydroxyethyl starch

[0160] g) hetastarch (Hespan, Hextend)

[0161] h) high molecular dextran (Hyskon, Dextran 70)

[0162] i) extracellular matrix proteins

[0163] j) fatty acid binding proteins or peptides

[0164] In another embodiment, the second compound is an organic or unorganic compound, used for better distribution in soil or water, for improving treating cells of plants or algae as depicted in the tenth aspect of the invention.

[0165] In a further preferred embodiment, a third, fourth, fifth or sixth compound, each with a different high binding affinity and / or specificity against target antigens depicted in the first, second, third, fourth, fifth, sixth, seventh, eighth and ninth aspects of this invention can be connected to generate an

[0166] a) ABC targeting one antigen with two or more binders to improve its avidity

[0167] b) ABC targeting two or more antigens to improve binding to different cell clones and / or tissues and / or preventing tumor escape thru down regulation of individual antigens

[0168] c) ABC with one or more target antigen binders and one or more compounds activating the target cell for a higher elimination rate of dormant tumor stem cells

[0169] d) ABC with an additional detection tag for diagnostics

[0170] e) ABC targeting one or more antigens on target cells and one or more antigens on nonmalign cells like T-cells, B-cells, Nk-cells, fibroblast, macrophages or dendritic cells. In a twelfth aspect of the invention, a method can be used for generating the ABC depicted in the eleventh aspect of this invention is provided selected from the group consisting of a) a method using chemical synthesis

[0171] b) a method using click chemistry

[0172] c) a method using site-directed mutagenesis

[0173] d) a method using native chemical ligation

[0174] e) a method using ketoacid-hydroxylamine ligation

[0175] f) a method using serine / threonine ligation

[0176] g) a method using subtiligase-mediated ligation

[0177] h) a method using sortase-mediated ligation

[0178] i) a method using butelase-mediated ligation

[0179] j) a method using tubulin-tyrosine ligase mediated ligation

[0180] k) a method using intein-mediated ligation

[0181] In a thirteenth aspect of the invention, the ABPs and ABCs depicted in the first, second and eleventh aspects of the invention, are binding to target antigens depicted in the first, second and ninth aspects of the invention, on non-malign cells depicted in the tenth aspect of the invention for use in

[0182] a) stimulating or inhibiting the growth of target cells

[0183] b) stimulating or inhibiting the migration of target cells

[0184] c) stimulating or inhibiting the transition of target cells from one compartment into another compartment

[0185] d) stimulating or inhibiting the growth of target cell axons

[0186] e) stimulating or inhibiting the release of target cell vesicles f) stimulating or inhibiting target cell metabolism

[0187] g) stimulating or inhibiting cell dormnancy

[0188] In a fourteenth aspect of the invention, the ABPs or ABCs depicted in the eleventh and thirteenth aspects of the invention, are binding to target antigens depicted in the ninth aspects of the invention, on plant cells or algae for use in

[0189] a) agriculture for human or animal food production

[0190] b) botanical work

[0191] c) synthesis of biofuels or organic precursors

[0192] d) synthesis of proteins, peptides or amino acids

[0193] e) synthesis of human or animal food additives

[0194] Other objects, features, advantages and aspects of the present invention will become apparent to those skilled in the art from the following description and appended claims. It should be understood, however, that the following description, appended claims, and specific examples, which indicate preferred embodiments of the application, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following.

[0195] DETAILED DESCRIPTION OF THE INVENTION

[0196] The present invention relates to novel combinations which comprise an ABP and a modulator compound for use as medicament.

[0197] In addition, the present invention relates to novel antigens, of human, or animal, or plant origin relocated and expressed on the extracellular side of the plasma membrane of malign and nonmalign cells and can be used for diagnostics and therapy for human and veterinarian medicine or for agricultural purpose.

[0198] In another addition, the present invention relates to novel antigen binding compounds (ABC) for use as medicament or supplement. All components (ABP, ABC and modulator compound) will now be described in detail.

[0199] Combinations of ABPs and modulator compounds

[0200] In the present invention, a 3D co-culture model was used to test combinations of ABPs according to the present invention with modulator compounds.

[0201] According to a first aspect, a combination is provided, wherein the combination comprises an antigen binding protein (ABP) or an isolated nucleic acid comprising a sequence encoding the ABP and

[0202] a modulator compound

[0203] for use as medicament, wherein the ABP comprises at least a first antigen binding domain capable of binding to an extracellular Ras antigen and wherein the modulator compound is selected from the group consisting of

[0204] a) an All-trans-retinoic acid (ATRA) based modulator,

[0205] b) a modulator of the cholesterol and triglyceride synthesis or metabolism, preferably Simvastatin, Fluvastatin, Lapaquistat, or Bemfivastatin

[0206] c) an immunomodulatory drug targeting cereblon, preferably Lenalidomide, Iberdomide, HOMO-Protac cereblon degrader 1, or Eragidomide,

[0207] d) a modulator of monomeric GTP-binding proteins, preferably the Ras family, preferably the Ras signaling complex, preferably NSC-70220, SAH-SOS1A TFA, Adagrasib, BI-2852, MRTX-1133, RMC-0331 or SOS1 -activator 1,

[0208] e) a kinase modulator, preferably of the Rho kinase pathway, preferably Narciclasine, f) a tyrosine kinase modulator, preferably of the JAK signaling pathway, preferably Ruxolitinib,

[0209] g) a modulator of esterases, preferably of the phosphodiesterase pathway, preferably Anagrelide,

[0210] h) a lipid, preferably a lipid part of a cell membrane

[0211] i) a fatty acid, preferably a saturated fatty acid or unsaturated fatty acid

[0212] j) a modulator of the gut microbiome, and

[0213] k) a combination of two or more of a) to k).

[0214] “Combination” according to the present invention means that the at least two components of the combination, an ABP and a modulator compound, are administered in a timely manner but not necessarily together. It is possible that first an ABP is administered and then a modulator compound. It is also possible that first a modulator compound is administered and then an ABP. Both components can also be administered essentially together.

[0215] According to the present invention, the term “capable of” binding to shall refer to a binding domain that is able to bind to a certain antigen, or is specific for a particular antigen, i.e. is specifically binding to said antigen. Moreover, the term binding domain “capable of binding” shall further refer to a binding domain that is specific for a particular antigen.

[0216] ABPs

[0217] The ABPs are preferably part of a combination or composition or kit. However, it is also intended that the ABP are used without a modulator compound. The ABP comprises at least a first antigen binding domain capable of binding to an extracellular Ras antigen. In one preferred embodiment, said extracellular Ras antigen is not part of, or is not restricted to, a human leukocyte antigen (HLA)-peptide complex, or specific HLA alleles, presented on the cell surface by the major histocompatibility complex (MHC). In another embodiment, said ABP comprises one or more additional antigen binding domain(s) that is capable of binding to antigen(s) present on a mammalian T-cell, preferably a human cluster of differentiation 3 (CD3) antigen or a human T cell receptor (TCR). In a further preferred embodiment, the first antigen binding domain comprises an amino acid sequence having a sequence identity of at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% sequence identity, to an amino acid sequence selected from SEQ ID NO: 20 to 31, 66 to 71, 134 to 178 and 290 to 396, or, in each case independently, optionally with no more than ten, nine, eight, seven, six, five, four, preferably no more than three, two or one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.

[0218] Thus, the present invention relates to an ABP having a binding domain for Ras, which mediates tumor specificity. Moreover, the ABP of the present invention preferably also has a binding domain for CD3, which enables the binding to T cells. Most preferably, the present invention relates to an ABP having a binding domain for Ras and a binding domain for CD3. In a particularly preferred example, the present invention relates to a bispecific antibody having a binding domain for Ras and a binding domain for CD3.

[0219] Ras antigen is often used herein synonymously with Ras protein. When expressed on a cell and being present on a cell surface, the Ras antigen is a Ras protein. The inventors recently found extracellular accessible Ras protein on cancer cells. The inventor then developed bi-specific ABPs that are able to bind to Ras proteins on the cell surface of cancer cells and CD3 positive T cells, and, thereby, eliminate cancerous cells via a T cell-induced immune response. Importantly, the binding of the anti Ras directed bi-specific ABPs to the tumor cells is preferably not restricted to specific HLA alleles.

[0220] The ABP according to the present invention binds to an epitope displayed by one or more extracellular accessible domain(s) of a Ras protein. Importantly, extracellular accessible Ras protein provides a multitude of epitopes including an epitope covering a part of the Ras wildtype sequence or part of Ras mutated sequence. Thus, the ABPs of the present invention can target various cells that aberrantly express mutant Ras or wildtype Ras proteins. Therefore, the ABPs of the present invention are highly beneficial over ABPs of the prior art, which target patientspecific RAS neoantigens in complex with MHC. Such RAS neoantigens are the result of mutations during oncogenesis, and allow individualized therapies, i.e. therapies for a small HI_A matched patient group because such neoantigens are expressed specific in a certain set of HLA alleles.

[0221] Importantly, the ABPs of the present invention target extracellular accessible Ras protein that preferably differs largely in its tertiary structure from Ras epitopes presented by short peptide fragments in complex with HLAs. Therefore, the ABPs of the present invention preferably differ from ABPs targeting Ras neoantigenes in complex with HLAs. Importantly, the ABPs of the present invention allow targeting extracellular accessible Ras protein in aggressive tumors on Ras mutated cancer cells from the cell outside.

[0222] In one preferred embodiment, the ABP of the present invention can recruit immune cells like natural killer (NK) cells or antigen presenting cells (like dendritic cells).

[0223] In a preferred embodiment, the extracellular Ras antigen is selected from a Kirsten rat sarcoma viral oncogene homolog (KRAS), such as KRAS4A or KRAS4B, a neuroblastoma RAS viral (v-RAS) oncogene homolog (NRAS), and a Harvey rat sarcoma viral oncogene homolog (HRAS). Of note, the two KRAS isoforms KRAS4A and KRAS4B arise from alternative RNA splicing. For HRAS, two isoforms arise from an alternate exon resulting in a frameshift and an early stop codon compared to wild type isoform 1. The encoded HRAS isoform 2 has a shorter and distinct C-terminus compared to wild type isoform 1. Thus, there is a total of five different Ras Proteins expressed in human cells, and the extracellular Ras antigen according to the present invention can be any of these.

[0224] The present invention further relates to an ABP, wherein the ABP comprises at least a first antigen binding domain capable of binding to an extracellular Ras antigen, a paralogue, an orthologue or other variant thereof.

[0225] In the present invention, the Ras antigen is of murine or human origin, preferably is a human Ras antigen or protein.

[0226] In another preferred embodiment, which can be combined with any and all other specifically preferred embodiments and aspects of the present invention, the first antigen binding domain of the ABP according to the present invention is capable of binding to an extracellular human Ras antigen. Preferably the binding is not restricted to specific HLA alleles.

[0227] Particularly preferred is that the extracellular Ras antigen comprises, preferably consists of, the amino acid sequence according to any one of SEQ ID NOs: 1, 3, 5, 7, 9 or 179 to 182, or comprises, preferably consists of, a sequence having at least 75% sequence identity, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence identity with any one of SEQ ID NOs: 1, 3, 5, 7, 9, or 179 to 182.

[0228] Even more preferred is that the extracellular Ras antigen comprises, preferably consists of, the amino acid sequence according to any one of SEQ ID NOs: 179 to 182, or comprises, preferably consists of, a sequence having at least 75% sequence identity, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence identity with any one of SEQ ID NOs: 179 to 182. SEQ ID NO: 179 corresponds to the amino acid sequence of KRAS4A, wherein the protease amino acids have been removed. SEQ ID NO: 180 corresponds to the amino acid sequence of KRAS4B, wherein the protease amino acids have been removed. SEQ ID NO: 181 corresponds to the amino acid sequence NRAS, wherein the protease amino acids have been removed. SEQ ID NO: 182 corresponds to the amino acid sequence HRAS, wherein the protease amino acids have been removed. Thus, the sequences according to any one of SEQ ID NOs: 179 to 182 corresponds to the respective wild type Ras proteins, after they have been post-translationally modified. The removal of the protease amino acids in any of the respective wild type Ras proteins is important for enabling the binding of the proteins to the cell membrane.

[0229] According to one preferred embodiment, the present invention thus pertains to an ABP, wherein the ABP comprises at least a first antigen binding domain specifically binding to an extracellular Ras antigen according to any one of SEQ I D NOs: 1, 3, 5, 7, 9, or 179 to 182.

[0230] In another particularly preferred embodiment, which can be combined with any and all other specifically preferred embodiments and aspects of the present invention, the extracellular antigen belongs to the RAS superfamily and shown in Table 1.

[0231] Table 1: Alternative target antigens with possible extracellular expression on the cell surface for ABP and / or ABC of this invention. Genes belonging to the RAS superfamily divided into the five major families Ras, Rho, Arf / Sara, Ran and Rab. Also genes belonging to the Go subfamily and genes belonging to the Src family kinase containing a membrane-targeting region at their N-terminus, which is myristoylated and sometimes palmitoylated, similar to the HVR regions of KRAS, NRAS and HRAS. All sequences were retrieved from the online databases on December 02, 2024.

[0232] Gene name NCBI Gene ID UniProt Number

[0233] KRAS 3845 P01116

[0234] P01116-1

[0235] P01116-2

[0236] G3V5T7

[0237] G3V4K2

[0238] A0A8I5KQ21

[0239] A0A8I5KR86

[0240] A0A8I5KQU3

[0241] A0A8I5KUB5

[0242] A0A8I5KYH6

[0243] A0A8I5KXN3

[0244] HRAS 3265 P01112

[0245] P01112-1

[0246] P01112-2

[0247] A0A804HJ06

[0248] A0A0J9YXG8

[0249]

[0250] A0A804HKM6

[0251] A0A8C8MQR2 ERAS 3266 Q7Z444 RALA 5898 P11233

[0252] H7C3P7 C9JPE8 RALB 5899 P11234

[0253] P11234-1 P11234-2 P11234-3 C9J6B1 C9JQB3 RRAS 6237 P10301 RRAS2 22800 P62070

[0254] P62070-1 P62070-2 P62070-3 P62070-4 E9PK85 MRAS 22808 014807

[0255] 014807-1 014807-2 C9J8Q6 RIT1 6016 Q92963

[0256] Q92963-1 Q92963-2 Q92963-3 A0A494C0S1 V9GYC3 RIT2 6014 Q99578

[0257] Q99578-1 Q99578-2 A0A3B3ITB4 K7EMR8

[0258]

[0259] RAP1A 5906 P62834 RAP1B 5908 P61224

[0260] P61224-1 P61224-2 P61224-3 P61224-4 E7ESV4 B7ZB78 F5GZG1 F5GX62 F5GYB5 F5H7Y6 F5H004 RAP2A 5911 P10114 RAP2C 57826 Q9Y3L5

[0261] A0A087X2C3 RAP2B 5912 P61225 DIRAS1 148252 095057

[0262] K7EN06 DIRAS2 54769 Q96HU8 A0A1B0GVC3 DIRAS3 9077 095661 RASD1 51655 Q9Y272

[0263] Q9Y272-1 Q9Y272-2 RASD2 23551 Q96D21 RASL10B 91608 Q96S79 RASL10A 10633 Q92737

[0264] Q92737-1 Q92737-2 NKIRAS1 28512 Q9NYS0 G5E9P3 NKIRAS2 28511 Q9NYR9 Q9NYR9-1

[0265]

[0266] Q9NYR9-2

[0267] Q9NYR9-3 Q9NYR9-4 H7BXP1 K7ERG2 RERG 85004 Q96A58 Q96A58-1 Q96A58-2 F5H252 RASL11B 65997 Q9BPW5 RASL11A 387496 Q6T310 RASL12 51285 Q9NYN1 Q9NYN1-1 Q9NYN1-2 Q9NYN1-3 GEM 2669 P55040 RRAD 6236 P55042 J3KRG9 J3KSM6 REM1 28954 075628 REM2 161253 Q8IYK8 Q8IYK8-1 Q8IYK8-2 RHEB 6009 Q15382 RHEBL1 121268 Q8TAI7 Q8TAI7-1 Q8TAI7-2 F8W1T5 RAC1 5879 P63000 P63000-1 P63000-2 A0A994J6T1 RAC2 5880 P15153

[0268] B1AH80

[0269]

[0270] B1AH77

[0271] B1AH78 RAC3 5881 P60763

[0272] J3KSC4 J3QLK0 RHOG 391 P84095 RHOJ 57381 Q9H4E5

[0273] Q9H4E5-1 Q9H4E5-2 G3V476 G3V4H1 RHOQ 23433 P17081 CDC42 998 P60953

[0274] P60953-1 P60953-2 Q5JYX0 A0A494BZX6 A0A590UJK8 A0A494C1M1 A0A8Q3WLC5 A0A8Q3SI43 RHOU 58480 Q7L0Q8

[0275] Q7L0Q8-1 Q7L0Q8-2 RHOV 171177 Q96L33 RHOH 399 Q 15669

[0276] D6RG23 D6RA52 RHOA 387 P61586

[0277] C9JX21 C9JNR4 A0A7I2YQV1 A0A7I2V3G1 RHOC 389 P08134

[0278]

[0279] Q5JR05 Q5JR07 Q5JR08 E9PQH6 E9PN11 RHOB 388 P62745 RHOD 29984 000212 E9PIG5 RHOF 54509 Q9HBH0 Q9HBH0-1 Q9HBH0-2 F5GXB1 V9GY67 RND3 390 P61587 Q53RZ3 E9PFH1 RND2 8153 P52198 RND1 27289 Q92730 H0YHG7 RHOBTB1 9886 094844 RHOBTB2 23221 Q9BYZ6 Q9BYZ6-1 Q9BYZ6-2 Q9BYZ6-3 A0A8I5KV41 RHOT1 55288 Q8IXI2

[0280] Q8IXI2-1 Q8IXI2-2 Q8IXI2-3 Q8IXI2-4 Q8IXI2-5 Q8IXI2-6 Q8IXI2-7 H7BXZ6

[0281]

[0282] RHOT2 89941 Q8IXI1

[0283] Q8IXI1-1 Q8IXI1-2 A0A8V8TM48 BLK 640 P51451 FGR 2268 P09769

[0284] Q5TGY6 FRK 2444 P42685

[0285] P42685-1 P42685-2 FYN 2534 P06241

[0286] P06241-1 P06241-2 P06241-3 HCK 3055 P08631

[0287] P08631-1 P08631-2 P08631-3 P08631-4 J3KPD6 H0Y3C5 LCK 3932 P06239

[0288] P06239-1 P06239-2 P06239-3 E9PAP0 E9PJ92 E9PKQ8 LYN 4067 P07948

[0289] P07948-1 P07948-2 E5RJ37 SRC 6714 P12931

[0290] P12931-1

[0291]

[0292] P12931-2 P12931-3 A0A8I5KYU4 YES1 7525 P07947 J3QRU1 RAN 5901 P62826 J3KQE5 B5MDF5 F5H018 RABL2A 11159 Q9UBK7 Q9UBK7-1 Q9UBK7-2 Q9UBK7-3 B7ZBD5 B7ZBD4 RABL2B 11158 Q9UNT1 Q9UNT1-1 Q9UNT1-2 Q9UNT1-3 A8MXF6 RABL3 285282 Q5HYI8 F8WDC7 C9JXM3 IFT22 64792 Q9H7X7 Q9H7X7-1 Q9H7X7-2 Q9H7X7-3 RAB1A 5861 P62820 P62820-1 P62820-2 P62820-3 E7END7 RAB1B 81876 Q9H0U4

[0293] E9PLD0

[0294]

[0295] RAB35 11021 Q 15286

[0296] Q 15286-1 Q 15286-2 F5H7F8 F5H157 RAB13 5872 P51153

[0297] A0A087WWB9 RAB8A 4218 P61006

[0298] P61006-1 P61006-2 RAB8B 51762 Q92930

[0299] H0YNE9 RAB10 10890 P61026 RAB12 201475 Q6IQ22

[0300] RAB3A 5864 P20336

[0301] M0R257 S4R3Q3 RAB3C 115827 Q96E17 RAB3B 5865 P20337 RAB3D 9545 095716 RAB40A 142684 Q8WXH6 RAB40B 10966 Q12829

[0302] H0YFJ5 RAB40C 57799 Q96S21

[0303] Q96S21-1 Q96S21-2 H3BTC6 H3BPA5 H3BNV8 H3BME4 RAB15 376267 P59190

[0304] P59190-1 P59190-2

[0305]

[0306] A0A2R8Y7G7

[0307] A0A2R8YFB8 G3V562 G3V196 RAB44 401258 Q7Z6P3 RAB27A 5873 P51159

[0308] P51159-1 P51159-2 H3BVH7 H3BS49 H3BN55 RAB27B 5874 000194

[0309] K7EJ38 RASEF 158158 Q8IZ41

[0310] Q8IZ41-1 Q8IZ41-2 RAB26 25837 Q9ULW5 Q9ULW5-1 Q9ULW5-2 H3BQ97 RAB37 326624 Q96AX2 Q96AX2-1 Q96AX2-2 Q96AX2-3 Q96AX2-4 A8MSP2 A8MTC6 B7Z3L0 A0A9H3ZVF6 RAB2A 5862 P61019

[0311] P61019-1 P61019-2 E9PKL7 RAB2B 84932 Q8WUD1

[0312]

[0313] Q8WUD1-1

[0314] Q8WUD1-2 A0A3B3ITL1 RAB4A 5867 P20338

[0315] A0A087WYT5 RAB4B 53916 P61018

[0316] P61018-1 P61018-2 M0R0X1 RAB14 51552 P61106

[0317] X6RFL8 A0A994J4B9 A0A994J451 RAB11A 8766 P62491

[0318] P62491-1 P62491-2 H3BSC1 B4DQU5 H3BMH2 RAB11B 9230 Q 15907

[0319] Q 15907-1 Q 15907-2 RAB25 57111 P57735 RAB39A 54734 Q 14964 RAB39B 116442 Q96DA2 RAB42 115273 Q8N4Z0 Q8N4Z0-1 Q8N4Z0-2 RAB19 401409 A4D1S5 A4D1S5-1 A4D1S5-2 RAB43 339122 Q86YS6 Q86YS6-1 Q86YS6-2

[0320]

[0321] RAB30 27314 Q15771

[0322] Q15771-1 Q15771-2 H0YDK7 E9PS06 E9PNB9 E9PMJ1 RAB33A 9363 Q 14088 RAB33B 83452 Q9H082 A0A494C0Z5 RAB18 22931 Q9NP72 Q9NP72-1 Q9NP72-2 Q9NP72-3 A0A8C8NLQ3 B7Z4P9 Q5W0J0 RAB17 64284 Q9H0T7 Q9H0T7-1 Q9H0T7-2 C9J0T6 H7C1P7 RAB5A 5868 P20339

[0323] P20339-1 P20339-2 RAB5C 5878 P51148

[0324] P51148-1 P51148-2 F8VVK3 K7ENY4 RAB5B 5869 P61020

[0325] P61020-1 P61020-2 F8VUA5

[0326]

[0327] RAB22A 57403 Q9UL26 RAB31 11031 Q 13636 RAB21 23011 Q9UL25 RAB20 55647 Q9NX57 RAB24 53917 Q969Q5

[0328] F8W8H5 RAB6A 5870 P20340

[0329] P20340-1 P20340-2 P20340-3 P20340-4 RAB6C 84084 Q9H0N0 RAB6B 51560 Q9NRW1 Q9NRW1-1 Q9NRW1-2 J3KR73 RAB41 347517 Q5JT25

[0330] Q5JT25-1 Q5JT25-2 RAB34 83871 Q9BZG1

[0331] Q9BZG1-1 Q9BZG1-2 Q9BZG1-4 E7ES60 K7EIF2 C9JY26 P0DI83 A0A1B0GTQ2 A0A1B0GWB1 C9JBG0 Q96PJ7 A0A1C7CYW6 RAB36 9609 095755

[0332] 095755-1

[0333]

[0334] 095755-2 RAB29 8934 014966

[0335] 014966-1 014966-2 014966-3 RAB32 10981 Q 13637

[0336] RAB38 23682 P57729

[0337] H0YDB7 RAB23 51715 Q9ULC3 RAB28 9364 P51157

[0338] P51157-1 P51157-2 P51157-3 H0Y9S6 IFT27 11020 Q9BW83 Q9BW83-1 Q9BW83-2 B1AH58 H0Y6C7 F5GZ09 RAB7A 7879 P51149

[0339] A0A6Q8PH84 A0A6Q8PGE6 A0A6Q8PG52 C9J8S3 C9IZZ0 RAB7B 338382 Q96AH8 A0A096LP44 RAB9B 51209 Q9NP90 RAB9A 9367 P51151 ARF1 375 P84077

[0340] A0A8V8TQC0 A0A8V8TQP8

[0341]

[0342] A0A8V8TNZ0

[0343] A0A8V8TNZ5 ARF3 377 P61204

[0344] P61204-1 P61204-2 F5H0C7 ARF4 378 P18085

[0345] C9JPM4 C9JAK5 ARF5 381 P84085

[0346] C9J1Z8 ARF6 382 P62330 ARFRP1 10139 Q 13795 Q 13795-1 Q 13795-2 Q 13795-3 Q 13795-4 SARA1 56681 Q9NR31 Q9NR31-1 Q9NR31-2 Q5SQT8 H0Y5E8 SAR1B 51128 Q9Y6B6 D6RDB2 D6RD69 GNAL 2774 P38405

[0347] P38405-1 P38405-2 P38405-3 K7EQ80 K7EPE2 GNAS 2778 095467 095467-1 Q5JWF2-1

[0348]

[0349] Q5JWF2-2

[0350] Q5JWF2-3 P63092-1 P63092-2 P63092-3 P63092-4 Q5JWF2 P63092 Q5JWE9 H0Y7E8 A0A804HIH4 A0A590UJY2 A0A590UJS2 A0A590UJQ9 A0A590UJX6 A0A590UK28 A0A590UK00 A0A590UJF0 A0A7I2V5R6 P84996 GNAI1 2770 P63096

[0351] P63096-1 P63096-2 A0A3B3ITM0 A0A3B3IUA8 GNAI3 2773 P08754 GNAI2 2771 P04899

[0352] P04899-1 P04899-2 P04899-3 P04899-4 P04899-5 P04899-6 GNA01 2775 P09471

[0353]

[0354] P09471-1

[0355] P09471-2

[0356] H3BTM2

[0357] H3BNR5

[0358] A0A1W2PP38

[0359] A0A1W2PPG6

[0360] GNAT1 2779 P11488

[0361] C9JCV8

[0362] GNAT2 2780 P19087

[0363] GNAT3 346562 A8MTJ3

[0364] GN AZ 2781 P 19086

[0365] GNA11 2767 P29992

[0366] K7EL62

[0367] GNAQ 2776 P50148

[0368] GNA14 9630 095837

[0369] GNA15 2769 P30679

[0370] GNA12 2768 Q03113

[0371] Q03113-1

[0372] Q03113-2

[0373] Q03113-3

[0374] E9PC54

[0375] GNA13 10672 Q 14344

[0376] Q 14344-1

[0377] Q 14344-2

[0378]

[0379] In one embodiment, the extracellular Ras antigen is a mutant Ras antigen according to any one of SEQ ID NOs: 11 to 19, 77 to 119, or 183 to 223. In one preferred embodiment, the targeted protein is a mutant Ras protein expressed on the extracellular site of a cancer cell. Thus, an ABP and / or ABC of the present invention can target Ras mutated oncogenic cells.

[0380] In a further embodiment, the extracellular antigen is a mutant version of any antigen shown in Table 1. In another preferred embodiment, the cells targeted by the ABP and / or ABC of the present invention are malignant cancer cells and / or Ras mutated cells.

[0381] In another preferred embodiment, the malignant cells targeted by the ABP and / or ABC of the present invention have no mutation in Ras, but show Ras cellular signaling dependency. Thus, Ras is important for the tumor cell survival and proliferation.

[0382] In a further embodiment, the ABP and / or ABC according to the present invention binds to an extracellular accessible Ras antigen with an ECso higher than 0.01 nM, preferably higher than 0.1 nM, even more preferably higher than 1 nM, even more preferably higher than 10 nM, even more preferably higher than 20 nM, and most preferably higher than 30 nM.

[0383] In a further preferred embodiment, the ABP and / or ABC enhances a cell-mediated immune response, such as the immune response mediated by an activated cytotoxic T-cell (CTL) to a mammalian cell expressing said extracellular accessible Ras antigen.

[0384] In a specific embodiment, the preferred one or more additional antigen binding domain(s) of the ABP and / or ABC enhances a cell-mediated immune response, such as that mediated by an activated cytotoxic T-cell (CTL), to a mammalian cell expressing said extracellular Ras antigen.

[0385] Thus, the preferable one or more additional antigen binding domain(s) of the ABP that is capable of binding to antigen(s) present on a mammalian T-cell, preferably the human cluster of differentiation 3 (CD3) antigen or a human T cell receptor (TCR), preferably increases the activity of immune cells, such as T-cells.

[0386] In one specific embodiment, the specific T cell response is induced by a specific anti-CD3 binding of the at least one second antigen binding domain of the ABP according to the present invention to antigens present on a human cluster of differentiation 3 (CD3) antigen.

[0387] In a preferred embodiment, the antigen binding domain(s) that is capable of binding to antigen(s) present on a mammalian T-cell, preferably a human cluster of differentiation 3 (CD3) antigen, comprises an amino acid sequence according to SEQ ID No: 48 or 49. In another specific embodiment, the one or more additional antigen binding domain(s) of the ABP and / or ABC enhances a cell-mediated immune response, such as that mediated by an activated natural killer (NK) cell, to a mammalian cell expressing said extracellular Ras antigen.

[0388] In another specific embodiment, the one or more additional antigen binding domain(s) of the ABP and / or ABC enhances a cell-mediated immune response, such as that mediated by an activated natural killer T cell (NKT cell), to a mammalian cell expressing said extracellular Ras antigen.

[0389] In another specific embodiment, the one or more additional antigen binding domain(s) of the ABP and / or ABC enhances a cell-mediated immune response, such as that mediated by an activated regulatory T cell (TReg), to a mammalian cell expressing said extracellular Ras antigen.

[0390] In another specific embodiment, the one or more additional antigen binding domain(s) of the ABP and / or ABC enhances a cell-mediated immune response, such as that mediated by an activated antigen-presenting cell (APC) or accessory cell, to a mammalian cell expressing said extracellular Ras antigen.

[0391] In a further preferred embodiment, the antigen binding domain capable of binding to an extracellular Ras antigen, preferably provided that said extracellular Ras antigen is not part of a, or is not restricted to, human leukocyte antigen (HI_A)-peptide complex, or specific HLA alleles, presented on the cell surface by the major histocompatibility complex (MHC), comprises an amino sequence according to any one of SEQ ID Nos: 20 to 31, or 66 to 71, or 134 to 178.

[0392] In one embodiment, the anti CD3 binder of the present invention was derived from the diL2K clone. In another embodiment, the anti CD3 binder of the present invention was derived from the LICHT1 clone. The anti-CD3 antibodies according to the present invention are herein sometimes referred to as diL2K and / or LICHT1. In one embodiment, the anti-CD3 antibodies diL2K and / or LICHT 1 bind to the epsilon chain of the CD3 receptor complex. In a further embodiment, the ABP of the present invention increases type-l cytokine secretion by said immune cells, for example T-cells or CTLs, such as one or more cytokines independently selected from the list consisting of: IFN-gamma, IL-2 and TNF-alpha.

[0393] Another embodiment relates to the ABP according to the present invention, wherein said ABP is selected from the group consisting of: an immunoglobulin molecule, such as an IgG, IgE, IgD, IgA, or IgM immunoglobulin, preferably an IgG immunoglobulin, a monoclonal antibody, a chimeric antibody, a bispecifc ABP, such as a bispecific antibody, a CDR-grafted antibody, a humanized antibody, a single domain antibody, a hemibody antibody, a single-chain KeyLock-antibody, a diabody, a single chain diabody, a variable domain of the antibody heavy chain or antibody light chain, a multispecific antibody, a chimeric antigen receptor (CAR), and a fragment of an antibody, such as a fragment of a monoclonal antibody, for example a single chain variable fragment (scFv), (scFv)2, a Fv, a disulfide linked Fv, Fab, Fab', F(ab')2 or a scFv-Fc, preferably wherein said ABP is a bispecific antibody or a CAR. The ABP can also be selected from the group of alternative protein binders including monobodies (derived from fibronectin type III), anticalins (derived from lipocalins), affibodies (derived from immunoglobulin-binding protein A) or DARPins (Designed Ankyrin Repeat Proteins). These alternative binders usually have a "constant" scaffold and a "variable" site for target antigen binding. Many types of alternative scaffolds are based on proteins with repeating motifs like leucine-rich repeats (LRRs), ankyrin repeats (ARs), Armadillo repeats (Arms), and tetratri co peptide repeats (TPRs).

[0394] The invention also includes novel dual antigen restricted ABP constructs, such as antibodies termed hemibodies, or antibodies using prodrug-activating chain exchange for a targeted immunotherapy of patients with cancer.

[0395] In one embodiment, the ABP comprises a heavy chain immunoglobulin constant domain selected from the group consisting of:

[0396] a human IgM constant domain,

[0397] a human lgG1 constant domain,

[0398] a human lgG2 constant domain,

[0399] a human lgG3 constant domain,

[0400] a human lgG4 constant domain,

[0401] a human IgE constant domain, a human IgA constant domain,

[0402] and human IgD constant domain,

[0403] an IgG constant domain variant with one or more mutations altering binding strength to Fc neonatal receptor, Fc gamma receptors, or C1 q.

[0404] A further embodiment pertains to the ABP according to the present invention, wherein one, preferably two, heavy chain variable domain(s) and one, preferably two, light chain variable domain(s), each comprise an antibody framework having at least a portion of a human antibody consensus framework sequence.

[0405] In another embodiment, the ABP comprises two heavy chain immunoglobulin constant domains, wherein both constant domains have specific mutations to reduce or abrogate the binding to the Fc gamma receptors (such as effector silent mutations), comprises the mutations L234A, L235A and P329A (numbering using Eu numbering), and a mutation to abrogate glycosylation of the constant domain (such as aglycosylation mutations), comprises the mutation N297A (numbering using Eu numbering) according to any one of SEQ ID NOs: 42, 43, and 120 to 133. The corresponding mutations in SEQ ID NO: 42 are 22A, 23A, 85A and 117A, and in SEQ ID NO: 43 are 14A, 15A, 77A and 109A. In a particularly preferred embodiment, the ABP is a bispecific antibody, and comprises at least one additional antigen binding domain that binds to a human cluster of differentiation 3 (CD3) antigen. Accordingly, the present invention provides T cell-engaging antibodies with a Ras binding domain. Importantly, the antibodies of the present invention are thereby able to redirect cytotoxic T cells against tumor cells.

[0406] In another particularly preferred embodiment, the ABP is an antibody with a toxic payload directly labeled to it. These Antibody-Drug Conjugates (ADCs) can carry a toxic payload like ozogamicin / calicheamicin, Vedotin (Monomethylauristatin E), Maytansinoide, camptothecin, auristatin orTesirin (SG-3199). The toxic payload can also be a radioactive label like Yttrium-90, lodine-131, Samarium-153, Lutetium-177, Astatine-211, Lead-212 / bismuth-212, Radium-223, Actinium-225 orThorium-227.

[0407] In a further embodiment, the ABP and / or ABC is used to carry a nucleic acid, such as an RNA molecule, like a siRNA molecule, into RAS expressing cells. In another particularly preferred embodiment, the ABP is part of an oncolytic virus, for a virus-mediated immune response, such as that mediated by DNA or RNA viruses, to a mammalian cell expressing said extracellular Ras antigen.

[0408] Another preferred embodiment relates to a fragment of an ABP according to the present invention, such as a fragment of a bispecific antibody that comprises at least a first antigen binding domain capable of binding to an extracellular Ras antigen, preferably provided that said extracellular Ras antigen is not part of a, or is not restricted to, HLA-peptide complex, or specific HLA alleles, presented on the cell surface by MHC, and preferably comprises at least one additional antigen binding domain that binds to a human cluster of differentiation 3 (CD3) antigen.

[0409] In a further embodiment, said ABP and / or ABC according to the present invention is isolated and / or substantially pure.

[0410] Yet another embodiment relates to the ABP and / or ABC according to the present invention, wherein said ABP is not cell membrane permeable.

[0411] A further embodiment relates to the ABP according to the present invention, wherein said ABP comprises an effector group.

[0412] In one embodiment, the ABP and / or ABC according to the present invention is labelled.

[0413] Another embodiment pertains to the ABP according to the present invention, wherein said ABP is Fc receptor binding attenuated.

[0414] A further embodiment pertains to the ABP according to the present invention, wherein the ABP comprises at least a first antigen binding domain capable of binding to an extracellular Ras antigen, preferably provided that said extracellular Ras antigen is not part of a, or is not restricted to, HLA-peptide complex, or specific HLA alleles, presented on the cell surface by MHC, and wherein said ABP optionally comprises one or more additional antigen binding domain(s) that is capable of binding to antigen(s) present on a mammalian T-cell, preferably a human cluster of differentiation 3 (CD3) antigen or a human T cell receptor (TCR), comprising: (i) a heavy chain variable domain comprising the CDRH1 region set forth in SEQ ID NO: 50 or 53, the CDRH2 region set forth in SEQ ID NO: 51 or 54, and the CDRH3 region set forth in SEQ ID NO: 52 or 55, or wherein in each case independently the CDRH1, CDRH2 and / or CDRH3 comprise a sequence having no more than three or two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to SEQ ID NO: 50 or 53, SEQ ID NO: 51 or 54, or SEQ ID NO: 52 or 55, respectively; or comprising a CDRH1, CDRH2 or CDRH3 sequence having at least 75% sequence identity, preferably at least 80% sequence identity, more preferably at least 90% sequence identity, and most preferably at last 95% sequence identity with SEQ ID NO: 50 or 53, SEQ ID NO: 51 or 54, or SEQ ID NO: 52 or 55; and

[0415] (ii) a light chain variable domain comprising the CDRL1 region set forth in SEQ ID NO:56 or 59, the CDRL2 region set forth in SEQ ID NO: 57 or 60, and the CDRL3 region set forth in SEQ ID NO: 58 or 61 or wherein in each case independently CDRL1, CDRL2 and / or CDRL3 comprise a sequence having no more than three or two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to SEQ ID NO: 56 or 59, SEQ ID NO: 57 or 60, or SEQ ID NO: 58 or 61, respectively; or comprising a CDRL1, CDRL2 or CDRL3 sequence having at least 75% sequence identity, preferably at least 80% sequence identity, more preferably at least 90% sequence identity, and most preferably at last 95% sequence identity with SEQ ID NO: 56 or 59, SEQ ID NO: 57 or 60, or SEQ ID NO: 58 or 61.

[0416] A further embodiment pertains to the ABP according to the present invention, wherein the heavy chain variable region comprises the amino acid sequence having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at last 95% sequence identity, to an amino acid sequence selected from SEQ ID NO: 62 and / or 64, or, in each case independently, optionally with no more than ten, nine, eight, seven, six, five, four, preferably no more than three, two or one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences; and / or wherein the light chain variable region comprises the amino acid sequence having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at last 95% sequence identity, to the amino acid sequence selected from SEQ ID NO: 63 and / or 65, or, in each case independently, optionally with no more than ten, nine, eight, seven, six, five, four, preferably no more than three, two or one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences. In this embodiment, the heavy chain variable region and the light chain variable region is referring to the anti-CD3 binding domain. A further embodiment pertains to the ABP according to the present invention, wherein the ABP comprises at least an antigen binding domain from an antibody variable heavy chain and variable light chain capable of binding to an extracellular Ras antigen, preferably provided that said extracellular Ras antigen is not part of, or is not restricted to, a HLA-peptide complex, or specific HLA alleles presented on the cell surface and wherein said Ras binding ABP optionally comprises:

[0417] (i) a heavy chain variable domain comprising the CDRH1 region set forth in any one of SEQ ID NOs: 135, 149, 162, 292, 300, or 338, the CDRH2 region set forth in SEQ ID NOs: 137, 151, 164, 293, 301, or 339, and the CDRH3 region set forth in any one of SEQ ID NOs: 139, 153, 166, 294, 302, or 340, or wherein in each case independently the CDRH1, CDRH2, and / or CDRH3 comprise a sequence having no more than three or two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to any one of SEQ ID NOs: 135, 149, 162, 292, 300, or 338, SEQ ID NOs: 137, 151, 164, 293, 301, or 339, or SEQ ID NOs: 139, 153, 166, 294, 302, or 340, respectively; or comprising a CDRH1, CDRH2 or CDRH3 sequence having at least 75% sequence identity, preferably at least 80% sequence identity, more preferably at least 90% sequence identity, and most preferably at last 95% sequence identity with any one of SEQ ID NOs: 135, 149, 162, 292, 300, or 338, SEQ ID NOs: 137, 151, 164, 293, 301, or 339, or SEQ ID NO: 139, 153, 166, 294, 302, or 340, respectively; and

[0418] (ii) a light chain variable domain comprising the CDRL1 region set forth in any one of SEQ ID NOs: 142, 156, 169, 295, 303, or 341, the CDRL2 region set forth in any one of SEQ ID NOs: 144, 158, 171, 296, 304, or 342 and the CDRL3 region set forth in any one of SEQ ID NOs: 146, 160, 173, 297, 305, or 343 or wherein in each case independently CDRL1, CDRL2, and / or CDRL3 comprise a sequence having no more than three or two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to any one of SEQ ID NOs: 142, 156, 169, 144, 158, 171, 146, 160, 173, 295, 303, 341, 296, 304, 342, 297, 305, or 343, respectively; or comprising a CDRL1, CDRL2, and / or CDRL3 sequence having at least 75% sequence identity, preferably at least 80% sequence identity, more preferably at least 90% sequence identity, and most preferably at last 95% sequence identity with any one of SEQ ID NOs: 142, 156, 169, 144, 158, 171, 146, 160, 173, 295, 303, 341, 296, 304, 342, 297, 305, or 343 respectively. The amino acid residues, CDRs and FRs were determined according to the IMGT numbering system and Kabat by IgBLAST set forth in any one of SEQ ID NOs: 134 to 174, 292 to 297, 300 to 305, and 338 to 343.

[0419] Yet another embodiment pertains to the ABP according to the present invention, for an individual antibody the amino acids in the antibody CDR regions, which are in direct contact with the Ras antigen, preferably provided that said Ras antigen is not part of a, or is not restricted to, a HLA-peptide complex, or specific HLA alleles presented on the cell surface, can change significantly, depending on the type of Ras antigen bound, mutated or not, as seen in the Tables below.

[0420] Table 2: Predicted binding epitopes in p21Ras

[0421] Predicted binding epitopes in p2LRas

[0422]

[0423] PCC01 (G12V-34) PCC02 (G13D-18) p21RasG12VStrong binding Strong binding

[0424] YKLVWGAVGVGKSA YKLVWGAVGVGKSA

[0425] p21Rasbi3DWeak binding Moderate binding

[0426] YKLVWGAGDVGKSA YKLVWGAGDVGKSA

[0427] P21 wild type Weak binding Moderate binding

[0428] YKLVWGAGGVGKSA YKLVWGAGGVGKSA

[0429]

[0430] Table 3: Binding of different p21Ras proteins

[0431] The binding of dil Terent p2 IRas proteins with KGH-R1 scFv and the amino acids involved | P21Ras protein VH VI.

[0432] C DRs Amino acids CDRs Amino acids I KRAS CDR3 Arg 100, SerlO? CDR1; Seri 71 NRASQfilKCDR3 His 103, Arg 109 t ’DRl I y-. Ion. Seri cG.

[0433] I I Thrl70, Seri 71 KRASG12SCDR1, CDR3 Tyr34, ArglOO.; CDRL CDR2 | Serl71. Tyri?3.

[0434] Glyl06, Serl07, i Serl95

[0435] Seri 08

[0436] KRASG12VCDR1, CDR3 Tyr34. SerlO?, ( 1)R1. ( DR2 1 yr J ” 3. \'a i I Serl07, Serl08 j Serl95 NRASG12CCDR1. CDR2. Ser32, Asp33, i;

[0437] Gly56. Tyr59,

[0438] Hisl03, Tyrl04.

[0439] Arg 109

[0440] HRAS CDR1, CDR2, Ser32, Asp33, ( I)1<1 i Seri 71. Tyrl?3.

[0441] Asp55, l'yr?9. 1 x rl ’5 Serl05, Argl09

[0442] NRAS CDR2, CDR3 Ser54, Asp55.

[0443] Ser58, Tyr59,

[0444] His 103, Tyrl04. i 1

[0445] NRASGGIR CDR2, CDR3 Ser54. Asp55, i CDR1 i Seri 71. TyrI73 Tyr59, Tyrl04,

[0446] KRASG13DCDR3 Glyl06. ArglOO ( J )R 1. ( I)R2 Seri" 1. < ily 1 ’2.

[0447] j Tyrl73. Seri 74.

[0448]

[0449] I> rl 75. I c-.ilM?

[0450] Table 4: Predicted amino acids in p21Ras in contact with PCC03

[0451] Predicted Amino acids in p21Ras in contact with PCC03 (KGH-R1) vjild type K-Ras Asp47, Argl61, Argl64, Lysl65, Aspl54, Prol40, Glnl50, Phel41, Glnl31, Argl35, Lysl28 N-RasQG1KTyrl66, Aspl08, Aspl07, Arg97, Tyrl37, Ilel39, Thrl58, Glyl62, Aspl54, ArglGl, Asp47, Argl64, Val45 K-Rasfi,7STyrl57, Aspl53, Glrl50, Thrl44, Glul43, Glnl31, Argl35, Prol40, ArglGl, Gly48, Asp47, ArglG4, LyslG5

[0452]

[0453] K-RasG12VLysl28, Aspl32, Glul43, Glnl31, Glnl50, Aspl54, Thrl58, Lysl65, Argl49, Vall52, Argl61, Asp47, Val45

[0454] Yet another embodiment pertains to the ABP according to the present invention, comprising at least one antibody heavy chain having an amino acid sequence with at least 80% sequence identity to, or having no more than twenty, fifteen, ten, nine, eight, seven, six, four, preferably three or two, more preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to, a sequence selected from selected from SEQ ID NOs: 66, 68, 70, 391, 393, or 395; and / or comprising at least one antibody light chain having an amino acid sequence with at least 80% sequence identity to, or having no more than twenty, fifteen, ten, nine, eight, seven, six, four, preferably three or two, more preferably no more than one amino acid substitution(s), deletion(s) or insertion(s), or no amino acid change, compared to a sequence selected from selected from SEQ ID NOs: 67, 69, 71, 392, 394, or 396. In this embodiment, the heavy chain variable region and the light chain variable region is referring to the anti-Ras binding domain.

[0455] A further embodiment pertains to the ABP according to the present invention, wherein the ABP comprises at least a fibronectin type III domain (FN3) binder, also known as a monobody, capable of binding to an extracellular Ras antigen, preferably provided that said extracellular Ras antigen is not part of, or is not restricted to, a HI_A-peptide complex, or specific HLA alleles presented on the cell surface and wherein said Ras binding ABP optionally comprises:

[0456] (i) With four different antigen binding loops (BC, DE, FG and CD) similar to antibody CDR regions comprising the BC loop region set forth in SEQ ID NOs: 175, 307, 312, 349, 354, or 359 the DE loop region set forth in SEQ ID NOs: 176, 309, 314, 351, 356, or 361, the FG loop region set forth in SEQ ID NOs: 177, 310, 315, 352, 357, or 362, and the CD loop region set forth in SEQ ID NOs: 178, 308, 313, 350, 355, or 360, orwherein in each case independently BC, DE, FG and CD comprise a sequence having no more than three or two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to any one of SEQ ID NOs: 175, 176, 177, 178, 307 to 310, 312 to 315, 349 to 352, 354 to 357, or 359 to 362, respectively; or comprising a BC, DE, FG and CD sequence having at least 75% sequence identity, preferably at least 80% sequence identity, more preferably at least 90% sequence identity, and most preferably at last 95% sequence identity with SEQ ID NOs: 175, 176, 177, 178, 307 to 310, 312 to 315, 349 to 352, 354 to 357, or 359 to 362.

[0457] (ii) Yet another embodiment pertains to the ABP according to the present invention, comprising at least one monobody having an amino acid sequence with at least 80% sequence identity to, or having no more than twenty, fifteen, ten, nine, eight, seven, six, four, preferably three or two, more preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to, a sequence selected from selected from SEQ ID NOs: 25, 26, 306, 311, 348, 353, or 358.

[0458] A further embodiment pertains to the ABP according to the present invention, wherein the ABP comprises at least a single variable domain of a heavy chain (VHH) antibody binder, also known as nanobody, capable of binding to an extracellular Ras antigen, preferably provided that said extracellular Ras antigen is not part of, or is not restricted to, a HI_A-peptide complex, or specific HLA alleles presented on the cell surface and wherein said Ras binding ABP optionally comprises:

[0459] (i) a single heavy chain variable domain comprising the CDR1 region set forth in any one of SEQ ID NOs: 317, 321, 325, 329, 333, or 345, the CDR2 region set forth in SEQ ID NOs: 318, 322, 326, 330, 334, or 346, and the CDR3 region set forth in any one of SEQ ID NOs: 319, 323, 327, 331, 335, or 347, or wherein in each case independently the CDR1, CDR2, and / or CDR3 comprise a sequence having no more than three or two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to any one of SEQ ID NOs: 317, 321, 325, 329, 333, 345, 318, 322, 326, 330, 334, 346, 319, 323, 327, 331, 335, or 347 respectively; or comprising a CDR1, CDR2 or CDR3 sequence having at least 75% sequence identity, preferably at least 80% sequence identity, more preferably at least 90% sequence identity, and most preferably at last 95% sequence identity with any one of SEQ ID NOs: 317, 321, 325, 329, 333, 345, 318, 322, 326, 330, 334, 346, 319, 323, 327, 331, 335, or 347, respectively; and

[0460] (ii) Yet another embodiment pertains to the ABP according to the present invention, comprising at least one nanobody having an amino acid sequence with at least 80% sequence identity to, or having no more than twenty, fifteen, ten, nine, eight, seven, six, four, preferably three or two, more preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to, a sequence selected from selected from SEQ ID NOs: 316, 320, 324, 328, 332, or 344. A further embodiment pertains to the ABP according to the present invention, wherein the ABP comprises at least an affinity binder based on a structurally robust protease inhibitor scaffold, also known as affimer, capable of binding to an extracellular Ras antigen, preferably provided that said extracellular Ras antigen is not part of, or is not restricted to, a HI_A-peptide complex, or specific HLA alleles presented on the cell surface and wherein said Ras binding ABP optionally comprises:

[0461] (i) a single chain variable domain comprising the VR1 region set forth in any one of SEQ ID NOs: 364, 367, or 370, and the VR2 region set forth in SEQ ID NOs: 365, 368, or 371, or wherein in each case independently the VR1, and / or VR2 comprise a sequence having no more than three or two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to any one of SEQ ID NOs: 364, 367, 370, 365, 368, or 371, respectively; or comprising a VR1, or VR2 sequence having at least 75% sequence identity, preferably at least 80% sequence identity, more preferably at least 90% sequence identity, and most preferably at last 95% sequence identity with any one of SEQ ID NOs: 364, 367, 370, 365, 368, or 371, respectively; and (ii) having an amino acid sequence with at least 80% sequence identity to, or having no more than twenty, fifteen, ten, nine, eight, seven, six, four, preferably three or two, more preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to, a sequence selected from selected from SEQ ID NOs: 363, 366 or 369.

[0462] A further embodiment pertains to the ABP according to the present invention, wherein the ABP comprises at least a designed ankyrin repeat protein, also known as DARPin, capable of binding to an extracellular Ras antigen, preferably provided that said extracellular Ras antigen is not part of, or is not restricted to, a HI_A-peptide complex, or specific HLA alleles presented on the cell surface and wherein said Ras binding ABP optionally comprises:

[0463] (i) a single chain variable domain having an amino acid sequence with at least 80% sequence identity to, or having no more than twenty, fifteen, ten, nine, eight, seven, six, four, preferably three or two, more preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to, a sequence selected from selected from SEQ ID NOs: 372 or 373. A further embodiment pertains to the ABP according to the present invention, wherein the ABP comprises at least a charge-neutralized variants of the Sso7d protein (UniProt P39476) from the hyperthermophilic archaeon Sulfolobus solfataricus, capable of binding to an extracellular Ras antigen, preferably provided that said extracellular Ras antigen is not part of, or is not restricted to, a HI_A-peptide complex, or specific HLA alleles presented on the cell surface and wherein said Ras binding ABP optionally comprises:

[0464] (i) a single chain variable domain having an amino acid sequence with at least 80% sequence identity to, or having no more than twenty, fifteen, ten, nine, eight, seven, six, four, preferably three or two, more preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to, a sequence selected from selected from SEQ ID NOs: 374, 375 or 376.

[0465] For one embodiment, the sequences of two FN3 based ABP, with the individual Ras binding loops and the amino acid differences are seen in the Table 5 below: Table 5. Amino acid sequences for PCC06 and PCC07

[0466] Amino acid sequences for PCC06 and PCC07

[0467] FN3 binding loops (underlined) and the amino acids in direct contact with RAS (bold)

[0468] GSVSSVPTKLEWAATPTSLLISWDAPAVTVDYYVITYGETGGNSPVQKF EVPGSKSTATISGLKPGVDYTITVYAWGWHGQVYYYMGSPISINYRT SSVPTKLEVVAATSLLISWDAPAVTVDYYVITYGETGGNSPVQKFEVPGS KSTATISGLKPGVDYTITVYAWGWHGOVYYYMGSPISINYRT

[0469] Individual antigen binding loops of PCC06 and PCC07

[0470] DYYV I _

[0471] GNSPV _

[0472] KFE _

[0473] WGWHGQVYYYMG _

[0474] Difference between PCC06 and PCC07

[0475] Amino acid inserts (grey box)

[0476] i >" A SS VPTKLEVVAA I P 1SLLISWDAPAVTVDYYVITYGETGGNSPVQKFE VPGSKSTATISGLKPGVDYTITVYAWGWHGQVYYYMGSPISINYRT

[0477] SSVPTKLEVVAATSLLISWDAPAVTVDYYVITYGETGGNSPVQKFEVPGS KSTATISGLKPGVDYTITVYAWGWHGQVYYYMGSPISINYRT

[0478] Predicted binding sites in p21Ras for PCC06 and PCC07

[0479] The a4-P6-a5 interface (amino acids 126 -167) DTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYR DTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRKHK ESRQAQDLARSYGIPYIETSAKTRQGVEDAFYTLVREIRQHK

[0480]

[0481] Yet another embodiment pertains to the ABP according to the present invention, comprising a human protein or a part of a human protein with a known binding site to a Ras antigen, wherein the human protein is binding to Ras in its active and / or inactive state, preferably wherein the binding site is a direct binding site.

[0482] In one embodiment, the part of a human protein binding to an extracellular Ras antigen, preferably provided that said extracellular Ras antigen is not part of, or is not restricted to, a HI_A-peptide complex, or specific HLA alleles presented on the cell surface, has an amino acid sequence with at least 80% sequence identity to, or having no more than twenty, fifteen, ten, nine, eight, seven, six, four, preferably three or two, more preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to, a sequence selected from any one of SEQ ID NOs: 23, 24, 27, 28, 29, 30, 31, and 377 to 390. In one embodiment, these proteins are referred to as “alternative Ras binders”.

[0483] In one embodiment, the ABP according to the present invention comprises an amino acid sequence with at least 80% sequence identity to, or having no more than twenty, fifteen, ten, nine, eight, seven, six, four, preferably three or two, more preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to, a sequence selected from any one of SEQ ID NOs: 23, 24, 27, 28, 29, 30, 31 and 377 to 390.

[0484] A further embodiment pertains to the “alternative Ras binders” for ABP according to the present invention, wherein the ABP comprises at least one antigen binding domain capable of binding with one, two, three, four or preferable more than five amino acids to Ras antigen, wherein the at least one antigen binding domain alone or in a bipartite complex with another Ras binding protein, or in a tripartite complex with another Ras binding protein and the membrane, or in a multipartite complex with one or more Ras binding proteins and / or the membrane, and wherein said amino acids in the ABP optionally comprises:

[0485] (i) a domain comprising the RBD-CRD region (amino acids 52 to 188) of the human RAF1 protein (UniProt P04049) as set forth in SEQ ID NO: 23, optionally wherein one or multiple amino acids as depicted in Table A and B are in contact with KRAS residues, or wherein one or multiple amino acids as depicted in Table C are in contact with the membrane, or wherein one or multiple amino acids as depicted in Table D are in contact with KRAS residues in a tripartite complex comprised of RBD-CRD, KRAS and the membrane, or (ii) a domain comprising the CDC25H region (amino acids 780 to 1019) of the human SOS1 protein (UniProt Q07889) as set forth in SEQ ID NO: 24, optionally wherein one or multiple amino acids as depicted in Table E are in contact with KRAS residues, or

[0486] (iii) a domain comprising the CDC25 region (amino acids 1038 to 1270) of the human RASGRF1 protein (UniProt Q13972) as set forth in SEQ ID NO: 29, optionally wherein one or multiple amino acids as depicted in Table F are in contact with Ras residues in a tripartite complex with Ras, Sos1 and RasGRF1, or (iv) a domain comprising the RAS binding region (amino acids 274 to 364) of the human RASSF5 protein (UniProt Q8WWW0-1) as set forth in SEQ ID NO: 377, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0487] (v) a domain comprising the RAS binding region (amino acids 201 to 363) of a splice variant of the human RASSF5 protein (UniProt Q8WWW0-2) as set forth in SEQ ID NO: 378, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0488] (vi) a domain comprising the RAS binding region (amino acids 201 to 363) of the human RASSF1 protein (UniProt Q9NS23-1) as set forth in SEQ ID NO: 379, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0489] (vii)a domain comprising the RAS binding region (amino acids 176 to 264) of the human RASSF2 protein (UniProt P50749-1) as set forth in SEQ ID NO: 380, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0490] (viii) a domain comprising the RAS binding region (amino acids 6 to 89) of the human RASSF7 protein (UniProt Q02833-1) as set forth in SEQ ID NO: 381, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0491] (ix) a domain comprising the RAS binding region (amino acids 19 to 91) of the human ARAF protein (UniProt P10398-1) as set forth in SEQ ID NO: 382, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0492] (x) a domain comprising the RAS binding region (amino acids 19 to 148) of the human ARAF protein (UniProt P10398-1) as set forth in SEQ ID NO: 383, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0493] (xi) a domain comprising the RAS binding region (amino acids 151 to 232) of the human BRAF protein (UniProt P15056) as set forth in SEQ ID NO: 384, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0494] (xii)a domain comprising the RAS binding region (amino acids 151 to 320) of the human BRAF protein (UniProt P15056) as set forth in SEQ ID NO: 385, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0495] (xiii) a domain comprising the RAS binding region (amino acids 56 to 131) of the human RAF1 protein (UniProt P04049-1) as set forth in SEQ ID NO: 386, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0496] (xiv) a domain comprising the RAS binding region (amino acids 1235 to 1451) of the human NF1 protein (UniProt P21359-1) as set forth in SEQ ID NO: 387, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0497] (xv) a domain comprising the RAS binding region (amino acids 748 to 942) of the human RASA1 protein (UniProt P20936-1) as set forth in SEQ ID NO: 388, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0498] (xvi) a domain comprising the RAS binding region (amino acids 302 to 512) of the human RASA4 protein (UniProt 043374-1) as set forth in SEQ ID NO: 389, optionally wherein one or multiple amino acids are in contact with RAS residues, or

[0499] (xvii) a domain comprising the RAS binding region (amino acids 938 to 1130) of the human RGS12 protein (UniProt 014924-1) as set forth in SEQ ID NO: 390, optionally wherein one or multiple amino acids are in contact with RAS residues.

[0500] Tables A to F (from PCT / EP2024 / 065774, the disclosure of which is hereby incorporated by reference in its entirety.) show amino acids of an ABP according to the present invention, which can bind Ras (obtained from crystal structures). Table A

[0501] Binding type KRAS RBD PCC04

[0502] H-Bonds UniProt: P01116 UniProt: P04049 SEQ ID No: 23

[0503] 1 D33 K84 Iglliflliiiifi

[0504] 2 E37 V69

[0505] 3 E37 R67 Mf|g®lii®il®ilil

[0506] 4 D38 T68

[0507] 5 D38 R89 iRSTiiaaaaaii

[0508] 6 S39 R67

[0509] 7 S39 R89 |R3igij|M

[0510] 8 R41 Q66

[0511] 9 R41 N64

[0512] Salt-bridge

[0513] 1 E31 K84

[0514] 2 D33 K84

[0515] 3 E37 R67

[0516] 4 D38 R89 |R3IgSl|^^

[0517] Non-bonded

[0518] interactions

[0519] 1 124 V88

[0520] 2 Q25 V88 lYSS®®®®®!®®®®®®®®®®

[0521] 3 Q25 K87

[0522] 4 V29 K84

[0523] 5 E31 K84 |K32||III||^

[0524] 6 D33 K84

[0525] 7 136 V69

[0526] 8 136 T57 aiiBiiliiiiii

[0527] 9 E37 T68

[0528] 10 E37 T69 llsi®®ffi®®®ffi®®fflffi®®!®i

[0529] 11 E37 R67

[0530] 12 E37 R59 iRllflllM

[0531] 13 D38 R67

[0532] 14 D38 T68 iTgllM

[0533] 15 D38 R89 l: Wi®®®®l®®®®®®®®®®i

[0534] 16 S39 R67

[0535] 17 S39 R89 iR3iiiJiii^

[0536] 18 S39 Q66 Wilii®il®il®il®i:

[0537] 19 Y40 Q66

[0538] 20 Y40 R89 iMTi®®®®®®®®®®®®®«

[0539] 21 Y40 V88 8i3iii®®®®s®®®®®®®®i

[0540] 22 R41 Q66 isiiiiiiiggiiii

[0541] 23 R41 N64 lNi2fgi|gililiW

[0542] 24 R41 K65

[0543]

[0544] 25 L56 R67 BiB®i®i|®i®®i®®i|

[0545] Table A shows the amino acids interactions present at the bipartite complex KRAS-RAFI(RBDCRD) for the interface KRAS - RBD from a crystal structure, the corresponding amino acids in PCC04 (SEQ ID NO: 23) and the type of binding between the amino acids. The amino acids in KRAS involved are conserved across all four RAS isoforms (KRas4b, KRas4a, NRas, HRas). Table B

[0546] Binding type KRAS CRD PCC04

[0547] H-Bonds UniProt: P01116 UniProt: P04049 SEQ ID No: 23

[0548] 1 K42 1178 1126

[0549] 2 K42 VI 80 V128

[0550] 3 Q43 Hl 39 H87

[0551] 4 V45 S177 S125

[0552] 5 D47 El 74 iEg2i®i®®®®i®®®®i®®

[0553] 6 G48 El 74 Bg2|®®®|®®®®®®®®®s

[0554] 7 G48 R143 R91

[0555] 8 R149 1178 1126

[0556] 9 D153 T178 1126

[0557] Non-bonded

[0558] interactions

[0559] 1 L23 1178 1126

[0560] 2 124 T182 1130

[0561] 3 N26 KI 79 K127

[0562] 4 R41 T182 1130

[0563] 5 K42 1182 1130

[0564] 6 K42 1178 1126

[0565] 7 K42 V180 V128

[0566] 8 Q43 Hl 39 H87

[0567] 9 Q43 1138 186

[0568] 10 Q43 F141 F89

[0569] 11 V44 S177 S125

[0570] 12 V44 El 74 B122I®®®®®®®®®®®®®?

[0571] 13 V45 Fl 63 Fill

[0572] 14 V45 El 74 IE®2®®®®®®®®®®®®®®

[0573] 15 V45 E174 |E122®®®®®®®®®®®®®®

[0574] 16 146 El 74 lEBSill®®®®®®®®®®®

[0575] 17 D47 R143 R91

[0576] 18 G48 F163 Fill

[0577] 19 G48 1178 1126

[0578] 20 G48 KI 79 K127

[0579] 21 R149 1178 1126

[0580] 22 R149 Hl 75 Hl 23

[0581] 23 D153 1178 1126

[0582] 24 D153 H175 H123

[0583] 25 Y157 1178 1126

[0584] 26 Y157 El 74 Big®®®®®®®®®®®®®?

[0585] 27 Y157 H175 Hl 23

[0586]

[0587] 28 Y157 S177 S125

[0588] Table B shows the amino acids interactions present at the bipartite complex KRAS-RAFI(RBDCRD) for the interface KRAS - CRD from a crystal structure, the corresponding amino acids in PCC04 (SEQ ID NO: 23) and the type of binding between the amino acids. The amino acids in KRAS involved are conserved across all four RAS isoforms (KRas4b, KRas4a, NRas, HRas). Table C

[0589] CRD loop PCC04

[0590] UniProt: P04049 SEQ ID No: 23 143 RKTFLKLAF 151 91 RKTFLKL AF 99

[0591]

[0592] 157 KFLLNGFR 164 105 KFLLNGFR 112

[0593] Table C shows the amino acids present at the membrane interaction of CRD loop residues in the structure of KRAS-RAFI(RBDCRD) complex, and the corresponding amino acids in the PCC04 sequence (SEQ ID NO: 23).

[0594] Table D

[0595] RBD-CRD PCC04

[0596] UniProt: P04049 SEQ ID No; 23 D80 D28

[0597] F99 illiliiM

[0598] L101 L49

[0599] A110 A58

[0600] N115

[0601] T116 T64

[0602] G123 G71

[0603] E124 E72

[0604] L126 L74

[0605]

[0606] C152 C100

[0607] Table D shows the amino acids interactions present at the tripartite complex comprised of RBD-CRD, KRAS and the membrane (Nanodisc), and the corresponding amino acids in the PCC04 sequence (SEQ ID NO: 23). Table E

[0608] KRAS CDC25 PCC05

[0609]

[0610] Table E shows the intermolecular interacting residue pairs between SOS1 and Kras4B, for the CDC25 domain and RAS with GTP- and / or GDP bound to from a crystal structure. Here, “T” and “D” denote GTP and GDP. SOS1TDdenotes GTP- and GDP-bound KRas4B interacting with SOS1 at the REM allosteric and CDC25 catalytic sites, respectively. The corresponding amino acids in the PCC05 binder (SEQ ID NOs: 24) are included, the amino acids not part of the PCC05 sequence (-). Table F

[0611] Stable interactions established during MD trajectories of Ras-RasGRFl complex

[0612]

[0613] Ras RasGRF1 PCC010

[0614] 1BKD, 1XD2 PDB ID: 2IJE SEQ ID No: 29

[0615] Tyr64 Y1189 Y152

[0616] Tyr64 LI 190 LI 53

[0617] Tyr64 LI 194 LI 57

[0618] Tyr40 F1199 F162

[0619] Gln61 Y1189

[0620] Gln61 T1195 T158

[0621] Ala59 LI 194 1 / 157

[0622] Ala59 V1198 VI6I

[0623] Ala59 F1199 1’162

[0624] Serl7 El 202 El 65

[0625] Serl7 V1198 VI 61

[0626] Alai 8 El 202 El 65

[0627] Tyr32 G1203 G166

[0628] Tyr32 F1199 F162

[0629] Gly60 LI 194 LI 57

[0630]

[0631] Lysl47 El 202 El 65

[0632] Table F shows the hot-spot amino acids in the Ras-RasGRF1 complex important for a stable interaction using a Molecular Dynamics (MD) simulation. Of note, the numbering for the RasGRF1 residues in the original report is 11 numbers shorter as the corresponding numbers in UniProt (Q13972), but the amino acid sequence is the same [8], The RasGRF1 numbers in this Figure are corrected for the UniProt numbers. The corresponding amino acids in the PCC010 binder (SEQ ID NOs: 29) are included.

[0633] A further particularly preferred embodiment pertains to the ABP according to the present invention, wherein the ABP comprises at least an antigen binding domain capable of binding to an extracellular Ras antigen, preferably provided that said extracellular Ras antigen is not part of, or is not restricted to, a HLA-peptide complex, or specific HLA alleles presented on the cell surface, and wherein said Ras binding ABP optionally comprises a diabody format set forth in SEQ ID NO: 228 to 239, wherein in each case independently comprise a sequence having no more than three or two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to SEQ ID NO: 228 to 239, respectively; or comprising a sequence having at least 75% sequence identity, preferably at least 80% sequence identity, more preferably at least 90% sequence identity, and most preferably at last 95% sequence identity with SEQ ID NO: 228 to 239.

[0634] In another embodiment, the ABP is a Chimeric Antigen Receptor (CAR) comprising from N-terminus to C-terminus (i) an extracellular domain comprising the at least first antigen binding domain, (ii) an extracellular hinge domain, (iii) a transmembrane domain, and (iv) a cytoplasmic domain, optionally wherein the ABP is a CAR which is expressed by a T cell (CAR T cell).

[0635] Optionally, the extracellular hinge domain, the transmembrane domain and the cytoplasmic domain comprise the amino acid sequence having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at last 95% sequence identity, to an amino acid sequence selected from SEQ ID NO: 72 or, in each case independently, optionally with no more than ten, nine, eight, seven, six, five, four, preferably no more than three, two or one, amino acid substitution(s), insertion(s) or deletion(s) compared to this sequence.

[0636] In a preferred embodiment, the CAR according to the present invention specifically binds to an extracellular Ras antigen according to the present invention.

[0637] In another embodiment, the CAR molecule according to the present invention is a CAR molecule of the first, second, third, fourth, fifth or next generation, optionally comprising one additional antigen binding domain, such as an scFv antigen binding domain, VHH, DARPIN, or variable NAR-domain.

[0638] In one embodiment, the CAR according to the present invention is a dual-antigen CAR.

[0639] In one embodiment, when the CAR molecule is expressed in a T cell, such as a CD3+ T cell or a CD4+ T cell or CD8+ T cell, the T cell demonstrates activity against at least 2 different cancer types, at least 3 different cancer types, at least 4 different cancer types, at least 5 different cancer types, at least 6 different cancer types, at least 7 different cancer types, at least 8 different cancer types, at least 9 different cancer types, or at least 10 different cancer types.

[0640] Importantly, the novel anti-Ras directed immunotherapy of the present invention can be used for cell-based therapies like chimeric antigen receptors (CARs) transfected T cells for treating different types of Ras-dependent cancers. In one embodiment, CAR-T cells have activity against Ras tumor antigens.

[0641] In the present invention it is also intended that the combination comprises an isolated nucleic acid comprising a sequence encoding for an ABP according to the present invention. The ABP may also be a CAR.

[0642] According to a further embodiment, said isolated nucleic acid comprises a sequence encoding for an antigen binding fragment or a monomer, such as a heavy or light chain, of an ABP according to the present invention.

[0643] In one embodiment, the isolated amino acid relates to the amino acid sequence of SEQ ID NO. 72 or their complementary sequences or sequences that have at least 95 % sequence identity with said sequence.

[0644] The isolated nucleic acid may be comprised in an expression construct, preferably further comprising promoter and / or terminator sequences for expressing the ABP according to the present invention. The isolated nucleic acid and / or the expression construct may be comprised in a recombinant (host) cell.

[0645] According to a further embodiment, said expression construct is for expressing an antigen binding fragment or a monomer, such as a heavy or light chain, of an ABP according to the present invention, or for a CAR according to the present invention.

[0646] In one embodiment, the ABPs according to the present invention have protein linkers connecting the different Ras binding domains, connecting the Ras binding domains with the antibody constant domains, connecting the Ras binding domain with the CAR hinge or transmembrane domain, or connecting the detection and / or purification tags with the ABP.

[0647] In yet another embodiment, the protein linker comprises an amino acid sequence having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at last 95% sequence identity, to an amino acid sequence selected from SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, or 41, or, in each case independently, optionally with no more than ten, nine, eight, seven, six, five, four, preferably no more than three, two or one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.

[0648] Four exemplary protein linkers between the Ig constant domain and Ras binders have an amino acid sequence selected from:

[0649] SEQ ID NO 36: GA

[0650] SEQ ID NO 37: AS

[0651] SEQ ID NO 38: GS

[0652] SEQ ID NO 39: GG

[0653] Of note, a protein linker comprising the amino acid sequence of SEQ ID NO 38 (GS) can also be a protein linker between a His tag and Ras binders.

[0654] In one embodiment, the ABP according to the present invention, has one, two, or more protein tags for detection and / or purification.

[0655] In a further embodiment, the protein tag is a His tag and / or a CL7 tag.

[0656] In yet another embodiment, the protein tag comprises an amino acid sequence having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at last 95% sequence identity, to an amino acid sequence selected from SEQ ID NO: 32, 33, 45, 46 or, in each case independently, optionally with no more than ten, nine, eight, seven, six, five, four, preferably no more than three, two or one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.

[0657] In one embodiment, the protein tag is a protein tag for detection and / or purification, wherein the protein tag has a specific proteolytic cleavage site to be cut off in the production and purification process from the final ABP.

[0658] In another embodiment, the proteolytic cleavage site comprises an amino acid sequence having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at last 95% sequence identity, to an amino acid sequence selected from SEQ ID NO: 47 or, in each case independently, optionally with no more than ten, nine, eight, seven, six, five, four, preferably no more than three, two or one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences. In one embodiment, the ABP according to the present invention, has two tags.

[0659] In another embodiment, the ABP according to the present invention, has a His tag and a CL7 tag.

[0660] In yet another embodiment, the expression construct encoding for the ABP according to the present invention, has a His tag and a CL7 tag.

[0661] In one embodiment, the CL7 tag is cleaved during the final purification of the ABP according to the present invention, and one, two, three, four, five, six, seven, eight, nine, or ten amino acids remain on the N-terminus on the ABP according to the present invention. In a preferred embodiment, the CL7 tag is cleaved during the final purification of the ABP according to the present invention, and two amino acids remain on the N-terminus on the ABP according to the present invention.

[0662] In yet another embodiment, the His tag of the expression construct is also present in the ABP according to the present invention, preferably the antibody according to the present invention. The His tag can be used for the detection of the ABP according to the present invention in serum or tissue.

[0663] In one embodiment, the ABP according to the present invention, or the CAR according to the present invention, have a protein secretion leader to express the CAR on the cell surface and / or for extracellular secretion.

[0664] In another embodiment, the protein secretion leader has an amino acid sequence having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at last 95% sequence identity, to an amino acid sequence selected from SEQ ID NO: 44 or, in each case independently, optionally with no more than ten, nine, eight, seven, six, five, four, preferably no more than three, two or one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.

[0665] The present invention further pertains to a nucleic acid construct (NAC) comprising an isolated nucleic acid according to the present invention and one or more additional sequence features permitting the expression of the encoded ABP, or a component of said ABP (such as an antibody heavy chain or light chain) in a (host) cell. The invention also relates to a recombinant host cell expressing an ABP according to the present invention, or comprising an isolated nucleic acid according to the present invention, or an expression construct according to the present invention, which is preferably selected from effector cells of the immune system, such as lymphocytes, for example CD8 positive cytotoxic lymphocytes, CD4 positive T cells, T helper cells, or Th17 T cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, killer dendritic cells, B cells, yb T cells, and a lymphocyte preparation containing NK cells and NKT mast cells. Thus, the present invention further pertains to a host cell transformed with an isolated nucleic acid according to the present invention, or an expression construct according to the present invention.

[0666] In a preferred embodiment, the recombinant host cell expresses an ABP capable of binding a Ras antigen, wherein the Ras antigen comprises, preferably consists of, the amino acid sequence according to any one of SEQ I D NOs: 1, 3, 5, 7, 9, 11 to 19, 77 to 119, or 179 to 223, or comprises, preferably consists of, a sequence having at least 75% sequence identity, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence identity with any one of SEQ I D NOs: 1, 3, 5, 7, 9, 11 to 19, 77 to 119, or 179 to 223.

[0667] In one example, said recombinant host cell is a Chinese Hamster Overay (CHO) cell, a Human Embryonic Kidney (HEK) cell, a Pichia pastoris cell, a Saccharomyces cerevisiae cell, a Hansenula polymorpha cell, a Schizosaccharomyces pombe cell, a Leishmania tarentolae cell, a Spodoptera frugiperda cell, or a Trichopulsia ni High Five cell.

[0668] In one example, said recombinant host is a prokaryote cell, is an Escherichia coli cell, a Caulobacter crescentus cell, a Lactobacillus zeae cell, a Lactococcus lactis cell, a Bacillus brevis cell, a Bacillus subtilis cell, a Bacillus megaterium cell, a Caulobacter crescentus cell, or a Corynebacterium species cell like a Corynebacterium glutamicum cell.

[0669] A further embodiment pertains to a therapeutically effective amount of the above recombinant host cell for use in the treatment of a proliferative disorder, such as cancer, and / or for use in adoptive, target-cell specific immunotherapy, in a subject in need thereof. Preferably, the cancer is selected from one or more of brain tumor, oesophageal cancer, mouth cancer, tongue cancer, thyroid cancer, lung cancer, stomach cancer, pancreatic cancer, liver cancer, bile duct cancer, kidney cancer, colon cancer, rectal cancer, prostate cancer, bladder cancer, cervical cancer, epithelial cell cancer, skin cancer, leukaemia, lymphoma, myeloma, myeloid malignancies, lymphoid malignancies, breast cancer, ovarian cancer, neuroendocrine carcinoma, endometrial cancer, vaginal cancer, blood-related cancer, uterine cancer, testicular cancer, glioma, bone sarcoma, cervix cancer, Synovial cancer, and sarcoma, more preferably wherein the cancer is neuroendocrine carcinoma, for example high-grade poorly differentiated neuroendocrine carcinoma (NEC-G3). One particularly preferred example of a cancer to be treated is NEC-G3 of the gastrointestinal tract or small cell lung cancer (NEC-G3 lung carcinoma).

[0670] Yet another embodiment of the present invention pertains to an engineered immune cell (genetically modified cell) expressing the ABP, wherein preferably the engineered immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer T cell, a y8T cell, and a NK cell. In a preferred embodiment, the cytotoxic T cell is a CD4+ T cell, a CD8+ T cell, a gamma delta T cell, or a natural killer cell.

[0671] The present invention also relates to a pharmaceutical composition comprising: (i) an ABP according to the present invention, or (ii) an isolated nucleic acid according to the present invention, or (iii) an expression construct according to the present invention, or (iv) a recombinant host cell according to the present invention, and a pharmaceutically acceptable carrier, stabilizer and / or excipient.

[0672] The present invention also relates to the inventive combination for use in a method of diagnosis, prevention and / or treatment of a proliferative disorder, such as cancer, and / or for use in adoptive, target-cell specific immunotherapy and / or for use in a method of diagnosis, prevention and / or treatment of a non-malignant disease. Preferably, the cancer is selected from one or more of brain tumor, oesophageal cancer, mouth cancer, tongue cancer, thyroid cancer, lung cancer, stomach cancer, pancreatic cancer, liver cancer, bile duct cancer, kidney cancer, colon cancer, rectal cancer, prostate cancer, bladder cancer, cervical cancer, epithelial cell cancer, skin cancer, leukaemia, lymphoma, myeloma, myeloid malignancies, lymphoid malignancies, breast cancer, ovarian cancer, neuroendocrine carcinoma, endometrial cancer, vaginal cancer, blood-related cancer, uterine cancer, testicular cancer, glioma, bone sarcoma, cervix cancer, Synovial cancer, and sarcoma, more preferably wherein the cancer is neuroendocrine carcinoma, for example high-grade poorly differentiated neuroendocrine carcinoma (NEC-G3). One particularly preferred example of a cancer to be treated is NEC-G3 of the gastrointestinal tract or small cell lung cancer (NEC-G3 lung carcinoma).

[0673] In a preferred embodiment, theABPs of the present invention used in the combination or alone are bispecific antibodies for use in the treatment of different types of cancer, such as cancer characterized by (a) mutated Ras gene(s), or cancers with no mutation in Ras gene(s) but with increased activation of the Ras signaling cascade(s).

[0674] In a preferred embodiment, the therapeutic compounds of the present invention are tri-specific or multi-specific antibodies for use in the treatment of different types of cancer, such as cancer characterized by (a) mutated Ras gene(s), or cancers with no mutation in Ras gene(s) but with increased activation of the Ras signaling cascade(s).

[0675] Importantly, target-cell specific immunotherapy is a very promising treatment strategy for, e.g., the treatment of cancer. When using target-cell specific immunotherapy, endogenous cells of the patient are used to destroy cancer cells via induction of an immune response, whereas treatment strategies for cancer not based on immunotherapy often use external compounds to destroy cancer cells. It is often difficult to control targeting using such external compounds, and such treatment strategies, therefore, risk harming healthy cells. Therefore, target-cell specific immunotherapeutic treatment strategies are highly advantageous over the use of external compounds.

[0676] In a preferred embodiment, the cancer is metastatic, stage III cancer, or stage IV cancer, optionally wherein the cancer is a Ras-dependent cancer.

[0677] In another embodiment, which can be combined with any aspect and any other embodiment of the present invention, the compounds or combinations of the present invention are for use in the diagnosis, prevention and / or treatment of RASopathies. RASopathies are a heterogenic group of developmental conditions, in which germline mutations in the Ras gene induce an overactivation of the RAS-MAPK signaling cascade. The result of an overactivation of the RAS-MAPK signaling cascade can be a malformation of the heart or other organs, an impairment of growth or developmental defects, and an enhanced risk for cancer.

[0678] In a preferred embodiment, which can be combined with any aspect and any other embodiment of the present invention, a disorder to be diagnosed, prevented and / or treated by any of the compounds of the present invention is a RAS driven disorder. Thus, the present invention provides strategies to target various RAS driven disorders with ABPs from the extra cellular site.

[0679] [2] In one embodiment, the combination according to the present invention or the ABP of the present invention is for use in modulating a cell-mediated immune response in a subject, optionally wherein the modulating the immune response is an inhibition of a cell-mediated immune response.

[0680] The inhibition of a cell-mediated immune response may be an inhibition of proliferation of an immune cell, such as a lymphocyte, and / or is an inhibition of cytokine expression in an immune cell, such as a lymphocyte.

[0681] The inhibition of a cell mediated immune response may be a reduction of proliferation / activity of effector memory T cells and / or increase of proliferation / activity of regulatory T cells (TREGs).

[0682] Another aspect relates to a diagnostic use of the combination or ABPs of the present invention to detect cells aberrantly expressing Ras protein on the surface of cells in vitro (for example by ELISA) or in vivo (for example by PET-CT with a Ras ligand) or in vitro by novel methods as disclosed infra.

[0683] A further aspect of the present invention relates to a method for the diagnosis, prevention and / or treatment of a proliferative disorder, such as cancer, in a subject, comprising the administration of a therapeutically effective amount of a combination according to the present invention, an ABP according to the present invention, an isolated nucleic acid according to the present invention, an expression construct according to the present invention, a recombinant host cell according to the present invention, or a pharmaceutical composition according to the present invention, to the subject, wherein the cancer is preferably selected from one or more of brain tumor, oesophageal cancer, mouth cancer, tongue cancer, thyroid cancer, lung cancer, stomach cancer, pancreatic cancer, liver cancer, bile duct cancer, kidney cancer, colon cancer, rectal cancer, prostate cancer, bladder cancer, cervical cancer, epithelial cell cancer, skin cancer, leukaemia, lymphoma, myeloma, myeloid malignancies, lymphoid malignancies, breast cancer, ovarian cancer, neuroendocrine carcinoma, endometrial cancer, vaginal cancer, blood-related cancer, uterine cancer, testicular cancer, glioma, bone sarcoma, cervix cancer, Synovial cancer, and sarcoma, more preferably wherein the cancer is neuroendocrine carcinoma, for example high-grade poorly differentiated neuroendocrine carcinoma (NEC-G3). One particularly preferred example of a cancer to be treated is NEC-G3 of the gastrointestinal tract or small cell lung cancer (NEC-G3 lung carcinoma).

[0684] A further aspect of the present invention relates to the use of a combination according to the present invention or an ABP according to the present invention, an isolated nucleic acid according to the present invention, an expression construct according to the present invention, a recombinant host cell according to the present invention, or a pharmaceutical composition according to the present invention for the manufacture of a medicament for the treatment of a proliferative disorder, such as cancer, wherein the cancer is preferably selected from one or more of brain tumor, oesophageal cancer, mouth cancer, tongue cancer, thyroid cancer, lung cancer, stomach cancer, pancreatic cancer, liver cancer, bile duct cancer, kidney cancer, colon cancer, rectal cancer, prostate cancer, bladder cancer, cervical cancer, epithelial cell cancer, skin cancer, leukaemia, lymphoma, myeloma, myeloid malignancies, lymphoid malignancies, breast cancer, ovarian cancer, neuroendocrine carcinoma, endometrial cancer, vaginal cancer, blood-related cancer, uterine cancer, testicular cancer, glioma, bone sarcoma, cervix cancer, Synovial cancer, and sarcoma, more preferably wherein the cancer is neuroendocrine carcinoma, for example high-grade poorly differentiated neuroendocrine carcinoma (NEC-G3). One particularly preferred example of a cancer to be treated is NEC-G3 of the gastrointestinal tract or small cell lung cancer (NEC-G3 lung carcinoma).

[0685] Yet another aspect of the present invention pertains to the use of a combination according to the present invention, an ABP according to the present invention, an isolated nucleic acid according to the present invention, an expression construct according to the present invention, a recombinant host cell according to the present invention, or a pharmaceutical composition for generating target-specific effector cells, in particular for generating cytotoxic T cells, or for killing tumor cells

[0686] Yet another aspect of the present invention relates to a method of modulating a cell-mediated immune response in a human cell that expresses an extracellular Ras antigen, comprising contacting said cell with a combination according to the present invention or an ABP according to the present invention, an isolated nucleic acid according to the present invention, an expression construct according to the present invention, a recombinant host cell according to the present invention, or a pharmaceutical composition according to the present invention, in the presence of an immune cell, such as a T-cell, thereby modulating, preferably enhancing, the cell-mediated immune response.

[0687] ABPs, preferably diabodies, can also have an effect on healthy cells. Thus, an ABP is provided for use in a method of diagnosis, prevention and / or treatment of a non-malignant disease. For example, healthy cells can proliferate upon treatment with ABCs of the present invention. This effect can be used in the field of regenerative medicine, for example. It can also be used in a method for enhancing wound healing in the treatment of aging or in lifestyle medicine.

[0688] The expression of functional extracellular Ras protein on non-malign cells opens new fields for use in humans or animals like for example: (i) Treating other disease than cancer like metabolic disease, hypercholesterinemia, hypertriglyceridemia, adrenoleukodystrophy, Diabetes type 1 or 2, Gaucher disease, Hereditary hemochromatosis, Lesch-Nyhan syndrome, obesity, acidbase imbalance, metabolic brain diseases, disorders of sodium, calcium or potassium metabolism, kidney disease, DNA repair-deficiency disorders, glucose metabolism disorders other than diabetes, hyperlactatemia, iron metabolism disorders, lipid metabolism disorders, malabsorption syndromes, metabolic syndrome X, inborn error of metabolism, mitochondrial diseases, phosphorus metabolism disorders, porphyrias, proteostasis deficiencies, metabolic skin diseases, wasting syndrome, chronic fatigue syndrome, water-electrolyte imbalance, wound healing or aging, (ii) Treat auto-immune disease of the integumentary system, of the digestive system, of the heart and vascular system, of the urinary system, of the nervous system, of the endocrine system, of the respiratory system, of the blood, of the reproductive system, of the eye, of the musculoskeletal system or to treat a combination of two or more of these systems, (iii) Treating neurological disorders like dementia of Alzheimer form, vascular dementia, Lewy body dementias, younger onset dementia, frontotemporal dementia, alcohol-related brain injury, virus infection associated dementia, chronic traumatic encephalopathy dementia, (iv) Treating forms of depression like major depression, persistent depressive disorders (formerly called dysthymia), bipolar disorder, seasonal affective disorder, perinatal depression or premenstrual dysphoric disorder, (v) Treating disease of the heart or blood vessels like systolic heart failure, diastolic heart failure, right-sided heart failure, congestive heart failure, mixed heart failure, heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, coronary heart disease, myocardial infarction (Ml), myocardial remodeling after Ml, genetic cardiomyopathy, heart valve disease, arterial hypertension, pulmonary hypertension or myocardial arrhythmias, (vi) To be used for chronic liver disease like alcoholic liver disease, non-alcoholic fatty liver disease, chronic viral hepatitis, alpha-1 antitrypsin deficiency, liver cirrhosis, primary biliary cirrhosis, primary sclerosing cholangitis or autoimmune hepatitis, (vii) To be used for chronic kidney disease like atypical hemolytic uremic syndrome, Alport syndrome, amyloidosis, APOL1-mediated kidney disease, cardiovascular-kidney-metabolic syndrome, complement 3 glomerulopathy (C3G), congenital abnormalities of the kidneys and urinary tract, kidney cystinosis, Fabry disease, focal segmental glomerulosclerosis, glomerulonephritis, goodpasture syndrome, granulomatosis with polyangiitis, hemolytic uremic syndrome, IgA nephropathy, interstitial nephritis, lupus nephritis, minimal change kidney disease, polycystic kidney disease, primary hyperoxaluria and kidney vasculitis, (vii) To be used in hematologic disorders like sickle cell anemia, thalassemia intermedia or major, immunthrombocytopenia, paroxysmal nocturnal hemoglobinuria or hemolytic anemias, (viii) To be used for fertility treatment, (ix) To be used in diagnostics of noncancer related disease, (x) To be used in vitro to stimulate or protect eukaryote cells in cell culture.

[0689] ABPs and / or ABCs can also activate or stimulate cells to proliferate. Using cells with a reporter plasmid and / or vector may then be used to detect and measure the activation of specific signaling cascades, for example to measure cell viability or activation, the latter for example with a reporter plasmid and / or vector resulting in light emission after activation.

[0690] Diabodies:

[0691] In one preferred embodiment, the ABP is a diabody. Twelve different bi-specificT cell recruiting Diabodies had been designed previously (Figure 1A; PCC01_D - PCC012_D; SEQ ID NOs: 228 to 239). Diabodies are small single-chain bi-specific antibodies. All 12 diabodies have shown high in vitro killing activity against tumor cells.

[0692] The predicted binding sites are shown in Figure 1B. The original scFv sequences for PCC01 (G12V-34, SEQ ID NOs: 20, 66, 67, 122, 134 to 147, 228) and PCC02 (G13D-18, SEQ ID NOs: 21, 68, 69, 123, 148 to 160, 229) are from a murine scFv combinatorial phage display library, extracted from mice immunized with Ras mutant peptides [9], The ScFv sequence (KGH-R1) used for PCC03 (SEQ ID NO: 22, 70, 71, 124, 161 to 174, 230) is from a murine scFv combinatorial phage display library, extracted from mice immunized with human wild-type K-Ras, N-Ras and H-Ras proteins

[0010] , The ScFv from clone KGH-R1 showed strong binding to human primary solid tumor tissues and its binding site with K-Ras, H-Ras and N-Ras was analyzed by molecular docking method

[0011] , The results showed that KGH-R1 ScFv binds to Ras proteins mainly through hydrogen bonding and salt bridges. The sequences for PCC06 (SEQ ID NOs: 25, 127, 175 to 178, 233) and PCC07 (SEQ ID NOs: 26, 128, 175 to 178, 234) are derivative from the same original monobody FN3 clone (NS1) [12,13], The NS1 selectively binds both H-Ras and K-Ras and potently inhibits H-Ras and K-Ras mediated cell signaling and transformation. The NS1 was isolated from the "side-and-loop" combinatorial library constructed in the phage display format against the H-Ras, H-Ras / GDP and H-Ras / GTP proteins. The NS1 monobody is a fibronectin type III domain (FN3) binder. A 1.4 A-resolution crystal structure of NS1 in complex with GDP-loaded H-Ras revealed interaction of NS1 with the a4-p6-a5 region within the so-called allosteric lobe, which lie on the surface of Ras protein.

[0693] The sequence for PCC04 (SEQ ID NOs: 23 and 231) is the known Ras binding domain (RBD-CRD) from the human protein RAF1 (P04049). There are several crystal structure data for the complex of KRAS with RAF1 (RBD-CRD) [14,15], The amino acids in KRAS involved in the binding are conserved across all four RAS isoforms (KRas4b, KRas4a, NRas, HRas). Of interest, the reported amino acids interactions present at the tripartite complex comprised of RBD-CRD, KRAS and the lipid membrane (artificial Nanodisc), are to some part different from the amino acids reported for the interactions between bipartite complex RBD-CRD and KRAS. This high flexibility of the RBD-CRD domain against different conformations of RAS is important. It is known for RAS to change its position and conformation in contact with the lipid membrane and bound signaling partners. Therefore, an ABP with the ability to bind different conformations of RAS at the cell membrane, will have an advantage over ABP targeting only a specific Ras conformation. This has been recently demonstrated for the present Ras targeting ABPs in the Diabody format, with the highest killing function for the PCC04 construct over the antibody and monobody based constructs (PCC01, PCC02, PCC03, PCC06 and PCC07) (PCT / EP2024 / 065774).

[0694] 2D cell culture

[0695] 2D cell culture models can for example be used to test the influence of compounds, such as a combination of an ABP and a modulator compound, on the viability of tumor cells. Thus, for example T cells and tumor cells are cultured together. Preferred cell ratios are: Effector to tumor (E: T) ratio 1:1, 1:3, 1:5, 1:10; 1:20 or 2:1, 3:1, 5:1, 10:1, 20:1, 50:1, 100:1

[0696] 3D model Different cell culture models can be used for testing anti-tumor activity of an ABP or a combination of the present invention. In one embodiment, a mix of human and animal cells is used. In another embodiment, only tumor cells are used. In another embodiment, tumor and effector cells, preferably T cells, NK cells or NKT cells, are used.

[0697] In order to better resemble the natural in vivo conditions, a new in vitro cell culture model was developed. In regular in vitro cell culture, tumor cells grow adherent, semi adherent or in suspension on the plastic surface in 2 dimensions (2D) (used in prior art PCT / EP2024 / 065774). In contrast, in vivo tumor cells grow in a complex tumor microenvironment (TME) in 3 dimensions. The importance of the TME is well known for tumor progression and has been shown to influence response to immune checkpoint blockade (ICB).

[0698] Thus, to study the effect of the TME on extracellular Ras targeting ABPs of the present invention, a new full-humanized in vitro co-culture assay resembling the complex human TME was developed (Figure 6A).

[0699] This co-culture model comprises tumor cells and at least one non-malignant cell selected from the group comprising or consisting of a fibroblast, an endothelial cell of a blood vessel, an endothelial cell of a lymphatic vessel, a tissue macrophage, a fat cell, an osteoblast, a chondrocyte, a smooth muscle cell, a preadipocyte, a pericyte, a mesenchymal stem cell, a melanocyte, a keratinocyte, hematopoietic progenitors, a dendritic cell, a skeletal muscle cell, a T cell and a B cell.

[0700] Preferred cell ratios are: Healthy to tumor (H: T) ratio 1:1, 1:3, 1:5, 1:10; 1:20, 1:50; 1:100 or 2:1, 3:1, 5:1, 7:1, 10:1, 15:1, 20:1, 30:1, 50:1, 100:1; 250:1, 500:1, 1000:1, 5000:1.

[0701] According to the present invention, an in vitro 3D cell culture model is provided comprising tumor cells and at least one non-malignant cell selected from the group comprising or consisting of a fibroblast, an endothelial cell of an arterial blood vessel, an endothelial cell of a venous blood vessel, an endothelial cell of a lymphatic vessel, a tissue macrophage, a fat cell, an osteoblast, a chondrocyte, a smooth muscle cell, a preadipocyte, a pericyte, a mesenchymal stem cell, a melanocyte, a keratinocyte, hematopoietic progenitors, a dendritic cell, a skeletal muscle cell, a T cell and a B cell, preferably wherein the at least non-malignant cell is a T cell, a fibroblast and an endothelial cell. The in vitro 3D cell culture model preferably further comprises the combination according to the present invention or the ABP according to the present invention for testing the influence of the combination on the viability and extracellular Ras expression of the at least one tumor cell.

[0702] The in vitro 3D cell culture model may further comprise a cellulose, preferably methylcellulose, and / or Matrigel®.

[0703] In one preferred embodiment, the in vitro 3D cell culture model comprises at least one tumor cell, at least one T cell, at least one fibroblast, at least one aortic endothelial cell, at least one lymphoid endothelial cell and a cellulose, preferably methylcellulose, and further comprises the combination according to the present invention for testing the influence of the combination on the viability and extracellular Ras expression of the at least one tumor cell.

[0704] Preferably, the cells are human cells.

[0705] In contrast to regular cell culture, tumor cells preferably grow in the co-culture assay 3 dimensionally (3D), forming cluster of cells and spheroids (Figure 6B). This is advantageous because spheroids recapitulate important tumor features including cellular heterogeneity, cell signaling pathways, cell-cell and cell- extracellular matrix (ECM) interactions, gene expression patterns similar to those of in vivo conditions and a tumor morphology composed of different cell layers.

[0706] There is another major advantage of this co-culture assay. This is preferably a full-humanized assay with different types of preferably healthy human cells, resembling to some part the complex human TME. This is advantageous because some cytokines, ligands, receptors or hormones produced by the surrounding stroma cells and affecting the tumor progression and resistance, are not cross reactive between human and mice.

[0707] Further, the co-culture assay using 6-well, 24-well, 96-well or 384-well plates can easily be expanded to hundreds or thousands of plates. This is important to investigate new extracellular Ras targeting formats alone or in combination with large inhibitor libraries to screen for combination partners in a timely manner. Moreover, this plate-based assay makes it possible to use a large variety of reagents and equipment using special plates (coated or not, transparent etc.), modified cells (reporter plasmids, gene knock-out or knock-in, resistant genes etc.), selected medium (supplements, pH, osmolality etc.), different oxygen conditions (from hypoxia to hyperoxia) and different analytic instruments (microscopy, luminescence or fluorescence sensor etc.). This is important to test and analyze the extracellular Ras expression, and the effect of modulating factors, in great detail.

[0708] Further, such an assay fulfills the “replace” requirements of the 3Rs principle (Re- place, Reduce, Refine) for animal testing. This is important to reduce the number of animals (mice) for this invention.

[0709] Co-culture assays, mimicking the complex TME in vitro, can for example be used for large-scale automated library screens for small molecule compounds (i.e. modulator compounds) or cellular-target genes modulating the extracellular Ras expression.

[0710] In a preferred embodiment, different concentrations of oxygen (02) are used for cell incubation. From hyperbaric 99% to regular ambient air oxygen concentration of 20,942 %. Or from regular 20,942 % to hypoxemia 0.01%. Low oxygen concentrations are advantageous, because they better reflect the physiological oxygen concentration in the human body ranges from 13-14% in arterial blood to 5-8% in venous blood, lung, liver, kidney, placenta, and bone marrow, to 4% in the brain, 3% in skeletal muscle, and 0.5-4.5% in lymphoid organs.

[0711] In another preferred embodiment, cell media with a pH different from the body plasma physiological pH (7.35 to 7.45) are used. In the body the physiological pH is different in different tissues. In the TME the pH can change dramatically with tumor pH values ranging between 5.55 and 7.69. Cell culture media can be used for example with a basic pH in the range between 7.5 and 12.0. Or media with an acidic pH in the range between 7.3 and 1.0.

[0712] Different artificial models can for example be used for analyzing Ras protein expression on lipid membranes, binders against Ras proteins or binders against other cell targets modulating the Ras expression on lipid membranes. For example, artificial lipid based membranes or artificial cells may be used. These could include one or more of polymersomes, proteinosomes, colloidosomes, liposomes, micelles, lipid membrane discs as well as membrane-vesicles. Modulator compounds

[0713] Modulator compounds used in combinations of the present invention are for example small molecule inhibitors (SMI) or fatty acids (lipids, also termed Lipid Mix). SMIs are preferably organic substances, preferably with a low molecular weight < 1000 daltons, that interfere with specific molecules required for cell growth and function. Surprisingly, all combinations with different SMIs from different chemical families with different structures, different targets and different mechanisms of action had a significantly better anti-tumor efficacy than ABPs alone. The tests were done with:

[0714] 1. All-trans-retinoic acid (ATRA) based modulator compounds (Figure 7)

[0715] The compound ATRA (DrugBank ID DB00755) used in combination with an extracellular Ras directed ABP, is a metabolite of vitamin A (DrugBank ID DB00162) and mediates the functions of cell growth, differentiation and development mainly through modulating gene expression. ATRA is a natural agonist of RAR nuclear receptors, with IC50s of 14 nM for RARa / p / y. Retinoic acid bind to PPARp / b with Kd of 17 nM. Retinoic acid acts as an inhibitor of transcription factor NFE2L2 through activation of retinoic acid receptor alpha.

[0716] Therefore, any compound inhibiting or modulating retinoid X receptors (RXRG Gene ID: 6258 UniProt P48443) (RXRB Gene ID: 6257 UniProt P28702) (RXRA Gene ID: 6256 UniProt P19793) and / or retinoic acid receptors (RARA Gene ID: 5914, UniProt P10276) (RARG Gene ID: 5916, UniProt P13631) (RARB Gene ID: 5915, UniProt 10826) and / or cellular retinoic acidbinding proteins (CRABP2 Gene ID: 1382, UniProt P29373) (CRABP1 Gene ID: 1381, UniProt P29762) and / or NFE2L2 (Gene ID: 4780, UniProt Q16236) can be used for this invention.

[0717] Examples for ATRA analogues are shown in Liang, C. et al. (2021) Overview of all-trans-retinoic acid (ATRA) and its analogues: Structures, activities, and mechanisms in acute promyelocytic leukaemia Eur J Med Chem. 220:113451: Metabolites of ATRA, hydrophobic unit-modified analogues, compounds with imidazole modifications in the hydrophobic part, compounds obtained by modifying the hydrophobic part of the molecule with adamantane, compounds which were obtained by replacing the hydrophobic part by a 4-methoxy-2,3,6-trimethylbenzene ring, a compound which was obtained by replacing the trimethyl cyclohexenyl ring of 9-cis-retinoic acid with a tetra-hydronaphthone ring, compounds obtained by connecting 9Z tetraenoic acid with a disubstituted cyclohexene ring, compounds with polar terminus modified analogues, Compounds whose terminal positions are substituted or fused with different groups, compounds with linker unit-modified analogues, compounds with receptor functional analogues, compounds with RARB selectivity or compounds with RXR selectivity.

[0718] As alternative, compounds inducing transcriptional activation and / or inhibition of cell differentiation-related genes, or compounds regulating one or more genes affected by ATRA in acute promyelocytic leukaemia (APL) cells (Table 6), can be used for this invention.

[0719] Table 6: Genes up-regulated by ATRA in LI937 cells (acute promyelocytic leukemia, APL) Gene ID Gene symbol Gene ID Gene symbol

[0720] 2268 FGR 638 BIK

[0721] 6688 SPI1 9516 LITAF

[0722] 1050 CEBPA 10397 NDRG1

[0723] 1053 CEBPE 7378 UPP1

[0724] 5585 PKN1 4046 LSP1

[0725] 7133 TNFRSF1B 3417 IDH1

[0726] 6503 SLA 5660 PSAP

[0727] 5294 PIK3CG 8870 IER3

[0728] 3659 IRF1 5292 PIM1

[0729] 241 ALOX5AP 2000 ELF4

[0730] 7127 TNFAIP2 6518 SLC2A5

[0731] 3198 HOXA1 10507 SEMA4D

[0732] 5916 RARG 3727 JUND

[0733] 3489 IGFBP6 952 CD38

[0734] 7150 TOP1

[0735]

[0736]

[0737] 2. Modulator compounds of the cholesterol and triglyceride synthesis and metabolism (Figures 8-11)

[0738] The modulator compounds Simvastatin (DrugBank ID DB00641) (Figure 8), Fluvastatin (DrugBank ID DB01095) (Figure 9), Lapaquistat (DrugBank Accession Number DB16215) (Figure 10) and Bemfivastatin (PubChem CID 11192585, CAS number 805241-79-6) (Figure 11), used in combination with an extracellular Ras directed ABP, are all modulators or inhibitors of the cholesterol and / or fatty acid synthesis. Simvastatin, Fluvastatin and Bemfivastatin are all cholesterol lowering drugs and belong to the group of statins. Simvastatin and Fluvastatin are competitive inhibitors of HMG-CoA reductase (HMGCR) (Gene ID: 3156, UniProt P04035). Bemfivastatin is an HMG-CoA Reductase (HMGCR) inhibitor enhancing the activity of liver extraction. HMG-CoA Reductase (HMGCR) is the rate-limiting enzyme for cholesterol synthesis in the conversion of HMG-CoA to mevalonate. In contrast, Lapaquistat acetate is a squalene synthase FDFT1 (Gene ID: 2222, UniProt P37268) inhibitor for decreasing plasma cholesterol and triglyceride levels, by lowering lipoproteins containing apoB100. Squalene is further downstream in the synthesis of cholesterol compared to HMG-CoA reductase.

[0739] Therefore, any compound inhibiting or modulating the synthesis of fatty acids and / or cholesterol, cholesterol oxygenated derivatives and / or cholesterol conjugated derivatives can be used for this invention.

[0740] Examples are shown in Table 7.

[0741] Table 7: Different inhibitors or modulators for HMG-CoA reductase (HMGCR) (Gene ID: 3156, UniProt P04035) or squalene (FDFT1 Gene ID: 2222, UniProt P37268) (SOLE Gene ID: 6713, UniProt Q 14534).

[0742] Name CAS (Chemical Abstracts Service) number Simvastatin 79902-63-9

[0743] Lovastatin 75330-75-5

[0744] Atorvastatin 134523-00-5

[0745] Rosuvastatin Calcium 147098-20-2

[0746] Mevastatin 73573-88-3

[0747] Pitavastatin 147511-69-1

[0748] Fluvastatin 93957-54-1

[0749] SR12813 126411-39-0

[0750] Meglutol 503-49-1

[0751] Monacolin J 79952-42-4

[0752] Bemfivastatin 805241-79-6

[0753] Clinofibrate 30299-08-2

[0754] HMG499 2416941-68-7

[0755] Rosuvastatin 287714-41-4

[0756]

[0757] P-Amyrin acetate 1616-93-9

[0758] Pravastatin 81093-37-0

[0759] S-2E 155730-92-0

[0760] L-157012 114801-28-4

[0761] P-Amyrin palmitate 5973-06-8

[0762] Ganomycin I 1191255-15-8

[0763] QH536 2754254-07-2

[0764] Crilvastatin 120551-59-9

[0765] L-669,262 130468-11-0

[0766] Cerivastatin 145599-86-6

[0767] Rawsonol 125111-69-5

[0768] Diallyl disulfide 2179-57-9

[0769] Diallyl Trisulfide 2050-87-5

[0770] Allicin 539-86-6

[0771] FIN56 1083162-61-1

[0772] Procyanidin B-53,3'-di-O-gallate 106533-60-2

[0773] Zaragozic acid D 155179-14-9

[0774] Liranaftate 88678-31-3

[0775] YM-53601 182959-33-7

[0776] SQLE-IN-1 1019169-83-5

[0777] Terbinafine 91161-71-6

[0778] FR194738 204067-52-7

[0779] YM-75440 780736-74-5

[0780] Zaragozic acid A 142561-96-4

[0781] Butenafine 101828-21-1

[0782] Alnusenone 508-09-8

[0783] BPH-1218 1426824-36-3

[0784] NB-598 Maleate 155294-62-5

[0785] NB-598 131060-14-5

[0786]

[0787] As alternative, any protein or protein complexes of the human cholesterol and / or lipid metabolism can be targeted with a modulating or inhibiting compound. Examples for these genes are squalene monooxygenase (SQLE) (Gene ID: 6713, UniProt Q14534), or mevalonate kinase (Gene ID: 4598, Q03426), or lanosterol synthase (Gene ID: 4047, UniProt P48449), or ACATI (Gene ID: 38, UniProt P24752), or HMGCS1 (Gene ID: 3157, UniProt Q01581), or GNPAT (Gene ID: 8443, UniProt 015228), or GPAM (Gene ID: 57678, UniProt Q9HCL2), or GPAT3 (Gene ID: 84803, UniProt Q53EU6), orAGPS (Gene ID: 8540, UniProt 000116), or PNPLA2 (Gene ID: 57104, UniProt Q96AD5), orACSS2 (Gene ID: 55902, Uniprot Q9NR19), or FASN (Gene ID: 2194, UniProt P49327) or SOD (Gene ID: 6319, UniProt 000767) or APOB (Gene ID: 338, UniProt P04114).

[0788] 3. Immunomodulatory drugs (IMiDs) targeting cereblon (Figures 12-15)

[0789] The compounds Lenalidomide (DrugBank ID DB00480) (Figure 12), Iberdomide (DrugBank Accession Number DB12101) (Figure 13), HOMO-Protac cereblon degrader 1 (CAS number 2244520-98-5) (Figure 14) and Eragidomide (DrugBank Accession Number DB19242) (Figure 15), used in combination with an extracellular Ras directed ABP, are all cereblon (CRBN) E3 ligase modulators or inhibitors.

[0790] Heterobifunctional degraders belonging to the family of proteolysis targeting chimeras (PROTACs) are a rapidly developing mode of therapeutic activity in which small molecules stabilize the interface between the E3 ubiquitin ligase and its “neosubstrate” protein that normally does not interact with the ligase, thereby promoting their ubiquitination and degradation in a drug-dependent manner. For example the HOMO-Protac cereblon degrader 1 (CAS number 2244520-98-5) was used for combination therapy of this invention (Figure 14). Therefore, any compound with function as PROTACs can be used for this invention.

[0791] Also, any compounds inhibiting or modulating the ubiquitin E3 ligase cereblon (Gene ID: 51185, UniProt Q96SW2) can be used in combination with an extracellular Ras directed ABP. Examples are shown in Table 8.

[0792] Table 8: Cereblon (CRBN) inhibitors or modulators and ligands for E3 ligase

[0793] Name CAS Number MedChemExpress (MCE®) numbering / clinicial trials CC-885 1010100-07-8

[0794] EM12-SO2F 2803819-61-4

[0795] SMART1 HY-W998345

[0796] Thalidomide 50-35-1

[0797] Pomalidomide 19171-19-8

[0798]

[0799] E3 ligase Ligand 32 2300099-98-1

[0800] BMS-986397 2564486-44-6

[0801] PLX-4545 2892065-45-9

[0802] PT- 179 2924858-25-1

[0803] Golcadomide 2379572-34-4

[0804] ZXH-1-161 2407654-51-5

[0805] TD-106 2250288-69-6

[0806] CO- 17369 1547162-46-8

[0807] Cereblon inhibitor 1 2672489-14-2

[0808] CRBN modulator-1 2407829-65-4

[0809] Cereblon inhibitor 2 2639380-62-2

[0810] CRBN ligand-9 55003-81-1

[0811] PROTAC CRBN ligand-2 HY- 158152

[0812] CRBN ligand-1 3032314-67-0

[0813] BWA-522 intermediate- 1 2241315-66-0

[0814] Avadomide 1015474-32-4

[0815] Golcadomide 2379572-34-4

[0816] Cemsidomide 2504235-67-8

[0817] BMS-986397 NCT04951778 Mezigdomide 2259648-80-9

[0818] CC-3060 444288-86-2

[0819]

[0820] As alternative, any protein or protein complexes of the human ubiquitin E3 ligase family can be targeted with a modulating or inhibiting compound. Examples are:

[0821] UBR5 (GenelD 51366, UniProt: 095071), XIAP (GenelD 331, UniProt P98170), ZFP91 (GenelD 80829, UniProt: Q96JP5), TRIM65 (GenelD 201292, UniProt: Q6PJ69), TRIM47 (GenelD 91107, UniProt: Q96LD4), TRIM39 (GenelD 56658, UniProt: Q9HCM9), TRIM3 (GenelD 10612, UniProt: 075382), TRIM25 (GenelD 7706, UniProt: Q14258), SIAH1 (GenelD 6477, UniProt: Q8IUQ4), RNF216 (GenelD 54476, UniProt: Q9NWF9), RNF40 (GenelD 9810, UniProt: 075150), RNF41 (GenelD 10193, UniProt: Q9H4P4), RNF6 (GenelD 6049, UniProt: Q9Y252), PDZRN3 (GenelD 23024, UniProt: Q9UPQ7), NEDD4 (GenelD 4734, UniProt: P46934), Mdm2 (GenelD 4193, UniProt: Q00987), CBL (GenelD 867, UniProt: P22681), BRCA1 (GenelD 672, UniProt: P38398) Another alternative are modulator compounds inhibiting or modulating the Wnt receptor signalling pathway through beta-catenin or the canonical Wnt receptor signaling pathway, important for cereblon (CRBN) activation and regulation in tumor cells.

[0822] 4. Modulators of the Ras signaling complex (Figure 16-22)

[0823] The modulator compounds NSC-70220 (CAS number 4551-00-2) (Figure 16), SAH-SOS1A TFA (2896737-31-6) (Figure 17) Adagrasib (DrugBank ID DB15568) (Figure 18), BI-2852 (CAS number 2375482-51-0) (Figure 19), MRTX-1133 (CAS number 2621928-55-8) (Figure 20), RMC-0331 (CAS number 2488788-52-7) (Figure 21), and SOS 1 -activator 1 (CAS number 2245237-53-8) (Figure 22), used in combination with an extracellular Ras directed ABP, are all modulators or inhibitors of targets in the Ras signaling complex.

[0824] Adagrasib (DrugBank ID DB15568) is a potent, orally-available, and mutation-selective covalent inhibitor of KRAS G12C. BI-2852 (CAS number 2375482-51-0) is a KRAS inhibitor for the switch l / ll pocket (Sl / I l-pocket) by structure-based agent design with nanomolar affinity. BI-2852 is mechanistically distinct from covalent KRASG12C inhibitors (binds to switch II pocket) and binds ten-fold more strongly to active KRASG12D versus KRASwt (740 nM vs 7.5 pM). MRTX-1133 (CAS number 2621928-55-8) is a noncovalent and selective KRAS G12D inhibitor. MRTX1133 optimally fills the switch II pocket and extends three substituents to favorably interact with the protein, resulting in an estimated KD against KRAS G12D of 0.2 pM. MRTX1133 prevents SOS1 -catalyzed nucleotide exchange and / or formation of the KRAS G12D / GTP / RAF1 complex, thereby inhibiting mutant KRAS-dependent signal transduction. MRTX1133 selectively inhibits KRAS G12D mutant, but not KRAS wild-type, tumor cells. RMC-0331 (CAS number 2488788-52-7) RMC-0331 (RM-023) is a potent, selective and orally bioavailable SOS1 inhibitor. RMC-0331 is a compound that blocks RAS activation via disruption of the RAS-SOS1 interaction. SOS 1 -activator 1 (CAS number 2245237-53-8) is a potent activator of SOS1-mediated nucleotide exchange with a Kd of 44 nM. SOS1 (Gene ID: 6654, UniProt Q07889) is a guanine nucleotide exchange factor that catalyzes the exchange of GDP for GTP on RAS. NSC-70220 (CAS number 4551-00-2) is a selective and allosteric SOS1 inhibitor. NSC-70220 inhibits allosteric site activation, and partially inhibited catalytic site activation and SAH-SOS1A TFA (2896737-31-6) is a peptide-based SOS1 / KRAS protein interaction inhibitor. SAH-SOS1A TFA binds to wild-type and mutant KRAS (G12D, G12V, G12C, G12S, and Q61H) with nanomolar affinity (EC50=106-175 nM). SAH-SOS1A TFA directly and independently blocks nucleotide association. Therefore, any compounds inhibiting or modulating the wild type Ras proteins (SEQ ID NOs: 1, 3, 5, 7, 9 or 179 to 182) and / or the mutant Ras proteins (SEQ ID NOs: 11 to 19, 77 to 119, or 183 to 223) can be used in combination with an extracellular Ras directed ABP. Examples for Ras direct or indirect inhibitors or modulators are shown in Table 9.

[0825] Table 9: Different inhibitors or modulators of Ras or farnesyltransferase or the PRMT5-MTA complex or SOS1 or SHP2 or SHP1

[0826] Name CAS Clinical study number

[0827] / Publications / Compound CID

[0828] A- 176120 185049-54-1

[0829] RAS / RAS-RAF-IN-1 2447039-81-6

[0830] RAS inhibitor Abd-7 2351843-48-4

[0831] RMC-6236 2765081-21-6

[0832] Rasarfin 674359-73-0

[0833] Ras modulator- 1 623935-08-0

[0834] RAS GTPase inhibitor 1 2252242-32-1

[0835] Pan-RAS-IN-7 2642135-72-4

[0836] K-Ras-IN-1 84783-01-7

[0837] Pan-RAS-IN-1 1835283-94-7

[0838] K- Ras- IN-4 3044773-77-2

[0839] Pan-RAS-IN-4 3024060-23-6

[0840] Pan-RAS-IN-3 3034588-80-9

[0841] Pan-RAS-IN-2 3034673-92-9

[0842] PROTAC K-Ras Degrader-4 2938169-99-2

[0843] K- Ras- IN-2 905794-70-9

[0844] K-Ras-PDEb-IN-1 1841464-21-8

[0845] PROTAC K-Ras Degrader-1 2378258-52-5

[0846] K-Ras- IN-3 3024991-82-7

[0847] PROTAC K-Ras Degrader-3 3043670-68-1

[0848] K-Ras ligand-Linker 2749492-84-8

[0849] Conjugate 1

[0850] K-Ras ligand-Linker 2741300-61-6

[0851] Conjugate 2

[0852]

[0853] K-Ras ligand-Linker 2378261-87-9 Conjugate 3

[0854] K-Ras ligand-Linker 2378261-83-5 Conjugate 4

[0855] Ftase inhibitor 1 149759-96-6 UCM-1336 1621535-90-7 ML-099 496775-95-2 MCP110 521310-51-0 SCH-53870 188480-50-4 G DC-6036- NH 2417918-80-8 L-731735 149756-20-7 KY1022 1029721-36-5 SOS1 activator 1 2245237-53-8 LUNA18 2676177-63-0 Pan-RAF kinase inhibitor 1 2648838-76-8 RMC-7977 2765082-12-8 Salirasib 162520-00-5 FTI-2153 344900-92-1 NSC1011 5335-97-7 LB42908 226927-89-5 L 731734 149786-89-0 NSC-658497 909197-38-2 LB42708 226929-39-1 XMU-MP-9 2251130-41-1 SOS1 agonist-1 2245237-61-8 Lonafarnib 193275-84-2 A- 176120 185049-54-1 XRP44X 729605-21-4 ADT-007 1945941-09-2 Kobe0065 436133-68-5 ML-097 743456-83-9 UC-857993 487001-04-7 Kobe2602 454453-49-7

[0856]

[0857] FTI-277 170006-73-2

[0858] RMC-0331 2488788-52-7

[0859] G12Si-1 2946593-42-4

[0860] P0P-3MB 1144114-27-1

[0861] SCH54292 188480-51-5

[0862] BAY-293 2244904-70-7

[0863] SML- 10-70-1 1536470-98-0

[0864] 6-CEPN 1054549-73-3

[0865] CP-609754 1190094-64-4

[0866] ARS-1323 1698024-73-5

[0867] KY1220 292168-79-7

[0868] ARS-1630 1698055-86-5

[0869] ZINC57632462 1286482-29-8

[0870] SOS1-IN-16 2930763-85-0

[0871] PROTAC SOS1 degrader-7 3036155-26-4

[0872] FTI-2148 251577-09-0

[0873] RMC-9805 2922732-54-3

[0874] L-739749 156511-34-1

[0875] KRAS inhibitor-22 2042365-57-9

[0876] XR 3054 247090-97-7

[0877] Glecirasib 2657613-87-9

[0878] Vociprotafib 2172652-48-9

[0879] FGTI-2734 1247018-19-4

[0880] SHP099 1801747-42-1

[0881] RMC-6291 2641998-63-0

[0882] Setidegrasib 2821793-99-9

[0883] H RS-4642 - NCT06620848

[0884] NCT06587061

[0885] MRTX1133 2621928-55-8

[0886] BI-3706674 JPRN-jRCT2030230343

[0887] NCT06056024

[0888] ERAS-4693 DOI: 10.1016 / j.taap.2023.116601 ERAS-5024

[0889]

[0890] BI-2865 2937327-93-8

[0891] BI-2493 2937344-16-4

[0892] FMC-376 NCT06244771

[0893] BBO-8520 2893809-51-1

[0894] MRTX9768 2629314-68-5

[0895] MRTX-1719 2630904-45-7

[0896] MRTX0902 2654743-22-1

[0897] MRTX-1257 2206736-04-9

[0898] MRTX-EX185 Compound CID: 163322337

[0899] Adagrasib 2326521-71-3

[0900] BI-2852 2375482-51-0

[0901] TH-Z827 2847881-81-4

[0902] TH-Z816 2847881-42-7

[0903] TH-Z835 2766209-50-9

[0904] TH-Z827 2847881-81-4

[0905] RMC-4550 2172651-73-7

[0906] RMC-3943 1801764-60-2

[0907] RMC-6291 2641998-63-0

[0908] RMC-4998 2642037-07-6

[0909] NSC-70220 4551-00-2

[0910] NSC-87877 56990-57-9

[0911] sos1-in-15 Compound CID: 165437863

[0912] sos1-in-14 Compound CID: 165413022

[0913] SOS1 activator 1 Compound CID: 134814234

[0914] SAH-SOS1ATFA 2896737-31-6

[0915] SAH-SOS1A 1652561-87-9

[0916]

[0917] As alternative, any protein or protein complexes of the Ras signaling pathway can be targeted with a modulating or inhibiting compound. Exemplary RAS pathway gene names are:

[0918] BRAF (GenelD: 673, UniProt P15056, H7C560, A0A2U3TZI2, A0A2R8Y8E0), ALK (GenelD: 238, UniProt Q9UM73, A0A087WZL3), AKT1 (GenelD: 207, UniProt P31749-1, P31749-2), AKT2 (GenelD: 208, UniProt P31751-1, P31751-2), BRCA2 (GenelD: 675, UniProt P51587, H0YE37, H0YD86, A0AAQ5BGN2), ERBB2 (GenelD: 2064, UniProt P04626-1, P04626-2, P04626-3, P04626-4, P04626-5, P04626-6), EGFR (GenelD: 1956, UniProt P00533-1, P00533-2, P00533-3, P00533-4), FGFR1 (GenelD: 2260, UniProt P11362-1, P11362-2, P11362-3, P11362-4, P11362-5, P11362-6, P11362-7, P11362-8, P11362-9, P11362-10, P11362-11, P11362-12), FGFR2 (GenelD: 2263, UniProt P21802-1, P21802-2, P21802-3, P21802-4, P21802-5, P21802-6, P21802-7, P21802-8, P21802-14, P21802-15, P21802-16, P21802-17), FGFR3 (GenelD: 2261, UniProt P22607-1, P22607-2, P22607-3, P22607-4), FGFR4 (GenelD: 2264, UniProt, P22455-1, P22455-2), FLT3 (GenelD: 2322, UniProt, P36888-1, P36888-2), GRB10 (GenelD: 2887, UniProt Q13322-1, Q13322-2, Q13322-3, Q13322-4), GRB2 (GenelD: 2885, UniProt P62993-1, P62993-2), KSR1 (GenelD: 8844, UniProt Q8IVT5-1, Q8IVT5-2, Q8IVT5-3, Q8IVT5-4), KSR2 (GenelD: 283455, UniProt Q6VAB6-1, Q6VAB6-2), MET (GenelD: 4233, UniProt P08581-1, P08581-2, P08581-3), MTOR (GenelD: 2475, UniProt P42345), NFKB1 (GenelD: 4790, UniProt P19838-1, P19838-2, P19838-3), PDGFRA (GenelD: 5156, UniProt P16234-1, P16234-2, P16234-3), PDGFRB (GenelD: 5159, UniProt P09619-1, P09619-2), PDPK1 (GenelD: 5170, UniProt 015530-1, 015530-2), RAF1 (GenelD: 5894, UniProt P04049-1, P04049-2), RALA (GenelD: 5898, UniProt P11233), RALB (GenelD: 5899, UniProt P11234-1, P11234-2, P11234-3), RASA1 (GenelD: 5921, UniProt P20936-1, P20936-2, P20936-3, P20936-4), RASA2 (GenelD: 5922, UniProt Q15283-1, Q15283-2), RASA3 (GenelD: 22821, UniProt Q14644-1, Q14644-2), RASGRF1 (GenelD: 5923, UniProt Q13972-1, Q13972-2, Q13972-3), RASGRF2 (GenelD: 5924, UniProt 014827), RHEB (GenelD: 6009, UniProt Q15382), ROCK1 (GenelD: 6093, UniProt Q13464), ROCK2 (GenelD: 9475, UniProt 075116), ROS1 (GenelD: 6098, UniProt P08922), SOS1 (GenelD: 6654, UniProt Q07889-1, Q07889-2), SOS2 (GenelD: 6655, UniProt Q07890-1, Q07890-2), STK11 (GenelD: 6794, UniProt Q15831-1, Q15831-2), STK3 (GenelD: 6788, UniProt Q13188-1, Q13188-2), STK4 (GenelD: 6789, UniProt Q 13043-1, Q 13043-2).

[0919] 5. Modulators of the Rho kinase pathway (Figure 23)

[0920] The compounds Narciclasine (CAS number 29477-83-6) (Figure 23) used in combination with an extracellular Ras directed ABP, modulates the ROCK, LIMK and cofilin signaling pathway, greatly increasing GTPase RhoA activity as well as inducing actin stress fiber formation in a RhoA-dependent manner. Narciclasine also broadly altered lipid metabolism via modulation of differential genes in pathways related to phospholipid metabolism. Therefore, any compounds inhibiting or modulating ROCK1 (Gene ID: 6093, UniProt Q13464) or ROCK2 (Gene ID: 9475, UniProt 075116) or LIMK1 (Gene ID: 3984, UniProt P53667) or LIMK2 (Gene ID: 3985, UniProt P53671) or RHOA (Gene ID: 387, UniProt P61586) can be used in combination with an extracellular Ras directed ABP of this invention.

[0921] 6. Modulators of the JAK signaling pathway (Figure 24)

[0922] The modulator compound Ruxolitinib (DrugBank ID DB08877) (Figure 24) used in combination with an extracellular Ras directed ABP, is an orally active and selective JAK1 / 2 inhibitor with IC50s of 3.3 nM and 2.8 nM in cell-free assays, and has 130-fold selectivity for JAK1 / 2 over JAK3.

[0923] Therefore, any compounds inhibiting or modulating the JAK kinase family, JAK1 (Gene ID: 3716, UniProt P23458) or JAK2 (Gene ID: 3717, UniProt 060674) or JAK3 (Gene ID: 3718, UniProt P52333) orTYK2 (Gene ID: 7297, UniProt P29597) can be used for this invention.

[0924] Examples for possible JAK inhibitors used in combination for this invention are Abrocitinib (DrugBank ID DB14973), Baricitinib (DrugBank ID DB11817), Delgocitinib (DrugBank Accession Number DB16133), Fedratinib (DrugBank ID DB12500), Filgotinib (DrugBank ID DB14845), Oclacitinib (DrugBank Accession Number DB11441), Peficitinib (DrugBank Accession Number DB11708), Pacritinib (DrugBank ID DB11697), Tofacitinib (DrugBank ID DB08895), Upadacitinib (DrugBank ID DB15091), Brepocitinib (CAS number 1883299-62-4), Cerdulatinib (DrugBank Accession Number DB15499), Momelotinib (DrugBank ID DB11763), Decernotinib (DrugBank Accession Number DB12566), Itacitinib (DrugBank Accession Number DB12154), Gandotinib (DrugBank Accession Number DB13040) and Gusacitinib (DrugBank Accession Number DB15670).

[0925] As alternative, any protein or protein complexes of JAK kinase down-stream binding partners can be targeted with a modulating or inhibiting compound. Examples are STAT1 (Gene ID: 6772, UniProt P42224), STAT2 (Gene ID: 6773, UniProt P52630), STAT3 (Gene ID: 6774, P40763), STAT4 (Gene ID: 6775, UniProt Q 14765), STAT5A(Gene ID: 6776, UniProt P42229), STAT5B (Gene ID: 6777, UniProt P51692) and STAT6 (Gene ID: 6778, P42226).

[0926] 7. Modulators of the phosphodiesterase pathway (Figure 25) The compound Anagrelide (DrugBank ID DB00261) (Figure 25) is an inhibitor of phosphodiesterase type III (PDE3) (IC50=36 nM).

[0927] Therefore, any compounds inhibiting or modulating the phosphodiesterase family, PDE1A (Gene ID: 5136, UniProt P54750), PDE1C (Gene ID: 5137, UniProt Q14123), PDE1B (Gene ID: 5153, UniProt Q01064), PDE3A(Gene ID: 5139, UniProt Q14432), PDE3B (Gene ID: 5140, UniProt Q 13370), PDE4A (Gene ID: 5141, UniProt P27815), PDE4B (Gene ID: 5142, UniProt Q07343), PDE4C (Gene ID: 5143, UniProt Q08493), PDE4D (Gene ID: 5144, UniProt Q08499), PDE2A (Gene ID: 5138, UniProt 000408), PDE5A (Gene ID: 8654, UniProt 076074), PDE11A (Gene ID: 50940, UniProt Q9HCR9), PDE6A (Gene ID: 5145, UniProt P16499), PDE6B (Gene ID: 5158, UniProt P35913), PDE6C (Gene ID: 5146, UniProt P51160), PDE10A(Gene ID: 10846, UniProt Q9Y233), PDE9A(Gene ID: 5152, 076083), PDE8A(Gene ID: 5151, UniProt 060658), PDE8B (Gene ID: 8622, UniProt 095263), PDE7A (Gene ID: 5150, UniProt Q 13946) or PDE7B (Gene ID: 27115, UniProt Q9NP56) can be used for this invention.

[0928] Examples for phosphodiesterase inhibitors which can be used for this invention are Milrinone (DrugBank ID DB00235), Amrinone (DrugBank ID DB01427), Enoximone (DrugBank ID DB04880), Tadalafil (DrugBank ID DB00820), Vardenafil (DrugBank ID DB00862), Cilostazol (DrugBank ID DB01166), Piclamilast (DrugBank ID DB01791), Rolipram (DrugBank ID DB01954), OSI-461 (DrugBank ID DB05415), Udenafil (DrugBank ID DB06267), Doxofylline (DrugBank ID DB09273), Trapidil (DrugBank ID DB09283), Pimobendan (DrugBankAccession Number DB11450), Mirodenafil (DrugBank Accession Number DB11792), PF-04447943 (DrugBank Accession Number DB11953), Vesnarinone (DrugBank Accession Number DB12082), Vinpocetine (DrugBank Accession Number DB12131), GSK-256066 (DrugBank Accession Number DB12137), Simendan (DrugBankAccession Number DB12286), MK-0873 (DrugBank Accession Number DB13029), Bucladesine (DrugBank Accession Number DB13242), Roflumilast (DrugBank ID DB01656), E-6005 (DrugBank Accession Number DB12776), Caffeine (DrugBank ID DB00201), Theophylline (DrugBank ID DB00277), Dyphylline (DrugBank ID DB00651), Pentoxifylline (DrugBank ID DB00806), Levosimendan (DrugBank ID DB00922), Dipyridamole (DrugBank ID DB00975), Papaverine (DrugBank ID DB01113), Aminophylline (DrugBank ID DB01223), Cilomilast (DrugBank ID DB03849), Ibudilast (DrugBank ID DB05266), Crisaborole (DrugBank ID DB05219), Zardaverine (DrugBank ID DB02918), Apremilast (DrugBank ID DB05676), Osoresnontrine (DrugBank Accession Number DB16274), Drotaverine (DrugBank ID DB06751), Avanafil (DrugBank ID DB06237), Sildenafil (DrugBank ID DB00203), Dovramilast (DrugBank Accession Number DB16242), Olprinone (DrugBank Accession Number DB16847), Zatolmilast (DrugBank Accession Number DB14790), Etazolate (DrugBank Accession Number DB05881), Ensifentrine (DrugBank ID DB16157).

[0929] 8. Compounds of fatty acids and cholesterol (Figure 26)

[0930] It was tested if the addition of lipids can increase the anti-tumor efficacy of the PCC04D Diabody (SEQ ID NOs: 23 and 231) in the co-culture assay (Figure 26). A mix containing different non-animal derived fatty acids including arachidonic acid (DrugBank ID DB04557), linoleic acid (DrugBank Accession Number DB14104), linolenic acid (DrugBank ID DB00132), myristic acid (DrugBank ID DB08231), oleic acid (DrugBank ID DB04224), palmitic acid (DrugBank ID DB03796) and stearic acid (DrugBank ID DB03193), as well as animal derived cholesterol (DrugBank ID DB04540) has been used (Lipid Mix). In two different tumor cell lines Panc-1 (Figure 26A) and MS-18 (Figure 26B), the addition of the Lipid mix caused a dose dependent increase in target cell killing by the PCC04D Diabody.

[0931] Therefore, different fatty acids can be used in combination therapy for this invention like shortchain fatty acids (SCFAs) with aliphatic tails of five or fewer carbons, or medium-chain fatty acids (MCFAs) with aliphatic tails of 6 to 12 carbons, or long-chain fatty acids (LCFAs) with aliphatic tails of 13 to 21 carbons, or very long chain fatty acids (VLCFAs) with aliphatic tails of 22 or more carbons. These can be saturated fatty acids or unsaturated fatty acids. Examples for different fatty acids are arachidonic acid (DrugBank ID DB04557), linoleic acid (DrugBank Accession Number DB14104), linolenic acid (DrugBank ID DB00132), myristic acid (DrugBank ID DB08231), oleic acid (DrugBank ID DB04224), palmitic acid (DrugBank ID DB03796) or stearic acid (DrugBank ID DB03193).

[0932] Further exemplary fatty acids are: Propionic acid, Valeric acid, Lauric acid, Myristic acid, Pentadecylic acid, Palmitic acid, Stearic acid, a-Linolenicacid, Eicosapentaenoic acid, Linoleic acid, Linolelaidic acid, y-Linolenic acid, Arachidonic acid, Palmitoleic acid, Oleic acid, Gondoic acid, Mead acid, Glycolic acid, Oxalic acid, Propiolic acid, pentanoic acid, pentanedioic acid, hexanoic acid, decanoic acid.

[0933] In addition, cholesterol (DrugBank ID DB04540) can be used in combination therapy for this invention. In addition, different metabolites of cholesterol can be used in combination therapy for this invention (Table 10).

[0934] Table 10: Metabolites of cholesterol and bile acids

[0935] Oxysterols

[0936] 4-beta-hydroxycholesterol (4P-OHC)

[0937] 5 alpha, 6 alpha-epoxycholesterol (a-epoxyC)

[0938] 5 beta, 6 beta-epoxycholesterol (P-epoxyC)

[0939] 5 alpha, 6 beta-epoxycholesterol (5a,6p-diOHC)

[0940] 7-hydroperoxycholesterol (7-OOHC)

[0941] 7-alpha-hydroxycholesterol (7a-OHC)

[0942] 7-alpha-hydroxy-4-cholesten-3-one (7a-OHC4)

[0943] 7-beta-hydroxycholesterol (7P-OHC)

[0944] 7-ketocholesterol (7-ketoC)

[0945] 7-dehydrocholesterol (7-DHC)

[0946] 24,25-epoxycholesterol (24,25-epoxyC)

[0947] 22-hydroxycholesterol (22-OHC)

[0948] 24-hydroxycholesterol (24 OHC)

[0949] 25-beta-hydroxycholesterol (25P-OHC)

[0950] 25-hydroxycholesterol (25-OHC)

[0951] 26-hydroxycholesterol (26-OHC)

[0952] Cholic acid 3a,7a,12a-trihydroxy-5p-cholan-24-oic acid Glycocholic acid N-(3a,7a,12a-Trihydroxy-5p-cholan-24- oyl)glycine

[0953] Taurocholic acid 2-(3a,7a,12a-Trihydroxy-5p-cholan-24- amido)ethane-1-sulfonic acid Deoxycholic acid 3a,12a-Dihydroxy-5p-cholan-24-oic acid Chenodeoxycholic acid 3a,7a-Dihydroxy-5p-cholan-24-oic acid Glycochenodeoxycholic acid N-(3a,7a-Dihydroxy-5p-cholan-24- oyl)glycine

[0954] Taurochenodeoxycholic acid 2-(3a,7a-Dihydroxy-5p-cholan-24- amido)ethane-1-sulfonic acid

[0955] Lithocholic acid 3a-Hydroxy-5p-cholan-24-oic acid

[0956]

[0957] Ursodeoxycholic acid 3a,7p-dihydroxy-5p-cholan-24-oic acid

[0958]

[0959] These fatty acids and / or cholesterol can be administrated as purified compounds. In addition, these fatty acids and / or cholesterol can be administrated through food and / or special diets.

[0960] 9. Modulation of the gut microbiome

[0961] Because fatty acids can improve the extracellular Ras directed therapy, short chain fatty acids (SCFAs) produced as bacterial metabolites of the gut microbiome can be important for this invention. This includes for example acetate (DrugBank Accession Number DB14511), propionate (DrugBank Accession Number DB19384) and butyrate (DrugBank ID DB03568). Therefore modulation of the gut microbiome can be used for this invention.

[0962] A method for this modulation is faecal microbiota transplant (FMT). FMT is the administration of stool preparation obtained from a healthy donor to a tumor patient (recipient). Another method for this modulation is faecal filtrate transplant (FFT). FTT aim is to remove the microbial cells but to retain the viral (bacteriophages) and microbial metabolites. Administrations of FMT and FFT are by special covered pills, or lower gastrointestinal endoscopy or with enema. Another method for this modulation is through special diets. Diets with plant foods (fruits, vegetables, wholegrains, legumes, and nuts) rich in naturally occurring fibres. Or fermented food, made through desirable microbial growth and enzymatic conversions of food components. Or prebiotics, defined as a substrate that is selectively utilised by host microorganisms conferring a health benefit. Or probiotics, defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host.

[0963] Another method for this modulation is phage therapy, using bacteriophages targeting specific bacteria strains in the gut. Another method for this modulation is the use of postbiotics which are functionally bioactive molecules produced by microbes that confers a health benefit on the host. Another method for this modulation is the use of specific antibiotics to reduce individual microbial strains detrimental to this invention. Another method for this modulation is the use of specific antibiotics to reduce or deplete the patients gut microbiome to improve the engraftment of the transplanted new microbiome.

[0964] Based on these data a large variety of different drugs, SMIs and other modulator compounds as well as different cellular targets and pathways for inhibition or modulation, can be used in combination therapies with extracellular Ras directed ABPs and / or ABCs of this invention. For example, modulator compounds can be (amongst others) any drug, chemical, peptide, protein, ligand and / or small molecule inhibitor. The combinations of the present invention can for example be used for humans or animals in the fields of cancer therapy, therapy of non-malignant disease, treatment of aging, in lifestyle medicine, in diagnostics and / or drug development.

[0965] The data testing many different combinations of the present invention demonstrate: (i) The extracellular Ras targeting ABP strategies can be modulated, (ii) This modulation can happen by the combination with a modulator compound, (iii) This combination therapy can increase the anti-tumor effect in a dose dependent manner, (iv) The use of a modulator compound is decreasing IC50 of the Ras targeting ABP and / or ABC.

[0966] Combinations comprising more than one modulator compound (i.e. same or different modulator compounds) and / or more than one ABP and / or ABC (same or different ABP and ABC) and / or more than one isolated nucleic acid (same or different isolated nucleic acids) comprising a sequence encoding the ABP are also possible. Such combinations can be advantageous in that they show a high anti-tumor activity.

[0967] In prior experiments, without extracellular Ras targeting ABPs, the effect of the individual modulator compounds on tumor cells was analyzed to determine a dose range, where the tested compound has reported target activity but only a small or minor negative effect on the tumor cells. If the anti-tumor effect of the compound alone is too high, a possible additional smaller benefit for the combination with extracellular Ras targeting ABP can be missed (data not shown).

[0968] ABPs and / or ABCs and modulator compounds can be used in a wide variety of concentrations. The person skilled in the art is aware how to test suitable concentrations in that the effect on the tumor cells, with or without T cells, is tested. Also, concentration gradients can be used. Toxicity assays are also standard in the art.

[0969] ABPs and / or ABCs are preferably in physiologic solutions for administration, like normal 0.9% NaCI solution, 1.3% saline solution, 0.45% half normal saline solution, 5% dextrose in water solution, lactate ringer solution, acetate ringer solution, hartmann solution, PlasmaLyte, Sterofundin ISO, ELO-M EL isoton, lonolyte, Isoplex, Gelaspan, Hextend, Tetraspan, Volulyte. The physiologic solutions have a preferred Osmolarity [mOsm L-1] between 277 and 309.

[0970] Possible routes of administration of the ABPs and / or ABCs are intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, intranodal or intrathecal. The administration can happen through fast injection (1 to 60 seconds long), slow injection (1 to 5 minutes), fast infusions (5 to 15 minutes), slow infusions (16 minutes to 8 hours) or continuous infusion (8 hours to 96 hours).

[0971] The administration of the modulator compounds can be together with the ABPs and / or ABCs administration, or before (10 minutes to 96 hours) or afterwards (10 minutes to 96 hours). The modulator compounds can also be administrated daily as continuous medication, or every two days, or three times a week, for example.

[0972] In vitro methods for detecting the expression of extracellular Ras proteins on the surface of cells

[0973] Development of diagnostic assays is essential for the safe and effective use of new therapeutics (companion diagnostics), for example to analyze before start of therapy, if the tumor cells are positive or negative for extracellular Ras expression. Also, only treating responders by testing for extracellular Ras expression before treatment, reduces costs. Such methods of the present invention are also preferably useful for large-scale and high-throughput drug, compound or targets screens.

[0974] In addition, such in vitro methods for detecting the expression of extracellular Ras proteins on cells can be used in screening assays in order to identify a compound (e.g. an SMI) or a target modulating extracellular Ras expression.

[0975] However, there are no prior art methods for detecting and / or measuring the expression of extracellular Ras proteins, in particular on the surface of living cells. There are three major challenges associated with such methods:

[0976] (i) Ras proteins are physiologically expressed at high amount in the cytosol of all nucleated cells. Extracellular Ras proteins, however, are expressed at low levels (Figure 29). This is a major risk for: (1) False positive results, if the intracellular Ras is detected and not separated from the extracellular Ras. (2) False negative results, if the detection of the intracellular Ras gives a background noise, similar or higher as the signal for the low extracellular Ras expression.

[0977] (ii) Extracellular Ras protein can also be expressed by dead or dying cells. It was shown herein that different human tumor cell lines express extracellular Ras protein on the surface of life and dead cells (Figure 29). The extracellular Ras expression on the dead cells can even be higher compared to the viable cells. In clinic and diagnostics, patient samples potentially arrive late during the day, or have to be shipped external for analysis. From the time point of extraction from the patient, the cell viability starts to deteriorate, increasing the dead or dying cell population. This is a major risk for: (1) False positive results, if the dead cell population is large and not clearly separated from the life population. (2) False negative, if the tumor cells are more susceptible to the ex vivo or shipping conditions and dye at higher rates as the surrounding healthy cells. (3) Analyzing failure if all or the majority of patient cells are dead. (iii) Regular compounds used in cell based detection methods can potentially cause false positive results even when used according to protocol (Figure 30). Fixatives used in such methods may bring Ras detection antibody inside the cells without disrupting the cell membrane or killing the cells.

[0978] Therefore, in vitro methods for the detection of extracellular Ras proteins on the surface of cells are important tools for the use of the combinations and ABPs of the present invention.

[0979] It is presently shown that using genetically modified cells expressing a small HiBiT tag fused to the amino-terminal end of KRAS or HRAS, extracellular Ras protein can be detected (Figure 31 and 34). This method is using a split-tag for protein-protein interaction. One half of the tag is expressed with Ras, the other half is added into the cell medium not able to penetrate inside the cells. Only extracellular Ras can successfully complement the split-tag halves, reconstituting the luciferase reporter for detection. Another additional important benefit of this used HiBiT tag system is, no additional washing step of the labeled cells is necessary for detection. This is a huge benefit for every large-scale and high-throughput screening assays using hundreds or thousands of plates with cells.

[0980] In the present invention, an in vitro method is provided for detecting the expression of predominantly extracellular Ras proteins on the surface of predominantly living cells selected from the group consisting of a) a method using a Ras protein labeling compound with substantially no penetration inside the cell and / or predominantly binding to the extracellular Ras protein b) a method using an agent blocking binding to intracellular Ras proteins and / or using a washing step to substantially remove binding to intracellular Ras proteins

[0981] c) a method using a Ras protein labeling compound as of a) and one or more labeling compounds binding to intracellular proteins not belonging to the Ras protein family d) a method using a dual-antigen protein-protein interaction (PPI) reporter

[0982] e) a method using cells expressing an altered Ras protein, preferably a Ras-tag fusion protein, wherein the tag can be detected on the extracellular side of the cell, optionally wherein the tag requires a second tag to be detectable (split-tag PPI)

[0983] f) a method using an ABP as defined herein

[0984] g) a method using 2D, 3D cell culture and / or spheroid or organoid cultures and / or stem cell-based embryo-like structures

[0985] h) a method combining two or more of a) to g).

[0986] The term “predominantly” in the sense of the present invention means “the most part of” or “most of”. Hence, “predominantly living cells” means that most cells are living cells. Detecting the expression of predominantly extracellular Ras shall have the meaning that for the most part, the detected Ras protein is extracellular.

[0987] An in vitro method is also provided for detecting modulation of cell activity induced by binding of ABPs or compounds to extracellular Ras on the surface of predominantly living cells selected from the group consisting of

[0988] a) a method using an agent to detect a cytokine level

[0989] b) a method using at least one electrode to detect a change in cells’ electrical potential c) a method using genetically modified cells with a reporter plasmid to detect and / or measure activation of a signaling cascade

[0990] d) a method combining two or more of a) to c).

[0991] Further, an in silico method is provided for detecting modulation of extracellular Ras on the surface of artificial cells selected from the group consisting of

[0992] a) a method using artificial cell membranes b) a method using artificial intelligence for calculating the extracellular Ras interface with small molecule compounds, with other proteins, with the lipid cell membrane and / or with the glycocalyx.

[0993] Tag

[0994] In another embodiment, labeling compounds against altered Ras protein can be used in an in vitro detection method. Altered in the sense of cellular Ras is preferably man-made genetically modified on the genomic and / or protein level. Altered preferably means a tag fused to a Ras protein to result in a Ras-tag fusion protein. In one embodiment, cells with a specific tag cloned into their genome are used. The tag is for example integrated in one or more of the Ras gene coding sequences. In another embodiment, the tag is integrated outside the Ras gene coding sequences. On the protein level the tag may then interact with Ras protein forming heterodimers.

[0995] The tag may be modified to be active only on the extracellular side. For example the tag has a protease cleaving site, specific for intracellular proteases. Or the tag has a binding site for proteasomal degradation in the cytosol. Or the tag is connected via a protease cleaving site to a cap protein which is protecting the tag from binding to a reporter, and the specific protease is added and / or present only in the extracellular solution.

[0996] In one embodiment, the tag is a small split-tag for PPI with no own reporter activity. One taghalf may be expressed with the Ras protein, the second tag-half may be expressed with a cell membrane receptor or linked to a reporter compound added into the cell medium and stay extracellular. Only for extracellular Ras protein there may be a tag-tag complementation and a positive signal from the reporter.

[0997] For example, this could be the HiBiT tag or SpyTag system (Nano-Gio® HiBiT) (SEQ ID NOs: 245 to 249). HiBiT is a small, 11-amino-acid, epitope tag (SEQ ID NO: 245) capable of complementation with the LgBiT partner (SEQ ID NO: 248). Alternative tags are also possible (SEQ ID NOs: 261 to 289).

[0998] The tag can be a split-protein for PPI with own reporter activity like fluorescent, luminescent or esterase. One half may be expressed with the Ras protein, the second half may be added into the cell medium or expressed by an extracellular protein. Only for the extracellular Ras there may be a complementation of the split-reporter, giving a signal for positive cells. Examples are split luciferase (1 / 2luc) complementation, or bimolecular luciferase complementation (BiLC) or split-GFP, split-RFP or split-YFP fluorescent protein complementation. Or a combination of complementation of split luciferase (CSL) and bioluminescence resonance energy transfer (BRET) approaches. Or a split BS2 esterase.

[0999] In some embodiments, reconstituted signals are driven by the colocalization of Ras protein with split-fragments on the cell surface. To improve the colocalization, the second fragment not specific for Ras protein, preferably has a specific targeting domain for the extracellular cell membrane, for example a domain against extracellular receptors, a ligand for a receptor or a domain against the cell glycocalyx.

[1000] The expression "comprise", as used herein, besides its literal meaning also includes and specifically refers to the expressions "consist essentially of" and "consist of". Thus, the expression "comprise" refers to embodiments wherein the subject-matter which "comprises" specifically listed elements does not comprise further elements as well as embodiments wherein the subject-matter which "comprises" specifically listed elements may and / or indeed does encompass further elements. Likewise, the expression "have" is to be understood as the expression "comprise", also including and specifically referring to the expressions "consist essentially of" and "consist of". The term "consist essentially of", where possible, in particular refers to embodiments wherein the subject-matter comprises 20% or less, in particular 15% or less, 10% or less or especially 5% or less further elements in addition to the specifically listed elements of which the subject-matter consists essentially of.

[1001] Optimizing methods for detecting extracellular Ras (Figures 33 and 34)

[1002] In the present invention, a culture model was used to test the impact of different protein linkers, connecting the isolated hypervariable regions (HVR) from two KRAS splice variants (SEQ ID NOs: 1 and 2) with the HiBiT tag (SEQ ID NO: 245). This was necessary because prior art reports the HVR of KRAS is sufficient for plasma membrane targeting of Ras proteins, but fail therein detecting Ras on the extracellular side of the plasma membrane

[0016] , It is presently shown that using a standard eukaryote vector pCEP4, expressing both major KRAS splice variants KRAS4a and KRAS4b HVR domains with an N-terminal HiBiT tag attached with no linker (SEQ ID NOs: 412 and 417), extracellular Ras protein is not detected (Figures 33A, B, C). Using a modified version of the pCEP4 vector, carrying 73 to 105 nucleotides of the respective 5’ and 3’ untranslated regions (UTR) of the two KRAS splice variants mRNA (SEQ ID NOs: 416 and 421), caused a small but detectable increased of extracellular expression for both KRAS4a and KRAS4b HVR domains with a HiBiT tag directly connected (Figure 33A). Using a short linker (SEQ ID NOs: 413 and 418) or a longer stiff linker forming an alpha-helix secondary structure (SEQ ID NOs: 415 and 420), increased the extracellular expression only for the KRAS4a splice variant (SEQ ID NOs: 418 and 420) in AsPC-1 cells (Figure 33A). In contrast, using a long and flexible glycine-serine linker (SEQ ID NOs: 414 and 419) increased significantly the extracellular expression for both KRAS splice variants in malign and nonmalign cells (Figure 33A, B, C). This is important for any method, using a tag fused to a cytoplasmic protein containing a PH domain and / or one or more PTM lipidation motifs. If the wrong linker is used, no target antigen can be detected on the extracellular cell membrane. Another additional important finding is that different splice variants of the same gene (SEQ ID NOs: 414 and 419) can be differently high expressed on the extracellular plasma membrane. For using gene array mRNA expression data for diagnostics or target discovery, this means not only the absolute gene expression is important, but also the expression of individual splice variants of the gene. Furthermore, any drug or compound modulating the expression of individual splice variants of a gene, can increase or decrease the extracellular expression of target antigens on the cell membrane and can be used in combination with the ABPs and ABCs of this invention for treating malign or non-malign disease.

[1003] Another important discovery is, the extracellular expression of cytoplasmic proteins HVR domains, containing a PH domain and / or one or more PTM lipidation motifs, is also a relevant mechanism in non-malign cells (Figures 33C and 34). This means for improving the safety of the ABPs and ABCs of this invention

[1004] a) binding to mutated antigen on malign cells with higher affinity and / or avidity as to the non-mutated antigen expressed on non-malign bystander cells will provide a significant benefit

[1005] b) or local activation in the malign tissue to a functional drug or compound will provide a significant benefit

[1006] c) or a combination with a second drug or compound, increasing or stabilizing the extracellular target antigen expression on malign cells and / or downregulating the expression on non-malign cells, will provide a significant benefit d) or a combination of 2 or more different ABPs and / or ABCs, targeting single rare antigens not expressed on non-malign cells and only on sub-clones of malign-cells but in combination on a wide malign population, will provide a significant benefit

[1007] e) or injecting them direct into the malign tissue or organ where the target cell population is located, will provide a significant benefit

[1008] An additional significant benefit of this discovery is, the ABPs and ABCs of this invention can be used for treating non-malign disease or stimulating healthy cells.

[1009] It is presently shown that cytoplasmic expressed full length KRAS4a wild type protein (SEQ ID NO: 444), with an N-terminal HiBiT-tag, is also detected on the extracellular surface of different malign and non-malign cells (Figure 34). These data validate the method and findings (Figures 33, 35, 36, 37, 38 and 39), the isolated plasma membrane attachment domains from cytosolic proteins, containing a PH domain and / or one or more PTM lipidation motifs, are sufficient to investigate their potential to relocate cytoplasmic proteins on the extracellular side of the plasma membrane of a cell.

[1010] Large screening assays with isolated protein domains for extracellular expression (Figures 35 to 38)

[1011] In the present invention, a culture model was used to test isolated protein domains with reported or predicted plasma membrane attachment motifs, containing one or more PTM lipidation motifs, from different human, animal and plant cells, for their extracellular expression on the plasma membrane of malign and non-malign cells.

[1012] Important is the finding, also the HVR domains of human HRAS and NRAS proteins (SEQ ID NOs: 426 and 427) are expressed on the extracellular side of the plasma membrane in different malign and non-malign cells (Figure 35). This will improve the treatment of leukemias and lymphomas, where the NRAS and HRAS genes are more expressed and more often mutated as the KRAS gene.

[1013] The three Ras proteins (KRAS, NRAS and HRAS) carry their HVR plasma membrane attachment motifs at the C-terminal end. Therefore, it is of great importance to discover also N-terminal located plasma membrane attachment motifs of different cytosolic proteins, cause extracellular expression on the cell membrane (Figure 36). This is the case for the human Abl 1 gene, a non-receptor tyrosine-protein kinase (SEQ ID NO: 428) and the human Bcr gene, a small GTP-binding protein with an intrinsic kinase activity (SEQ ID NO: 429). This is important, because aberrant regulation of tyrosine kinases has been linked to the onset and progression of a variety of diseases, including cancer, autoimmune disorders, and neurological disorders. For example the BCR:: ABL1 fusion protein, with a constitutively active Abl1 tyrosine kinase which signals thru multiple pathways which promote uncontrolled cell proliferation and survival, is found in most people with chronic myelogenous leukemia (CML), and in some people with acute lymphoblastic leukemia (ALL). If the N-terminal located plasma membrane attachment motif of Bcr is relocating the oncogenic BCR:: ABL1 protein on the extracellular side of the cell membrane, it can be targeted with ABPs or ABCs of this invention for cancer cell destruction. Another important finding is the extracellular expression of the N-terminal PM attachment domains from the two genes Fyn and Blk (SEQ ID NOs: 430 and 431), two closely related tyrosine kinases that belong to the Src-family kinases, a subset of non-receptor tyrosine kinases that play pivotal roles in diverse cellular signaling pathways. For instance, the involvement of Fyn has been established in the advancement of various cancer forms, such as breast cancer and glioblastoma. The oncogene Blk is expressed in T-cell and B-cell malignancies and in melanoma cells. Therefore, expression of Blk and Fyn on the membrane of malignant cells opens new ways for treatment with ABPs or ABCs of this invention. The nonreceptor tyrosine kinase Jak2 (SEQ ID NO: 432) is expressed at low levels on the extracellular plasma membrane. Jak2 is an attractive target for myeloid malignancies, often mutated in myeloid proliferative neoplasms. Another important finding is the extracellular expression of Src domain (SEQ ID NO: 433). Src a non-receptor protein tyrosine kinase, participating in signaling pathways that control a diverse spectrum of biological activities including gene transcription, immune response, cell adhesion, cell cycle progression, apoptosis, migration, and transformation. Although mutations in Src are rare, both overexpression and overactivation of Src have been observed in numerous cancer types, including those of the brain, mainly glioblastoma, as well as cancer of the liver, lung, colon, breast, bladder and pancreas cells what opens new ways for treatment with ABPs or ABCs of this invention.

[1014] Another important finding is a similar extracellular expression of N-terminal and C-terminal plasma membrane attachment domains, containing one or more PTM lipidation motifs, from different cytosolic proteins of animal origin (Figure 37). This is true for the Arl8 gene from drosophila melanogaster (fruit fly), an N-terminally PTM lipidation modified Arf-like GTPase (SEQ ID NO: 434). True for the Let-60 gene from caenorhabditis elegans, a C-terminally PTM lipidation modified GTP-binding protein with GTPase activity (SEQ ID NO: 435). True for the Rap1 gene from drosophila melanogaster, a C-terminally PTM lipidation modified Ras protein with intrinsic GTPase activity (SEQ ID NO: 436). True for the Ras64B gene from drosophila melanogaster, a C-terminally PTM lipidation modified Ras protein with intrinsic GTPase activity (SEQ ID NO: 437). And also true for the Ras85D gene from drosophila melanogaster, a C-terminally PTM lipidation modified Ras protein with intrinsic GTPase activity (SEQ ID NO: 438). This finding allows the ABPs and ABCs of this invention to be used on animal cells

[1015] For treating malign or non-malign disease in animals

[1016] For methods of diagnostics in animals

[1017] For killing a specific species of vertebrae animals

[1018] For killing a specific species of insects

[1019] For stimulating animal cells for agriculture purpose or food production

[1020] For stimulating animal cells for recombinant protein production

[1021] Another major important finding is a similar extracellular expression of plasma membrane attachment domains, containing one or more PTM lipidation motifs, from different cytosolic proteins of plant origin (Figure 38). This is true for the ARAC4 gene from arabidopsis thaliana (Mouse-ear cress), a C-terminally PTM lipidation modified Rho GTPase (SEQ ID NO: 439). True for the ARAC8 gene from arabidopsis thaliana (Mouse-ear cress), a C-terminally PTM lipidation modified Rac-like GTP-binding protein (SEQ ID NO: 440). True for the RAC1 gene from oryza sativa subsp. japonica (Rice), a C-terminally PTM lipidation modified Rac-like GTP-binding protein (SEQ ID NO: 441). True for the RAC4_1 gene from zea mays (maize), a C-terminally PTM lipidation modified Rac-like GTP-binding protein 4 (SEQ ID NO: 442). And also true for the RAB5A gene from oryza sativa subsp. japonica (Rice), a C-terminally PTM lipidation modified Ras-related GTP-binding protein (SEQ ID NO: 443). This finding allows the ABPs and ABCs of this invention to be used on plant cells

[1022] For stimulating plants and algae for agriculture purpose and food production

[1023] For stimulating plants and algae for botanical work

[1024] For stimulating plants and algae for recombinant protein, peptide or amino acid production

[1025] For stimulating and algae for production of biofuels and organic precursors For killing specific species of plants and algae

[1026] Dose dependent modulation of extracellular Ras expression with small molecule inhibitors (Figure 39)

[1027] In the present invention, a culture model was used to test the impact of small molecule inhibitors, not binding directly to Ras antigen but instead inhibiting an essential oncogene important for the activation of different signaling cascades, on the extracellular expression levels of Ras. For this, the human CM L cell line K562 was transfected with the human KRAS4a HVR domain connected to the HiBiT-tag (SEQ ID NO: 419) or the human KRAS4b HVR domain connected to the HiBiT-tag (SEQ ID NO: 414) and treated for a short amount of time with different Abl1 inhibitors over a large dose range, to block enzyme functions but not to induce apoptosis and the extracellular KRAS expression was analyzed.

[1028] Important is the finding for the inhibitor Asciminib, a STAMP inhibitor specifically targeting the ABL myristoyl pocket with very low inhibition of other kinase, inducing extracellular KRAS upregulation at high dose only for the KRAS4a transcript variant and in contrast only at low dose for the KRAS4b transcript variant (Figure 39A). Important is also the finding for Dasatinib, an inhibitor of the Abl1 kinase with much larger co-inhibition of other kinase like Src family kinases, KIT and PDGFR, inducing extracellular KRAS upregulation only at lower dose for both transcript variants (Figure 39B). This finding allows

[1029] Small molecule inhibitors to be used for upregulation of target antigens of this invention, in particular kinase inhibitors or tyrosine kinase inhibitors

[1030] In silico methods of this invention to be used, analyzing the entire or major target inhibition profiles of small molecule inhibitors over a large dose range, for the identification or development of drugs to be used for extracellular target antigen upregulation of this invention.

[1031] An additional significant implication of this discovery is, the need to closely monitor the serum or plasma concentrations of small molecule inhibitors used to upregulate the extracellular target antigen expression of this invention, in the patients. For many drugs, under continuous constant application, their serum concentration is not fixed. Instead, kidney function, liver function, serum albumin concentration or cytochrome P450 inhibition or activation by other comedications or food is modulating small molecule inhibitor serum levels. Especially for inhibitors, upregulating extracellular target antigen expression of this invention only at very low concentrations, there is a need for monitor their serum or plasma concentrations. Small molecule Ras inhibitors linked to albumin can be used as ABCs for killing cells or cells 40 to

[1032] In the present invention, a chemical synthesis method was used to generate novel fusion ABCs targeting extracellular Ras on the surface of cells. For this, different small molecule inhibitors (SMI) with reported binding and inhibition of KRAS protein were linked to human serum albumin (SEQ ID NO: 445) using click chemistry, creating a fusion molecule consisting of a SMI covalent connected via chemically functionalized polyethylene glycol (PEG) linkers to albumin (Figure 40A). Two PEG linkers were used, a short C27 PEG10 linker and a longer C55 PEG24 linker. Important is the finding for the KRAS_G12D_inhibitor_3, a KRAS G12D inhibitor with an IC50 of <500 nM, linked to albumin with anti-tumor efficacy against AsPC-1 cells for fusion-ABCs using the PEG 10 and PEG24 linkers (Figure 40B), and no effect was reported for albumin fused with the PEG10 or PEG24 linker without inhibitor against AsPC-1 cells (Figure 40C).

[1033] Important is also the finding for the ARS- 1323 inhibitor, a covalent inhibitor that covalently binds to the Switch-ll pocket (S-IIP) of the KRAS G12C mutant protein, linked to albumin with antitumor efficacy against K562 cells for fusion-ABCs using only the PEG24 linker (Figure 41A), and no effect was reported for albumin fused with the PEG10 or PEG24 linker without inhibitor against K562 cells (Figure 41 B).

[1034] Important is also the finding for the pan-KRAS-IN-2 inhibitor, a pan-inhibitor with IC50S < 10 nM for KRAS WT and mutants, linked to albumin with anti-tumor efficacy against MOLM-13 cells for fusion-ABCs using the PEG10 and PEG24 linkers (Figure 42A), and for the ARS-1323 inhibitor linked to albumin with anti-tumor efficacy against MOLM-13 cells for fusion-ABCs using only the PEG24 linker (Figure 42B), and no effect was reported for albumin fused with the PEG10 or PEG24 linker without inhibitor against MOLM-13 cells (Figure 42B).

[1035] Important and surprising is the finding for the pan-KRAS-IN-2 inhibitor linked to albumin with a positive stimulatory effect against Raji cells for fusion-ABC using the PEG24 linker (Figure 43A), and for the MRTX1133 inhibitor, a selective alkyne-based KRAS G12D inhibitor, linked to albumin with a positive stimulatory effect against Raji cells for fusion-ABC using the PEG24 linker (Figure 43B), and for the KRAS_G12D_inhibitor_3 linked to albumin with a positive stimulatory effect against Raji cells for fusion-ABC using the PEG10 linker in sharp contrast to the anti-tumor efficacy of the inhibitor alone (Figure 43C), and for the ARS-1323 inhibitor linked to albumin with a positive stimulatory effect against Raji cells for fusion-ABC using the PEG10 linker and with an anti-tumor efficacy for fusion-ABC using the PEG24 linker (Figure 43D), and no effect was reported for albumin fused with the PEG10 or PEG24 linker without inhibitor against Raji cells (Figure 43E).

[1036] Important is also the finding for the MRTX1133 inhibitor linked to albumin with a positive stimulatory effect against THP-1 cells for fusion-ABC using the PEG24 linker in sharp contrast to the anti-tumor efficacy of the inhibitor alone (Figure 44A), and no effect was reported for albumin fused with the PEG10 or PEG24 linker without inhibitor against THP-1 cells (Figure 44B).

[1037] Important is also the finding for the pan-KRAS-IN-2 inhibitor linked to albumin with anti-tumor efficacy against MS-18 cells for fusion-ABCs using the PEG10 and PEG24 linkers (Figure 45A), and no effect was reported for albumin fused with the PEG10 or PEG24 linker without inhibitor against MS-18 cells (Figure 45B).

[1038] ABP against extracellular Ras can stimulate cell migration (Figure 46)

[1039] In the present invention, a cell-culture model was used to test the effect of an ABP of this invention against viable cells in the absence of effector cells at different physiological and non-physiological oxygen concentrations. The oxygen concentrations for the majority of healthy human tissues are in the range of 3 to 10%, the median tumor oxygen concentrations are <2%, and the room oxygen concentration in regular cell culture is 21%. For this, AsPC-1 cells were cultured at 21%, 8% and 1% oxygen concentrations alone or together with the Ras binder PCC06 (SEQ ID NOs: 25, 127, 175 to 178, 233), and the cell migration was measured over 9 days. Important is the finding, the ABC increased cell migration at all three oxygen concentrations tested (Figure 46).

[1040] Extracellular Ras antigen is expressed on viable patient cells (Figure 47)

[1041] In the present invention, a FACS methode was used to investigate the expression of Ras antigen on the surface of primary patient cells. Important is the finding, Ras antigen is expressed on fresh isolated viable cells from a chronic myelomonocytic leukemia (CMML) patient whose malign cells have mutations in the genes CSF3R, DNMT3A, RLINX1 and TP53 (Figure 47A), and also Ras antigen is expressed on fresh isolated viable cells from a patient with myeloproliferative neoplasia (MPN) whose cells have mutations in the genes ASXL1, EZH2, JAK2 and NRAS (Figure 47B). DESCRIPTION OF THE FIGURES

[1042] 1A shows the Diabody format used in this invention All Diabodies use the same anti- CD3 binding variable-heavy (VH) and variable light (VL) chain domains (black filled ovals) from the UCHT1 clone (SEQ ID NO: 49) targeting the epsilon chain of the human CD3 receptor complex on T cells (SEQ ID NOs: 49, 53, 54, 55, 59, 60, 61, 64 and 65). The anti-CD3 VLand VH domains are connected via a flexible 20 amino acid long GS linker (grey solid lines) to the Ras ABP (white ovals). Every Diabody has a 6 His tag (SEQ ID NO: 33) connected via a two amino acid GS linker to the C-terminal end (black dotted line). Each Diabody has a different Ras specific ABP (PCC_01D to PCC_012D) (SEQ ID NOs: 20 to 31). The PCC01_D, PCC02_D and PCC03_D are using as Ras ABP the variable-heavy (VH) and variable light (VL) chains from Ras binding antibodies, connected via a 15 amino acid long flexible GS linker (black solid line). The PCC06_D and PCC07_D are using as Ras ABP a FN3 monobody domain. The PCC04_D, PCC05_D and PCC08D to PCC012_D are using as Ras ABP the isolated domains from human proteins with known Ras interaction.

[1043] 1B shows an overview of the predicted binding sites to Ras protein for all 12 ABPs (PCC01 to PCC012) used in this invention. On the bottom is the Ras secondary structure with the different domains. The amino acids are numbered. Hypervariable region (HVR) is important for attachment to the membrane. The black square filled boxes represent the ABP binding sites. Because of the complex tertiary and quaternary structure of Ras, most ABPs have contact with different domains of Ras. The binding site for PCC08 (containing the SOS2-CDC25H domain) was predicted to be similar to the binding site of PCC05 (containing the SOS1-CDC25H domain), due to lack of structural data for the SOS2-KRAS complex, but high similarity between the two CDC25H domains (86% for amino acids 780 - 1019). The binding site for PCC09 (containing the RASGRP3-CDC25 domain), PCC011 (containing the RASGRP1-CDC25 domain) and PCC012 (containing the RASGRP2-CDC25 domain) was predicted to be similar to the binding site of PCC05 (containing the SOS1-CDC25H domain), due to lack of structural data for the RASGRP3-, RASGRP2- and RASGRP1-KRAS complexes. For RASGRP1 the CDC25 domain consists of a compact bundle of 10 helices that forms the core of the structure. Two antiparallel and tightly packed helices that form a prominent hairpin protrude from this core. The structure of nucleotide-free Ras bound to the SOS-CDC25 domain showed that the switch 2 region of Ras docks on the helical bundle of the CDC25 domain. Based on the structural similarity of the CDC25 domains, empty Ras is expected to bind the CDC25 domain of RasGRPI. For RASGRP3 and RASGRP2 the binding to HRAS or NRAS is reported, but it is weaker compared to the binding to other members of the Ras family.

[1044] 2 shows expression of HLA- and HLA-I I complex on the surface of different wild type (WT) and HLA knock-down (KO) tumor cells using Flow cytometry. Figure 2A shows MM1. S wild type cells (top row) and MM1. S cells with HLA-I knock-down (bottom row). HLA-I (light blue, middle column) and HLA-II (red, right column) is highly expressed on the wild type cells. In contrast in the HLA-I knock-down cells, the HLA-I expression is significantly reduced, but not the HLA-II expression. Figure 2B vice versa in MM1. S HLA-II knock-down cells (top row), the HLA-II expression (red, right column) is completely lost, but not the HLA-I expression (light blue, middle column). In HLA-I and HLA-II double knock-down cells (bottom row), the HLA-I expression (light blue, middle column) is significantly reduced and the HLA-II expression completely lost (red, right column). Figure 2C shows MV-4-11 wild type cells (top row) and MV-4-11 HLA-II knock-down cells (bottom row). HLA-I (light blue, middle column) and HLA-II (red, right column) is highly expressed on the wild type cells. In contrast in the HLA-II knock-down cells, the HLA-II expression is completely lost, but not the HLA-I expression. Figure 2D shows K562 wild type cells (top row) and K562 HLA-I knock-down cells (bottom row). HLA-I (light blue, middle column) is highly expressed on the wild type cells but not HLA-II (red, right column). In contrast in the HLA-I knock-down cells, the HLA-I expression is significantly reduced and the HLA-II negativity unchanged. Figure 2E shows in the K562 HLA-II knockdown cells (top row), the negative HLA-II expression (red, right column) is not changed, and the HLA-I expression (light blue, middle column) stays the same. In K562 HLA-I and HLA-II double knock-down cells (bottom row), the HLA-I expression (light blue, middle column) is significantly reduced and the negative HLA-II expression unchanged (red, right column).

[1045]

[1046] and 5 shows in vitro killing in regular 2D cell culture for the different wild type (WT) and HLA knock down (KO) cell lines, analyzed in Figures 2 with Flow cytometry, using two different? cell activating Diabodies PCC04D (SEQ ID NO: 231) and PCC06D (SEQ ID NO: 233) against extracellular Ras. The Diabodies were utilized in a concentration gradient from 30 nM to 0.014 nM, using 1 to 3 dilution steps. The CD8+ T cell to tumor cell (E: T) ratio was 5 to 1. The viability of the luciferase positive tumor cells was measured after 48 hours incubation. Figure 3A shows extraordinary and high killing activity of a construct (PCC04D) against different HLA positive and negative MM1. S cells according to this invention. Against the wild type cells (black circle, solid line), against the HLA-I knock-down cells (white upright triangle, black spotted line), against the HLA-II knock-down cells (white upside-down triangle, black spotted line) and against the HLA-I+II double knock-down cells (white diamond, black spotted line). Importantly, these results show for the anti Ras directed ABPs of this invention, their binding and T cell engaging function is not restricted to HLA alleles presented on the tumor cell surface by the MHC. These results clearly indicate that said extracellular Ras antigen is not part of the HLA-peptide complex. Figure 3B shows analogous experiment to Figure 3A with extraordinary and high killing activity of a construct (PCC06D) against different HI_A positive and negative MM1. S cells in low nanomolar range. Against the wild type cells (black spot, solid line), against the HLA-I knock-down cells (white upright triangle, black spotted line), against the HLA-II knock-down cells (white upside-down triangle, black spotted line) and against the HLA-I+II double knock-down cells (white diamond, black spotted line). Again, these results show for the anti Ras directed ABPs of this invention, their binding and T cell engaging function is not restricted to HLA alleles presented on the tumor cell surface by the MHC. Figure 4A shows extraordinary and high killing activity of a construct (PCC04D) against HLA positive and negative MV-4-11 cells according to this invention. Against the wild type cells (black circle, solid line) and against the HLA-II knock-down cells (white upside-down triangle, black spotted line). Figure 4B shows analogous experiments to Figure 4A with extraordinary and high killing activity of a construct (PCC06D) against HLA positive and negative MV-4-11 cells according to this invention. Against the wild type cells (black circle, solid line) and against the HLA-II knockdown cells (white upside-down triangle, black spotted line). Again, these results show for the anti Ras directed ABPs of this invention, their binding and T cell engaging function is not restricted to HLA allel presented on the tumor cell surface by the MHC. These results clearly indicate that said extracellular Ras antigen is not part of the HLA-peptide complex. Figure 5 shows extraordinary and high killing activity of a construct (PCC04D) against different HLA positive and negative K562 cells according to this invention. Against the wild type cells (black circle, solid line), against the HLA-I knock-down cells (white upright triangle, black spotted line), against the HLA-II knock-down cells (white upside-down triangle, black spotted line) and against the HLA-I+II double knock-down cells (white diamond, black spotted line). Interestingly, the tumor cell killing was even higher in the HLA knock-down cells compared to the wild type cells in higher nM range. Importantly, these results again show for the anti Ras directed ABPs of this invention, their binding and T cell engaging function is not restricted to HLA alleles presented on the tumor cell surface by the MHC. These results clearly indicate that said extracellular Ras antigen is not part of the HLA-peptide complex. 6A shows an illustration of the regular 2D cell culture (top) used in the experiments depicted in Figures 3 to 5, 29 and 30, and the 3D co-culture assay (bottom) used in the experiments depicted in Figures 7 to 28. In regular 2D cell culture tumor cells (white cells with black spots) are mixed with healthy T cells (round grey cells). In 3D co-culture different nonmalign cells (grey cells) are mixed with the tumor cells. Also in the co-culture assay is methylcellulose (grey mesh) to form a semi-solid medium, and Corning Matrigel® a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma, a tumor rich in extracellular matrix proteins, including laminin, collagen IV, heparan sulfate proteoglycans and entactin / nidogen. Figure 6B shows human pancreatic cancer cell line Panc-1 in regular 2D cell culture medium (left), growing adherent mostly in monolayer with a spindle shape form. The Panc-1 cells growing in 3D co-culture medium (right), together with healthy human HLIAEC and NHDF cells, forming cell clusters.

[1047]

[1048] 7 to 25 and 27 to 28 show the extraordinary benefit for a combination therapy of different small molecule inhibitors (SMI) with anti Ras directed construct of this invention. For this, the 3D co-culture assay was used with human CD8 positive T cells, the Diabody PCC04D (SEQ ID NO: 231) and different human tumor cells (AsPc-1, Panc-1 and MS18). The Diabody was used in a low nM concentration gradient. Different controls were used to distinguish the anti-tumor effects of the respective single components concerning the Diabody, the SMI and the T cells, from the anti-tumor effect of the respective Diabody and SMI combination therapy. For this, the SMI effect on the tumor cells was analyzed at two different concentrations (horizontal dotted line, SMI low concentration only) (horizontal solid line, SMI high concentration only). The effect of a Diabody PCC04D (SEQ ID NO: 231) monotherapy against the tumor cells was also analyzed (grey dotted line, PCC_04 only). Tumor cells with CD8+ T cells (cells + T cells) and tumor cells only (cells only) were used as negative control to exclude possible T-cell response against cell line neoantigens. 6% DMSO in cell culture medium was used as positive control for tumor cell killing (cells + 6% DMSO). For the combination therapy, the Diabody was used in the same concentration gradient as in the PCC04 monotherapy together with the respective SMI at the same concentration as in the SMI only groups (black solid line with filled black rectangle, combination Diabody and SMI high concentration) (black dotted line with filled black circle, for combination Diabody and SMI low concentration).

[1049] 7A shows in AsPc-1 co-culture, for ATRA with PCC04D a significant increase in antitumor activity for the combination therapy, using ATRA in low concentration (black dotted line with filled black circle). ATRA low concentration (400 nM) and high concentration (2 pM). Importantly, these results show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with an ATRA based compound. Figure 7B shows analogous experiments to Figure 7A in Panc-1 co-culture, for ATRA with PCC04D a significant increase in anti-tumor activity for the combination therapy, using ATRA in high concentration (black solid line with filled black rectangle). ATRA low concentration (400 nM) and high concentration (2 pM). Importantly, these results again show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with an ATRA based compound.

[1050] 8A shows in AsPc-1 co-culture, for Simvastatin with PCC04D a significant increase in anti-tumor activity for the combination therapy, using Simvastatin in high concentration (black solid line with filled black rectangle). Simvastatin low concentration (2 pM) and high concentration (10 pM). Importantly, these results show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with a Statin based compound.

[1051] 8B shows analogous experiments to Figure 8A in Panc-1 co-culture for Simvastatin with PCC04D a significant increase in anti-tumor activity for the combination therapy, using Simvastatin in high concentration (black solid line with filled black rectangle). Simvastatin low concentration (2 pM) and high concentration (10 pM). Importantly, these results again show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with a Statin based compound.

[1052] 9A shows in AsPc-1 co-culture, for Fluvastatin with PCC04D a significant increase in anti-tumor activity for the combination therapy, using Fluvastatin in high concentration (black solid line with filled black rectangle). Fluvastatin low concentration (200 nM) and high concentration (1 pM). Importantly, these results show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with a Statin based compound.

[1053] 9B shows analogous experiments to Figure 9A in Panc-1 co-culture, for Fluvastatin with PCC04D a significant increase in anti-tumor activity for the combination therapy, using Fluvastatin in high concentration (black solid line with filled black rectangle). Fluvastatin low concentration (200 nM) and high concentration (1 pM). Importantly, these results again show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with a Statin based compound. 10 shows in AsPc-1 co-culture, for Lapaquistat with PCC04D at high concentration (4nM) a significant increase in anti-tumor activity for the combination therapy, using Lapaquistat in high concentration (black solid line with filled black rectangle) and low concentration (black dotted line with filled black circle). Lapaquistat low concentration (2 pM) and high concentration (10 pM). Importantly, these results show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with a Statin based compound.

[1054] 11 shows in AsPc-1 co-culture, for Bemfivastatin with PCC04D a significant increase in anti-tumor activity for the combination therapy, using Bemfivastatin in high concentration (black solid line with filled black rectangle) and low concentration (black dotted line with filled black circle). Bemfivastatin low concentration (2 pM) and high concentration (10 pM). Importantly, these results show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with a Statin based compound.

[1055] 12 shows in AsPc-1 co-culture, for Lenalidomide with PCC04D a significant increase in anti-tumor activity for the combination therapy, using Lenalidomide in high concentration (black solid line with filled black rectangle) and low concentration (black dotted line with filled black circle). Lenalidomide low concentration (2 pM) and high concentration (10 pM). Importantly, these results show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with a cereblon (CRBN) or E3 ligase based compound or inhibitor.

[1056] 13A shows in AsPc-1 co-culture, for Iberdomide with PCC04D a significant increase in anti-tumor activity for the combination therapy, using Iberdomide in high concentration (black solid line with filled black rectangle) and low concentration (black dotted line with filled black circle). Iberdomide low concentration (4 pM) and high concentration (20 pM). Importantly, these results show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with a cereblon (CRBN) or E3 ligase based compound or inhibitor.

[1057] 13B shows analogous experiments to Figure 13A in Pane- 1 co-culture for Iberdomide with PCC04D at high concentration (4nM) a significant increase in anti-tumor activity for the combination therapy, using Iberdomide in high concentration (black solid line with filled black rectangle) and low concentration (black dotted line with filled black circle). Iberdomide low concentration (4 pM) and high concentration (20 pM). Importantly, these results again show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with a cereblon (CRBN) or E3 ligase based compound or inhibitor.

[1058] 14 shows in AsPc-1 co-culture for HOMO-Protac cereblon degrader 1 with PCC04D at high concentration (4nM) a significant increase in anti-tumor activity for the combination therapy, using HOMO-Protac cereblon degrader 1 in high concentration (black solid line with filled black rectangle) and low concentration (black dotted line with filled black circle). HOMO-Protac cereblon degrader 1 low concentration (2 pM) and high concentration (10 pM). Importantly, these results show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with a cereblon (CRBN) or E3 ligase based compound or inhibitor.

[1059] 15 shows in AsPc-1 co-culture for Eragidomide with PCC04D a significant increase in anti-tumor activity for the combination therapy, using Eragidomide in high concentration (black solid line with filled black rectangle) and low concentration (black dotted line with filled black circle). Eragidomide low concentration (20 nM) and high concentration (100 nM). Importantly, these results show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with a cereblon (CRBN) or E3 ligase based compound or inhibitor.

[1060] 16 shows in AsPc-1 co-culture, for NSC-70220 with PCC04D a significant increase in anti-tumor activity for the combination therapy, using NSC-70220 in high concentration (black solid line with filled black rectangle) and low concentration (black dotted line with filled black circle). NSC-70220 low concentration (2 pM) and high concentration (10 pM). Importantly, these results show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with a Ras inhibiting or modulating compound.

[1061] 17 shows in AsPc-1 co-culture, forSAH-SOSIATFAwith PCC04D a significant increase in anti-tumor activity for the combination therapy, using SAH-SOS1ATFA in high concentration (black solid line with filled black rectangle). SAH-SOS1A TFA low concentration (4 pM) and high concentration (20 pM). Importantly, these results show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with a Ras inhibiting or modulating compound. 18A shows in AsPc-1 co-culture, for Adagrasib with PCC04D at high concentration (4nM) a significant increase in anti-tumor activity for the combination therapy, using Adagrasib in high concentration (black solid line with filled black rectangle) and low concentration (black dotted line with filled black circle). Adagrasib low concentration (600 nM) and high concentration (3 pM). Importantly, these results show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with a Ras inhibiting or modulating compound. Figure 18B shows analogous experiments to Figure 18A in MS-18 coculture, for Adagrasib with PCC04D a significant increase in anti-tumor activity for the combination therapy, using Adagrasib in high concentration (black solid line with filled black rectangle). Adagrasib low concentration (600 nM) and high concentration (3 pM). Importantly, these results again show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with a Ras inhibiting or modulating compound.

[1062] 19 shows in AsPc-1 co-culture for BI-2852 with PCC04D a significant increase in antitumor activity for the combination therapy, using BI-2852 in high concentration (black solid line with filled black rectangle) and low concentration (black dotted line with filled black circle). BI-2852 low concentration (4 pM) and high concentration (20 pM). Importantly, these results show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with a Ras inhibiting or modulating compound.

[1063] 20A shows in Panc-1 co-culture for MRTX-1133 with PCC04D at high concentration (2nM and 4nM) a significant increase in anti-tumor activity for the combination therapy, using MRTX-1133 in high concentration (black solid line with filled black rectangle). MRTX-1133 low concentration (600 nM) and high concentration (3 pM). Importantly, these results show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with a Ras inhibiting or modulating compound. Figure 20B shows analogous experiments to Figure 20A in MS-18 co-culture, for MRTX-1133 with PCC04D at high concentration (2nM and 4nM) a significant increase in anti-tumor activity for the combination therapy, using MRTX-1133 in high concentration (black solid line with filled black rectangle) and low concentration (black dotted line with filled black circle). MRTX-1133 low concentration (600 nM) and high concentration (3 pM). Importantly, these results again show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with a Ras inhibiting or modulating compound. 21 shows in AsPc-1 co-culture, for RMC-0331 with PCC04D at high concentration (2nM and 4nM) a significant increase in anti-tumor activity for the combination therapy, using RMC-0331 in low concentration (black dotted line with filled black circle). RMC-0331 low concentration (2 pM) and high concentration (10 pM). Importantly, these results show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with a Ras inhibiting or modulating compound.

[1064] 22 shows in AsPc-1 co-culture for SOS 1 -activator 1 with PCC04D a significant increase in anti-tumor activity for the combination therapy, using SOS1 -activator 1 in high concentration (black solid line with filled black rectangle) and low concentration (black dotted line with filled black circle). SOS 1 -activator 1 low concentration (400 nM) and high concentration (2 pM). Importantly, these results show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with a Ras inhibiting or modulating compound.

[1065] 23 shows in AsPc-1 co-culture, for Narciclasine with PCC04D at high concentration (2nM and 4nM) a significant increase in anti-tumor activity for the combination therapy, using Narciclasine in low concentration (black dotted line with filled black circle). Narciclasine low concentration (4 nM) and high concentration (20 nM). Importantly, these results show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with a Rho kinase pathway inhibiting or modulating compound.

[1066] 24 shows in AsPc-1 co-culture for Ruxolitinib with PCC04D a significant increase in anti-tumor activity for the combination therapy, using Ruxolitinib in low concentration (black dotted line with filled black circle). Ruxolitinib low concentration (4 pM) and high concentration (20 pM). Importantly, these results show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with a Jak signaling pathway inhibiting or modulating compound.

[1067] 25 shows in AsPc-1 co-culture for Anagrelide with PCC04D a significant increase in anti-tumor activity for the combination therapy, using Anagrelide in high concentration (black solid line with filled black rectangle) and low concentration (black dotted line with filled black circle). Anagrelide low concentration (4 pM) and high concentration (20 pM). Importantly, these results show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with a phosphodiesterase inhibiting or modulating compound. Figure 26A shows in Panc-1 co-culture, for a mixture of different lipids with PCC04D a significant increase in anti-tumor activity for the combination therapy. The lipid mix contained the fatty acids arachidonic, linoleic, linolenic, myristic, oleic, palmitic and stearic acid and cholesterol (Lipid Mixture 1, Chemically Defined, L0288, Sigma-Aldrich®), and was used in a concentration gradient from 2.0% to 0.06% (Concentration %). For this experiment two similar Panc-1 co-cultures were used, one with medium only containing the lipid mix gradient (solid line with filled black rectangles, Lipid Mix only) and one with medium containing the lipid mix gradient and a fix concentration of 5nM PCC04D (dotted line with filled black circles). The antitumor efficacy of the PCC04D as monotherapy was measured at 5nM in medium without lipid mix (small grey dotted horizontal line, 5nM PCC_04 only). Importantly, these results show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with different fatty acids and cholesterol. Figure 26B shows analogous experiments to Figure 26A in MS-18 co-culture, for a mixture of different lipids with PCC04D a significant increase in anti-tumor activity for the combination therapy. The same methods and experimental setup was used as described in Figure 26A. Importantly, these results again show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with different fatty acids and cholesterol.

[1068] Figure 27A shows in AsPc-1 co-culture, for the combination of three inhibitors (Fluvastatin, CC-90009 and MRTX-1133) with PCC04D at high concentration (1nM and 0.5nM) a significant increase in anti-tumor activity for the combination therapy (dotted line with filled black circles) compared to PCC04D monotherapy (solid line with filled black rectangle). The anti-tumor effect of the three inhibitor combination without Diabody was also analyzed (small dotted horizontal line, SMI-combi only). Fluvastatin was used with 500nM, CC-90009 with 500nM and MRTX-1133 also with 500nM. Importantly, these results show for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with more than two compounds targeting each different pathways. Figure 27B shows analogous experiments to Figure 27A in AsPc-1 co-culture, for the combination of three inhibitors (Fluvastatin, Lenalidomide, and Bl-2865) with PCC04D at high concentration (1nM and 0.5nM) a significant increase in anti-tumor activity for the combination therapy (dotted line with filled black circles) compared to PCC04D monotherapy (solid line with filled black rectangles). The anti-tumor effect of the three inhibitor combination without Diabody was also analyzed (small dotted horizontal line, SMI-combi only). Fluvastatin was used with 500nM, Lenalidomide with 500nM and BI-2865 also with 500nM. Importantly, these results show again for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with more than two compounds targeting each different pathways. Figure 27C shows analogous experiments to Figure 27A and 27B in AsPc-1 co-culture, for the combination of three inhibitors (Fluvastatin, Lenalidomide, and SOS1-activator 1) with PCC04D at high concentration (1nM and 0.5nM) a significant increase in antitumor activity for the combination therapy (dotted line with filled black circles) compared to PCC04D monotherapy (solid line with filled black rectangles). The anti-tumor effect of the three inhibitor combination without Diabody was also analyzed (small dotted horizontal line, SMI-combi only). Fluvastatin was used with 500nM, Lenalidomide with 500nM and SOS1 -activator 1 also with 500nM. Importantly, these results show again for the anti Ras directed ABPs of this invention, the increase in therapeutic efficacy in combination with more than two compounds targeting each different pathways.

[1069] 28A shows 9 different anti Ras Diabodies (SEQ ID NOs 228 to 239) against human MM1. S cells in the absence of human T cells (black bars = Antibody without T cells) and in the presence of human CD8+ T cells (grey bars = Antibody + T-cells). The Diabody concentration in the medium was 30 nM. No Diabody showed strong or moderate killing activity in the absence of T cells. But without T cells there was a strong stimulation of cells compared to untreated cells. The addition of T cells was able to abrogate this proliferative effect via tumor cell killing, for example for PCC04D, but not for all constructs. Importantly, these results indicate for the anti Ras directed ABPs of this invention, there is possible direct stimulatory effect on the target cells metabolism. Figure 28B shows analogous experiments to Figure 28A for 8 different anti Ras Diabodies (SEQ ID NOs: 228 to 239) against the murine C1498 cells in the absence of human T cells (black bars = Antibody without T cells) and in the presence of human CD8+ T cells (grey bars = Antibody + T-cells). The Diabody concentration in the medium was 30 nM. Again, no Diabody showed strong or moderate killing activity in the absence of T cells. But without T cells there was again a strong stimulation of cells compared to untreated cells. The addition of T cells was able to abrogate this proliferative effect via tumor cell killing, for example for PCC02D and PCC04D, but not for all constructs. Importantly, these results again indicate for the anti Ras directed ABPs of this invention, there is possible direct stimulatory effect on the target cells metabolism, also for different species.

[1070] 29A shows the extracellular Ras expression on HL-60 cells using Flow cytometry. To measure the expression on living and dead cells, a cell viability stain was used. The Ras expression on the dead cells (grey boxed quadrant) was higher compared to the living cells (black boxed quadrant). Importantly, these results indicate for any diagnostic test used to detect Ras expression on the cell surface, to gain on specificity it is beneficial to exclude or minimize the influence of the expression on the dead cell population. Notably, the correct binding of a Ras detection construct to its target, on a cell population not the primary focus for prognostic evaluation (dead cells), has the risk to cause false positive results for every standard and / or current diagnostic test used in clinic today. Figure 29B shows in analogous experiments to Figure 29A the extracellular Ras expression on Panc-1 cells using Flow cytometry. To measure the expression on living and dead cells, a cell viability stain was used. The Ras expression on the dead cells (grey boxed quadrant) was higher compared to the living cells (black boxed quadrant). Importantly, these results again indicate it is beneficial for any diagnostic test to exclude the Ras expression on the dead cells. Figure 29C shows in analogous experiments to Figure 29A and 29B the extracellular Ras expression on MS-18 cells using Flow cytometry. To measure the expression on living and dead cells, a cell viability stain was used. The Ras expression on the dead cells (grey boxed quadrant) was again higher compared to the living cells (black boxed quadrant). Importantly, these results again indicate it is beneficial for any diagnostic test to exclude the Ras expression on the dead cells.

[1071] 30 shows the false positive detection of extracellular Ras on K562 cells using a standard fixation method used for Flow cytometry. For this, cells were treated before antibody staining with CellCover fixative according to manufacturer’s protocol. For extracellular Ras detection (left side), the same antibody clone (Ras10) was used as in Figures 29A-C. To control for intra cellular proteins, an antibody against the cytoplasmic protein MPO was used (right side). Both antibodies were used on the freshly fixed cells with an extracellular staining protocol. To measure the expression on living cells, a cell viability stain was used. The fixation caused a significant high unspecific binding of anti Ras detection antibody to the vital cells. This is most likely caused by fixative effects against the cell membrane, not high enough to kill the cells, but high enough for binding of the Ras detection antibody to the much larger intracellular Ras population. The binding of the MPO antibody to the cells supports the detection of intracellular proteins. Importantly, these results indicate for any diagnostic test used to detect Ras expression on the cell surface, to gain on specificity it is beneficial to exclude or minimize the effect of the large intracellular Ras expression for analysis. Notably, the correct binding of a Ras detection construct to its target, on a cell population which is the primary focus for prognostic evaluation (living cells), has the risk to cause false positive results for standard and / or current diagnostic test, if steps or methods are used which are not specifically optimized for the difference in the extracellular versus intracellular Ras expression ratios. Therefore, these data again clearly indicate the need for new and specific methods to detect extracellular Ras expression by this invention.

[1072] Figure 31A shows an illustration for the novel HiBiT-tag expression method used to screen for extracellular Ras protein expression on the cell surface. Depicted is the nucleotide insertion of a Hi BiT tag into the DNA of three human RAS genes, KRAS (top), NRAS (middle) and HRAS (bottom). The DNA is shown in 5’ to 3’ orientation, with the ATG start codon (underlined), followed by the coding sequence for the amino acids (grey boxed), the point mutations used in the single-stranded oligodeoxynucleotides (ssODNs) (SEQ ID NOs: 252, 256, and 260) ordered as Ultramer® DNA Oligo (fat black) and the inserted HiBiT sequence (small cursive letters). Figure 31 B shows low expression of the KRAS-HiBiT-fusion (#1) on the cell surface of AsPc-1 cells. For this, the HiBiT tag was cloned separately into the three Ras genes, KRAS, NRAS and HRAS, using different CRISPR RNA (crRNA) (SEQ ID NOs: 250, 251, 253, 254, 255, 257, 258, and 259). Figure 31C shows in analogous experiments to Figure 31 B extraordinarily high expression of the KRAS-HiBiT-fusion (#2) on the cell surface of K562 cells. The same CRISPR RNAs (crRNA) were used as for Figure 31 B. Importantly, these results show novel detection methods using genetically modified cells, can be used to screen for extracellular Ras expression. Therefore, these data again clearly indicate the need for new and specific methods to detect extracellular Ras expression by this invention.

[1073] Figure 32A shows a possible in vitro method for detecting the expression of predominantly extracellular Ras proteins on the surface of predominantly living cells using a Ras-protein-labeling-compound (Ras-PLC) (grey pac-man circle) against extracellular Ras with substantially no penetration inside the cell. EpCAM represents a known transmembrane receptor (black square frustum), Ras proteins (grey tear-shaped). A reporter signal (white star) is directly labeled (A) to the Ras-PLC, or the reporter signal is indirectly labeled to the Ras-PLC (black arrow). Figure 32B shows another illustration of an in vitro method for detecting the expression of predominantly extracellular Ras proteins on the surface of predominantly living cells. Using in addition to the Ras-PLC (grey pac-man circle), one or more labeling-compounds against intracellular proteins not belonging to the Ras targets (grey triangles and grey hexagon), for detection of cells with damaged membranes and / or to preferably exclude them from analysis. Ras proteins (grey tear-shaped). A reporter signal (white 5 arm star) is directly labeled (A) to the Ras-PLC, and two other reporter signals (white 7 arm star and white 14 arm star) are directly labeled to the labeling-compounds against intracellular proteins not belonging to the Ras targets (B and C). Figure 32C shows another illustration of an in vitro method for detecting the expression of predominantly extracellular Ras proteins on the surface of predominantly living cells using a dual-antigen protein-protein interaction (PPI) reporter. For this, the first labeled protein is targeting Ras (grey pac-man circle) and the second labeled protein (grey triangle) is targeting the extracellular domain of a known membrane receptor, in this depicted case EpCAM (black square frustum), Ras proteins (grey tear-shaped). For detection, the reporter (white star) only gives a signal if both proteins bind to their respective targets in close proximity on the cell surface and reconstitute the reporter binding site (B-A).

[1074]

[1075] 32 D shows another illustration of an in vitro method for detecting the expression of predominantly extracellular Ras proteins on the surface of predominantly living cells using man-made genetically modified cells, expressing a Ras-tag fusion protein. Ras proteins (Ras, grey tear-shaped) fused with a tag (black dotted line). A detection compound, with preferably substantially no penetration inside the cell, will only detect extracellular Ras expression. Because there are modified tag-detection compounds with extremely high specificity and sensitivity, higher than the kd (dissociation constant) of regular antibodies, this method will be beneficial for large-scale screening assays detecting even small differences in extracellular Ras expression levels. Figure 32E shows another illustration of an in vitro method for detecting the expression of predominantly extracellular Ras proteins on the surface of predominantly living cells using man-made genetically modified cells, expressing two different tags each on a different antigen, for a split-tag PPI. One tag (black dotted line) is fused to the Ras proteins (Ras) and the second tag (grey dotted line) is fused with the extracellular part of a known cell membrane receptor, in this depicted case EpCAM (black square frustum). Only for extracellular Ras there is a tag-tag PPI complementation for the detection reporter (white star).

[1076] 33 shows expression of the two splice variants KRAS 4a (4a) (SEQ ID NOs: 417, 418, 419, 420, 421) and KRAS_4b (4b) (SEQ ID NOs: 412, 413, 414, 415, 416) isolated HVR domains (underlined), including the plasma membrane attachment motifs, connected to the HiBiT tag (black bold) (SEQ ID NO: 245) in different cell lines. For four constructs the HiBiT tag is connected direct to the Ras HVR domains (hi and hi UTR) (SEQ ID NOs: 412, 416, 417, 421). The UTR (black xxxx) stands for the untranslated mRNA sequences direct 5’ upstream of the ATG start codon, or direct 3’ downstream of the TAA stop codon for each of the KRAS transcript variants, for KRAS_4a (SEQ ID NO: 421) and KRAS_4b (SEQ ID NO: 416). In six constructs different linkers (grey box) are used to connect the HiBiT tag to the Ras HVR domains. For two constructs a short 4 amino acid long linker (h2) is used (SEQ ID NOs: 413 and 418), for two constructs a 25 amino acid long and flexible glycine-serin linker (h3) is used (SEQ ID NOs: 414 and 419), and for two constructs a 16 amino acid long but stiff linker (h4), forming an alpha-helix (grey letters), is used (SEQ ID NOs: 415 and 420). Cells were transfected with pCEP4 plasmids, expressing the different constructs, and three days later washed to exclude HiBiT tag in solution, and the extracellular cell surface HiBiT expression (grey bars) was analyzed, and the total cellular HiBiT expression in lysed cells (black bars) was analyzed. Native cells without plasmid transfection but same culture conditions were used as negative controls (native). A nested T-test two-tailed P value was used to compare the expression levels of transfected cells against untransfected cells (native). Figure 33Ain AsPC-1 tumor cells, there is a significant extracellular expression of the constructs using a long flexible GS-linker (4a_h3 and 4b_h3) and the extracellular expression for the KRAS4a transcript is higher compared to the KRAS4b transcript. A long but more stiffer linker (h4) and a short but flexible linker (h2) only increased the extracellular expression for the KRAS4a transcript (4a_h4), constructs with no linker (hi) did not show any extracellular expression for both transcript variants. Modification of the mRNA sequence, thru attaching gene splice variant specific UTR regions, did improve the extracellular expression for both transcript variants (4a_h1UTR and 4b_h1UTR).

[1077] Figure 33B in K562 leukemia cells, there is only a significant extracellular expression for the KRAS_4a transcript using a long flexible GS-linker (4a_h3) and lesser extracellular expression for the KRAS_4b transcript (4b_h3). Figure 33C in healthy human HaCaT cells, there is a significant extracellular expression for the KRAS_4a transcript using a long flexible GS-linker (4a_h3) and a lesser extracellular expression for the KRAS_4b transcript (4b_h3). Importantly, these results show for new methods to be used for detecting extracellular antigens of this invention, optimizing different parts of the methods, for example using long and more flexible linkers or optimizing the nucleotide mRNA sequence, is crucial for success. Also, different splice variants of the same gene can be significantly different expressed on the cell surface. In addition, important is the finding also non-malign cells (Figure 33C) can express Ras antigen on their cell surface, what is significant for therapy because it shows the need to optimize the ABPs and ABCs of this invention, or the methods used for their application, to reduce side effects on healthy tissues and to improve safety. But the Ras expression on healthy cells also opens new ways to treat non-malign cells with ABPs and ABCs of this invention, for example in the fields of non-malign disease or in the area of regenerative medicine. Figure 34 shows expression of the full length KRAS4a wild type protein (SEQ ID NO: 444) in different cell lines, with the HiBiT tag (SEQ ID NO: 245) connected via a long and flexible glycine-serine linker to its N-terminal end. In this linker is a short second tag included (ALFA tag) which also can be used for detection. A nested T-test two-tailed P value was used to compare the expression levels of transfected cells against untransfected cells (native). Figure 34A in all three cell lines KRAS is significantly expressed on the extracellular cell surface (grey bars) and also in the total cell lysate (black bars). This expression is higher in malign-cells (K562 and AsPC-1) compared to non-malign cells (HaCaT). Figure 34B using a WESTERN-blot method for protein detection in total cell lysates. In all three transfected cell lines the recombinant expressed full length KRAS protein (Kras4a_Full_WT) can be detected using an antibody specific for the N-terminal HiBiT tag. Native cells (K562 native cells), not transfected with plasmid, show no HiBiT tag expression. An antibody against actin was used to control for protein in the samples. A protein ladder shows the molecular weight in kilodalton (kDa). Importantly, these results confirm the extracellular expression of Ras protein is also an important incidence for the full-length protein in different malign and non-malign cell types. In addition, these results validate the expression methods, using isolated parts from proteins consisting of plasma membrane attachment domains with a PH domain and / or one or more PTM lipidation motifs, to screen for new cytoplasmic proteins with the capability to be relocated from the cytosol to the extracellular cell surface. Also important is the validation of the extracellular Ras expression in non-malign cells (HaCaT), which is lower compared to the malign cells (K562 and AsPC-1) opening a possible new therapeutic window for the ABPs and APCs of this invention.

[1078] Figure 35 shows for different cell lines, the expression of isolated HVR domains from the human HRAS gene (SEQ ID NO: 426) and the human NRAS gene (SEQ ID NO: 427) with the HiBiT tag connected via a long and flexible GS linker at their N-terminal end. As positive control the two KRAS splice variants KRAS4a_h3 and KRAS4b_h3 (SEQ ID NOs: 419 and 414) were used. To control for HiBiT tag extracellular expression, two isolated transmembrane domains from single pass cell surface receptors were used connected with the same HiBiT tag and linker, from the human BCMA gene (Gene ID: 608) with the HiBiT tag at the N-terminal end (SEQ ID NO: 422) and from the human CD38 gene (Gene ID: 952) with the HiBiT tag at the C-terminal end (SEQ ID NO: 423). To analyze if the transition thru the ER-GOLGI secretion pathway is increasing the extracellular Ras expression, the signal secretion leader of the human Histidine-rich glycoprotein (HRG) (UniProt P04196) was attached at the N-terminal end of the two KRAS splice variants KRAS4a_HRGsignal and KRAS4b_HRGsignal (SEQ ID NOs: 424 and 425). A nested T-test two-tailed P value test was used for comparing transfected cells against untransfected cells (native) * = p<0.5, ** = p<0.01, *** = p<0.001, **** = p<0.0001, ns = p>0.5 (not significant). Figure 35A in AsPC-1 cells, Figure 35B in K562 cells and Figure 35C in HaCaT cells, all four RAS proteins are significantly expressed on the cell surface (grey bars), and also for all control constructs with transmembrane domains (HiBiT_BCMA and CD38TM_HiBiT) the detection HiBiT tag is correctly expressed on the cell surface (grey bars). Importantly, these results show the extracellular expression of Ras protein is also the case for the HRAS and NRAS proteins. Important is the finding, the secretion of Ras proteins thru the ER-GOLGI complex is not increasing their extracellular expression, instead their expression is similar or even lower compared to their cytosolic expression. Again, extracellular Ras expression is observed in malign (AsPC-1 and K562) and non-malign (HaCaT) cells.

[1079] Figure 36 shows for different cell lines, the expression of isolated plasma-membrane binding domains from other human cytosolic proteins of the large RAS superfamily and the kinase family. All constructs had the HiBiT tag connected via a long and flexible GS linker at their C -terminal end, because their plasma membrane attachment domains were located at their N-terminal end in the native protein. A splice variant of the Abl1 protein (UniProt P00519-2) (SEQ ID NO: 428) was used, the Bcr protein (UniProt P11274) (SEQ ID NO: 429) was used, the Blk protein (UniProt P51451) (SEQ ID NO: 430) was used, the Fyn protein (UniProt P06241) (SEQ ID NO: 431) was used, the Jak2 protein (UniProt 060674) (SEQ ID NO: 432) was used and the Src protein (UniProt P12931) (SEQ ID NO: 433) was used. As positive control the two KRAS splice variants KRAS4a_h3 and KRAS4b_h3 (SEQ ID NOs: 419 and 414) were used. A nested T-test two-tailed P value test was used for comparing transfected cells against untransfected cells (native) * = p<0.5, ** = p<0.01, *** = p<0.001, **** = p<0.0001, ns = p>0.5 (not significant). Figure 36A in AsPC-1 cells, Figure 36B in K562 cells and Figure 36C in HaCaT cells, all the proteins are significantly expressed on the cell surface in varying levels (grey bars) and also in the total cell lysate (black bars). These are important findings, clearly confirming again human cytosolic proteins, with a N-terminal or C-terminal plasma membrane attachment domain, can be relocated from the cytosol to the extracellular side of the plasma membrane. This allows ABPs and ABCs of this invention to target proteins of the large RAS superfamily and the kinase family on the extracellular membrane of malign and non-malign cells. 37 shows for different cell lines the expression of isolated plasma-membrane binding domains from cytosolic proteins of animal origin of the RAS family. Constructs had the Hi BiT tag connected via a long and flexible GS linker at their C -terminal end (Arl8_H i BiT) or their N-terminal end (LET-60_HiBiT, Rap1_HiBiT, Ras64B_HiBiT and Ras85D_HiBiT). The Arl8 protein from drosophila melanogaster (UniProt Q9VHV5) (SEQ ID NO: 434) was used, the Let-60 protein from caenorhabditis elegans (UniProt P22981) (SEQ ID NO: 435) was used, the Rap1 protein from drosophila melanogaster (UniProt P08645) (SEQ ID NO: 436) was used, the Ras64B protein from drosophila melanogaster (UniProt P04388) (SEQ ID NO: 437) was used and the Ras85D protein from drosophila melanogaster (UniProt B3NZR4) (SEQ ID NO: 438) was used. As positive control the two KRAS splice variants KRAS4a_h3 and KRAS4b_h3 (SEQ ID NOs: 419 and 414) were used. A nested T-test two-tailed P value test was used for comparing transfected cells against untransfected cells (native) * = p<0.5, ** = p<0.01, *** = p<0.001, **** = p<0.0001, ns = p>0.5 (not significant). Figure 37A inAsPC-1 cells, Figure 37B in K562 cells and Figure 37C in HaCaT cells, all the proteins are significantly expressed on the cell surface in varying levels (grey bars) and also in the total cell lysate (black bars). These are important findings, clearly confirming also cytosolic proteins of animal origin, with a N-terminal or C-terminal plasma membrane attachment domain, can be relocated from the cytosol to the extracellular side of the plasma membrane. This allows ABPs and ABCs of this invention to target cytosolic proteins on the extracellular membrane of malign and non-malign animal cells.

[1080] 38 shows for different cell lines the expression of isolated plasma-membrane binding domains from cytosolic proteins of plant origin of the GTPase family. Constructs had the H i BiT tag connected via a long and flexible GS linker at their N-terminal end.

[1081] The Arac4 protein from arabidopsis thaliana (UniProt Q38919) (SEQ ID NO: 439) was used, the Arac8 protein from arabidopsis thaliana (UniProt Q9SU67) (SEQ ID NO: 440) was used, the Rac1 protein from oryza sativa subsp. japonica (UniProt Q9SSX0) (SEQ ID NO: 441) was used, the Rac4_1 protein from zea mays (UniProt A0A3L6DAC8) (SEQ ID NO: 442) was used and the Rab5A protein from oryza sativa subsp. japonica (UniProt Q0ILQ6) (SEQ ID NO: 443) was used. As positive control the two KRAS splice variants KRAS4a_h3 and KRAS4b_h3 (SEQ ID NOs: 419 and 414) were used. A nested T-test two-tailed P value test was used for comparing transfected cells against untransfected cells (native) * = p<0.5, ** = p<0.01, *** = p<0.001, **** = p<0.0001, ns = p>0.5 (not significant). Figure 38A inAsPC-1 cells, Figure 38B in K562 cells and Figure 38C in HaCaT cells, all the proteins are significantly expressed on the cell surface in varying levels (grey bars) and also in the total cell lysate (black bars). These are important findings, clearly confirming also cytosolic proteins of plant origin, with a plasma membrane attachment domain, can be relocated from the cytosol to the extracellular side of the plasma membrane. This allows ABPs and ABCs of this invention to target cytosolic proteins on the extracellular membrane of plant cells.

[1082] 39 shows for K562 leukemia cells, the changes of extracellular KRAS expression if the central driving oncogene BCR:: ABL1, responsible for the aberrant activation of different signaling cascades, is targeted. For this, BCR:: ABL1 positive K562 cells expressing the two KRAS splice variants KRAS4a_h3 and KRAS4b_h3 (SEQ ID NOs: 419 and 414) with the N-terminal HiBiT tag, were treated with the kinase inhibitors Asciminib, a potent and selective allosteric BCR:: ABL1 inhibitor binding to the myristoyl pocket of ABL1, and the kinase inhibitor Dasatinib, a potent ATP competitive dual Src / Bcr:: Abl1 inhibitor. Cells were only treated for a short time (10 minutes) to ensure BCR:: ABL1 inhibition but not to activate apoptosis. A nested T-test two-tailed P value test was used for comparing kinase inhibitor treated cells against kinase inhibitor untreated cells (untreated) * = p<0.5, ** = p<0.01, *** = p<0.001, **** = p<0.0001, ns = p>0.5 (not significant). To control for apoptotic cells, Kras transfected cells were treated with DMSO (DMSO-treated). Kras untransfected cells (native) were used as negative control. Figure 39A shows for the inhibitor Asciminib, at high inhibitor concentration a significant extracellular upregulation for the KRAS4a transcript (left side), but in contrast for the KRAS4b transcript (right side) no extracellular upregulation at high inhibitor concentrations but instead a high extracellular expression at low inhibitor concentrations. In apoptotic cells the extracellular KRAS expression is significantly down regulated for both KRAS transcripts. Figure 39B shows for the inhibitor Dasatinib, for KRAS4a transcript (left side) and the KRAS4b transcript (right side) only a significant upregulation of extracellular Ras at low inhibitor concentrations. This is important, clearly showing the co-treatment of malign cells with a potent drug, inhibiting central oncogenic pathways in the cells, can upregulate extracellular Ras expression at different concentrations. This is different for each inhibitor. Important is the finding, for some inhibitors a significant upregulation is only seen at low concentrations and lost at higher concentrations. This is important for drugs or compounds of this innovation for combination use with ABPs and ABCs of this innovation, to monitor the effective serum inhibitor concentrations in the patients and for some inhibitors to be used only at extreme low concentrations. Figure 40A shows on the left side on the top an illustration of the extracellular Ras targeting ABCs of this invention, using small molecule inhibitor (SMI) fused via PEG linker to the serum protein albumin. On the left side on the bottom is an illustration of the regular therapy with cell permeable SMI, binding to all Ras antigens inside and outside the cell. These novel ABCs of this innovation are redirecting the SMI efficacy from the intracellular to the extracellular space. On the right side is a SDS gel showing the produced ABCs (CC1 to CC10) with a PEG10 linker used for ligating the SMI to albumin, the ABCs (CC11 to CC12) with a PEG24 linker used for ligating the SMI to albumin, with the calculated respective concentrations, human serum albumin (HSA) as control for protein concentration, albumin only connected with the PEG10 (HSA_PEG10) or PEG 24 (HSA_PEG24) linkers and a protein ladder on the side.

[1083] Figures 40 to 45 show for various tumor and leukemia cell lines, the effect of different ABCs of this invention, consisting of an SMI ligated with a PEG10 or PEG24 linker to human serum albumin. To control for the sole SMI effect on the cells, inhibitor was used in solution (SMI). As negative control (neg.) SMI was used, treated with the same chemicals and purifications methods used for generating the ABCs, but without albumin. This control was included to ensure the observed effects of the ABCs is not caused by leftover unbound SMI in solution after the production process. Untreated cells were used to control for regular cell viability and set at 100%.

[1084] Figure 40B shows for AsPC-1 cells, treated with the compound CC7, KRAS_G12D_inhibitor 3 ligated with the PEG10 linker to albumin, and CC17, KRAS_G12D_inhibitor 3 ligated with the PEG24 linker to albumin, a target cell killing at high concentrations. No effect is observed for the negative control. Figure 40C shows for AsPC-1 cells, no effect of albumin ligated with the PEG10 linker (23-Albumin_PEG10_DMSO) and albumin ligated with the PEG24 linker (24-Albumin_PEG24_DMSO) on the cells. This is important, showing the observed effects of the ABCs is not caused by chemical modified albumin itself on the AsPC-1 cells. These are important findings, they prove a KRAS specific SMI can be ligated to a large serum protein, creating new functional ABCs of this invention, targeting extracellular Ras for target cell killing.

[1085] Figure 41A shows for K562 cells, treated with the compound CC3, ARS-1323 inhibitor ligated with the PEG10 linker to albumin, and CC13, ARS-1323 inhibitor ligated with the PEG24 linker to albumin, a dose dependent target cell killing for the ABC with the PEG24 linker (CC13). No effect is observed for the negative control. Surprisingly the SMI alone had no effect on the cells. These are important findings, they prove a KRAS specific SMI, with no significant efficacy against the target cells by itself, can obtain efficacy when ligated with proper linker to a large serum protein, creating a new functional ABC of this invention targeting extracellular Ras for target cell killing. Figure 41 B shows for K562 cells, no effect of albumin ligated with the PEG10 linker (23-Albumin_PEG10_DMSO) and albumin ligated with the PEG24 linker (24-Albumin_PEG24_DMSO) on the cells. This is important, showing the observed effects of the ABC is not caused by chemical modified albumin itself on the K562 cells.

[1086] Figure 42A shows for MOLM-13 cells, treated with the compound CC1, pan KRAS-IN-2 inhibitor ligated with the PEG10 linker to albumin, and CC11, pan KRAS-IN-2 inhibitor ligated with the PEG24 linker to albumin, a dose dependent target cell killing for both constructs. No effect is observed for the negative control. Figure 42B shows for MOLM-13 cells, treated with the compound CC3, ARS-1323 inhibitor ligated with the PEG10 linker to albumin, and CC13, ARS-1323 inhibitor ligated with the PEG24 linker to albumin, a dose dependent target cell killing for the ABC with the PEG24 linker (CC13). No effect is observed for the negative control and a minor killing effect for the SMI alone at high concentration. Figure 42C shows for MOLM-13 cells, no effect of albumin ligated with the PEG10 linker (23-Albumin_PEG10_DMSO) and albumin ligated with the PEG24 linker (24-Albumin_PEG24_DMSO) on the cells. This is important, showing the observed effects of the ABCs is not caused by chemical modified albumin itself on the MOLM-13 cells. These are important findings, providing again prove different KRAS specific SMI can be ligated to a large serum protein, creating new functional ABCs of this invention, targeting extracellular Ras for target cell killing.

[1087] Figure 43A shows for Raji cells, treated with the compound CC1, pan KRAS-IN-2 inhibitor ligated with the PEG10 linker to albumin, and CC11, pan KRAS-IN-2 inhibitor ligated with the PEG24 linker to albumin, a dose dependent stimulation of cell viability for ABC with the PEG24 linker (CC11). No effect is observed for the negative control and a minor killing effect for the SMI alone at high concentrations. These are important findings, providing prove a KRAS specific SMI ligated with the proper ligand to a large serum protein, is creating an ABCs of this invention with a novel effector function, stimulating cell viability thru binding to extracellular Ras on the target cells.

[1088] Figure 43B shows for Raji cells, treated with the compound CC5, MRTX1133 inhibitor ligated with the PEG10 linker to albumin, and CC15, MRTX1133 inhibitor ligated with the PEG24 linker to albumin, a dose dependent stimulation of cell viability for ABC with the PEG24 linker (CC15). No effect is observed for the negative control and for the SMI alone. These are again important findings, providing prove a KRAS specific SMI ligated with the proper ligand to a large serum protein, is creating an ABCs of this invention with a novel effector function, stimulating cell viability thru binding to extracellular Ras on the target cells.

[1089] Figure 43C shows for Raji cells, treated with the compound CC7, KRAS_G12D_inhibitor 3 ligated with the PEG10 linker to albumin, and CC17, KRAS_G12D_inhibitor 3 ligated with the PEG24 linker to albumin, a dose dependent stimulation of cell viability for ABC with the PEG10 linker (CC7). No effect is observed for the negative control and a dose dependent killing for the SMI alone. These are important findings, providing prove a KRAS specific SMI ligated with the proper ligand to a large serum protein, is creating an ABCs of this invention with a novel effector function, stimulating cell viability thru binding to extracellular Ras on the target cells.

[1090] Figure 42D shows for Raji cells, no effect of albumin ligated with the PEG10 linker (23-Albumin_PEG10_DMSO) and albumin ligated with the PEG24 linker (24-Albumin_PEG24_DMSO) on the cells. This is important, showing the observed effects of the ABCs is not caused by chemical modified albumin itself on the Raji cells.

[1091] Figure 44A shows forTHP-1 cells, treated with the compound CC5, MRTX1133 inhibitor ligated with the PEG10 linker to albumin, and CC15, MRTX1133 inhibitor ligated with the PEG24 linker to albumin, a dose dependent stimulation of cell viability for ABC with the PEG24 linker (CC15). No effect is observed for the negative control and a dose dependent killing for the SMI alone. These are important findings, providing again prove a KRAS specific SMI with killing function against a target cell, can be transformed into an ABC of this invention with target cell stimulating function thru the use of a proper ligand.

[1092] Figure 44B shows for THP-1 cells, no effect of albumin ligated with the PEG10 linker (23-Albumin_PEG10_DMSO) and albumin ligated with the PEG24 linker (24-Albumin_PEG24_DMSO) on the cells. This is important, showing the observed effects of the ABCs is not caused by chemical modified albumin itself on the THP-1 cells.

[1093] Figure 45A shows for MS-18 cells, treated with the compound CC1, pan KRAS-IN-2 inhibitor ligated with the PEG10 linker to albumin, and CC11, pan KRAS-IN-2 inhibitor ligated with the PEG24 linker to albumin, a dose dependent target cell killing for both constructs. No effect is observed for the negative control and a dose dependent killing for the SMI alone. These are important findings, providing again prove a KRAS specific SMI can be ligated to a large serum protein, creating a new functional ABCs of this invention, targeting extracellular Ras for target cell killing.

[1094] Figure 45B shows for MS-18 cells, no effect of albumin ligated with the PEG10 linker (23-Albumin_PEG10_DMSO) and albumin ligated with the PEG24 linker (24-Albumin_PEG24_DMSO) on the cells. This is important, showing the observed effects of the ABCs is not caused by chemical modified albumin itself on the MS-18 cells.

[1095] Figure 46 shows for AsPC-1 cells in a scratch assay, with cells growing at different oxygen concentrations, 21% O2 (black line), 8% O2 (light grey line) and 1% O2 (dark grey line), and cells growing in regular medium (square) or cells growing in medium containing the PCC_06 construct (triangle) (SEQ ID NOs: 25, 127, 175 to 178, 233), the effect on cell migration over 9 days. Under regular cell culture oxygen concentration of 21%, after 9 days the opening of the scratch area was reduced to 30% (black line with black squares) and with the PCC06 construct this area was further reduced (black line with black triangles). Under reduced oxygen concentration of 8%, after 9 days the opening of the scratch area was similar reduced to 30% (light grey line with light grey squares) and with the PCC06 construct this area was again further reduced (light grey line with light grey triangles). Under severe hypoxia conditions with 1% oxygen, after 9 days the opening of the scratch area was only reduced to 60% (dark grey line with dark grey squares) and with the PCC06 construct this area was further reduced (dark grey line with dark grey triangles). These are important findings, the binding of an ABP of this invention to target cells can stimulate cell migration under different conditions, proving evidence for a stimulatory effect of extracellular Ras binders on target cells in the absence of effector cells

[1096] Figure 47 shows the expression of Ras antigen on the surface of fresh isolated viable patient cells. For detection of Ras an anti-Ras detection antibody (PCCR10_lgGFITC) was used. Figure 47A shows on the peripheral blood cells of a patient with chronic myelomonocytic leukemia (CMML), whose malign cells have mutations in the genes CSF3R, DNMT3A, RUNX1 and TP53, extracellular Ras expression on the monocytes and polymorphonuclear neutrophils and not on the lymphocyte population.

[1097] Figure 47B shows on the peripheral blood cells of a patient with myeloproliferative neoplasia (MPN), whose malign cells have mutations in the genes ASXL1, EZH2, JAK2 and NRAS, extracellular Ras expression on the monocytes and polymorphonuclear neutrophils. An anti CD45 antibody (CD45-APC) was used to check for the expression of the hematopoietic cell surface marker CD45. These are important findings, proving prove the extracellular expression of Ras is not an artificial cell culture effect only seen on immortalized cell lines, instead Ras can also be found on the cell surface of fresh isolated leukemic cells from humans. This enables the ABPs and ABCs of this invention to be used for detecting and treating malign disease.

[1098] EXAMPLES

[1099] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the description, figures and tables set out herein. Such examples of the methods, uses and other aspects of the present invention are representative only, and should not be taken to limit the scope of the present invention to only such representative examples. Standard methods of molecular biology were used (see, e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (2001)).

[1100] The examples show:

[1101] 1. Isolation of human cells from the blood

[1102] CD8+ T cells, from healthy human donors, were used as effector cells to test the in vitro activity of the anti Ras antibodies. First, the mononuclear cells were separated from the peripheral blood through Ficoll density gradient centrifugation (Ficoll-Paque™ Plus, Cytivia) at 3000 rpm with acceleration at 5 and deceleration at 1 for 18 minutes. Afterward the separated peripheral blood mononuclear cells (PBMCs) were collected and washed with 40ml PBS. The PBMCs were then subjected to red blood cell lysis with 10 ml of Gibco’s ACK lysing buffer for 15 minutes at room temperature. Then, the PBMCs were washed once with 40 ml PBS and proceed for magnetic selection according to the user manual of the magnetic beads provided by the company (CD8+ T Cell Isolation Kit, human, Miltenyi). A negative selection was used to isolate the CD8+ T cell population from the CD8 negative PBMC cells. Briefly, the number of PBMNCs was first determined and 1x 108PBMNCs were resuspended in 400 pl of MACS buffer (PBS at pH 7.2 supplemented with 0.5% BSA and 2 mM EDTA). Then, 200 pl of MicroBeads (to deplete CD8 negative cells) was added to the PBMNCs suspension, mixed well and incubated for 15 minutes and then washed with 50 ml MACS buffer and resuspended in 500 pl of MACS buffer. The labelled PBMNC were then loaded onto their respective columns according to the cell number. All magnetic labelled CD8-negative cells were captured on to the magnetic column while the CD8-positive cells were eluted by washing the column with MACS buffer. The CD8-positive cells were then collected and cultivated in RPMI1640 supplemented with 10% (v / v) FBS, 1% (v / v) GlutaMAX™, 1% (v / v) penicillin-streptomycin-neomycin antibiotic mixture, 0.05 mM 2-mercaptoethanol and 50 lll / ml human IL-2 (# AF-200-02-100UG, ThermoFisher Scientific). In order to expand the cells, Gibco™ Dynabeads™ Human T-Activator CD3 / CD28 for T Cell Expansion and Activation was added to the CD8+ T cells with one bead to five cells ratio and the expansion was carried out for five days. After the 5 days, the CD3 / CD28 Dynabeads™ were captured and removed from the proliferating CD8 positive T cells, using a magnet. The CD8+ T cells were used directly for in vitro killing assays or cryopreserved in 50% (v / v) complete RPMI1640 supplemented with 45% (v / v) FBS and 10% (v / v) DM SO for future use.

[1103] 2. PCC antibody generation

[1104] Custom gene synthesis services including chemical synthesis was used (GeneArt®, Thermo Fisher Scientific Inc.). Each DNA plasmid had their specific restriction enzyme cloning sites at the end for subcloning into the expression plasmids. For the cloning of the Diabody constructs, a pMA-RQ plasmid was ordered containing the nucleotide sequence for the Diabody back bone with the split anti CD3 VL and VH domains (from the LICHT1 clone) already included (SEQ ID NO: 224). The 12 different Ras ABPs (PCC01 to PCC012, SEQ ID NOs: 20 to 31) were in the pCEP4 plasmid (SEQ ID NOs: 122 to 133). For sub-cloning their DNA sequences, amplification via PCR was used with specific primers for each construct (SEQ ID NOs: 251 to 274). For each PCR reaction was used: 36.25 pl water, 10 pl 5x Q5® reaction buffer, 1 pl dNTP mix (10 pM), 1.25 pl Primer-mix forward and reverse (10 pM), 1 pl ABP-DNA-plasmid, 0.5 pl Q5® High-Fidelity DNA polymerase (New England Biolabs GmbH) and with PCR cycling conditions (step-1 98°C for 1 minute, step-298°C for 15 seconds, step-360°C for 30 seconds, step-472°C for 3 minutes, step-5 is go-to step-2 for 5 more times, step-698°C for 15 seconds, step-7 66°C for 30 seconds, step-8 72°C for 3 minutes, step-9 is go-to step-6 for 38 more times, final step- 10 72°C for 3 minutes, then hold reaction at 12°C). The PCR product was loaded onto a 1% agarose gel and run for 1 hour at 80 volt. After the run, the individual bands were extracted using the MiniElute® Gel extraction kit (© QIAGEN 2013-24). Following restriction enzyme digestion of the Diabody backbone pMA-RQ plasmid and the gel extracted 12 ABPs (PCC01 to PCC012) with the FastDigest Restriction enzyme system from Thermo Scientific™. The DNA was cut with the FastDigest Ehel (Thermo Scientific™, #FD0444) and FastDigest BamHI (Thermo Scientific™, #FD0054). Then, the cut Diabody backbone was dephosphorylated using the FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific™, #EF0654). Subsequently, the ABPs were ligated into the Diabody backbone using theT4 DNA Ligase (New England BioLabs, #M0202S). For the ligation 30ng of linearized pMA-RQ vector DNA, 5 to 10ng DNA of PCC insert, 10xT4-ligase buffer and 5 U / pl T4 DNA Ligase, incubated at 16°C over night. The 5 pl ligation mixture was used for each construct for transformation of MachTI cells for plasmid preparation as descript in section (3) Production of plasmids in bacteria.

[1105] The 12 different Diabodies (according to any one of SEQ ID NOs: 228 to 239) were sub-cloned into the inducible prokaryote expression plasmid pColdIV (Takara Bio Group, #3364). The pCOLD system is using the cold-shock Protein A (cspA) promoter for expression of high-purity, high-yield recombinant protein in E. coli. The vector selectively induces target protein synthesis at a low temperature (12 to 16°C), a condition which suppresses the expression of host proteins and decreases protease activity. This results in high yields of target proteins. For higher yields and correctly folded recombinant ABP production, a bacterial expression and folding leader was used at the N-terminal domain (starting at the Vector multiple cloning site mRNA transcription ATG start codon), the trigger factor from bacterium Pseudoalteromonas distincta (UniProt F3BFD4). Only the amino acid 112 to 434 domain from the original protein (F3BFD4), without the ribosom binding site, was used in this invention (SEQ ID NO: 243). For higher purity of recombinant ABP production, the CL7 tag protein (SEQ ID NO: 45), connected via a 15 amino acid long flexible GS linker (SEQ ID NO: 40) to a 3C protease cutting site (SEQ ID NO: 47), was connected to the individual folding leaders (SEQ ID NOs: 240, 226 and 227).

[1106] To ensure the correct cloning, 20 pl (300 - 800 ng DNA) of each plasmid was send for DNA sequencing by LGC Genomics (Berlin, Germany), using the sequencing primers pCold Fwd (5‘-ACGCCATATCGCCGAAAGG-3‘; SEQ ID NO: 275) and pCold Rev (5‘-GGCAGGGATCTTAGATTCTG-3';; SEQ ID NO: 276).

[1107] 3. Production of plasmids in bacteria

[1108] The different pColdIV vectors carrying the respective individual final Diabody sequences (PCC01_D, PCC02_D, PCC03_D, PCC04_D, PCC05_D, PCC06_D, PCC07_D, PCC08_D, PCC09_D, PCC010_D, PCC011_D and PCC012_D, SEQ ID NOs: 228 to 239), were transformed separately into E. coli Maehl cells (Invitrogen, C862003). After short thawing E. coli cells on ice, 10 pL of plasmid Vector (10-100 ng DNA) was mixed with 90pL of cells by gentle pipetting and incubated for 20 minutes on ice. Afterwards, a heat-shock was performed at 42°C for 90 seconds followed by incubation on ice for 5 minutes. The cells were then transferred into 600 pL of 2xYT medium (Carl Roth, X966.3) and incubated at 37°C and 230 rpm for one hour. After a subsequent centrifugation at 13000 rpm for two minutes, 500 pL of the supernatant was discarded. The cells were resuspended in the remaining 200 pL of medium and plated out on LB (Carl Roth, 6673.1) agar plates containing a plasmid specific selection antibiotic (100 pg / mL Carbenicillin). The plates were then incubated upside down at 37°C overnight.

[1109] On the next day, a single cell colony was picked and inoculated in 5 mL of 2xYT medium (100 pg / mL Carbenicillin) and incubated at 37°C over-night. An aliquot of each over-night culture was stored at 4°C for short-term preservation. The over-night cultures were centrifuged at room temperature and 4500 g for 10 minutes after which the plasmids were isolated according to the QIAprep Spin Miniprep Kit (QIAGEN, 27115). For the elution step, 20 pl pre-warmed UltraPure Water (Invitrogen, 11538646) was added to the middle of the column membrane and incubated at room temperature for two minutes before centrifugation at 13000 rpm for three minutes. The elution step was repeated one more time to maximize the final plasmid DNA yield. To determine the concentration and purity of the plasmids, the products were analyzed photometrically at 260nm and 280 nm using the NanoDrop® 2000 (Thermo Scientific, ND-2000). The plasmids were stored at -20°C until further use. For large-scale plasmid amplifications, short-term preserved E. coli Maehl cultures, positive for the respective plasmid, were inoculated in 500 mL of 2xYT medium (100 pg / mL Carbenicillin) and incubate at 37°C and 230 rpm overnight. An aliquot (1 ml) of each overnight culture was mixed with the same volume of LB-medium (+ 30% glycerol) and stored at -80°C for long-term preservation. The overnight cultures (499 ml) were centrifuged at 5000 g and 4°C for 30 minutes after which the plasmids were isolated according to the NucleoBond Xtra Maxi Kit (Maschery Nagel, 740414.50) while considering low copy numbers. The final purified plasmids were reconstituted in UltraPure Water (Invitrogen, 11538646). To determine the concentration and purity of the plasmids, the products were analyzed photometrically at 260nm and 280 nm using the NanoDrop® 2000 (Thermo Scientific, ND-2000). The plasmids were stored at -20°C until further use. 4. Recombinant Diabody production in bacteria

[1110] For each Diabody the pColdIV plasmid was transfected into SHuffle® T7 Competent E. coli cells (New England Biolabs Inc.). Single bacteria clones were used for fermentation and production in a larger volume, using 2x YT medium supplemented with 100 pg / ml carbenicilin and 0.2% (w / v) glucose. Diabody protein production was induced adding 0.1 mM IPTG and incubated at 14°C for 18 - 20 hours. Afterward from the E. coli cell pellet the Diabodies were extracted using first a His-tag purification followed by a CL7 / IM7-purification (Trialtus Bioscience). For bacteria lysis the Emulsiflex C3 (Avestin®) was used. For this the bacteria suspension is loaded into the sample reservoir, with set air pressure to 60-80 psi, and run a total of three times thru the machine. The lysate was then centrifuged 3 times for 30-45 min at 5000 g and 4°C. The clarified supernatant was then used for His-tag purification. First the supernatant was transferred into dialysis tubes (Membra-Cel™ 14000 dalton, Carl Roth®) and dialyzed against 1* PBS (pH 7.4) at 4 °C overnight. The dialysate was then transferred into falcon tubes and incubated with Co-IMAC beads (400 pL of pure beads per 50 mL of supernatant, Agarose Bead Technology, 6BCL-QHNi-X) at 4°C overnight under constant rotation. The bead-protein suspension was then centrifuged at 800 g and 4°C for 30 minutes. The bead-protein pellet is resuspended in CO-IMAC loading buffer (50mM Na phosphate buffer (pH 7.5), 300mM NaCI, 10mM Imidazol (pH 8.0)) and centrifuged at 800 g and 4°C for 30 minutes. This was performed for two more times. Then the pellet was resuspended in 1ml Co-IMAC loading buffer and transferred onto a flow column (Themo Scientific, 29924). Afterwards, the column with the loaded beads was washed three times with 10 mL of cooled Co-IMAC loading buffer each. For the elution, 1 ml of cooled Cobalt-IMAC elution buffer (50mM Na phosphate buffer (pH 7.5), 300mM NaCI, 150mM Imidazol (pH 8.0)) was loaded while the column was closed with a stopper. After an incubation of 5 minutes, the elution fraction with the antibodies was collected. Afterward, the elution fraction was transferred into dialysis tubes (Membra-Cel™ 14000 dalton, Carl Roth®) and dialyzed against 1* PBS (pH 7.4) at 4 °C overnight. The dialysate was then transferred into a falcon tube with 200 pL of IM7-Beads added (Trialtus Bioscience®). The mix was incubated at 4°C overnight in rotation. The next day, the mixture was centrifuged at 800 g and 4°C for 20 minutes. The CL7-pellet was transferred onto a 10 mL column. The column was washed stepwise with 10 mL of different washing Buffers (Table 11). Table 11: List of buffers

[1111] Steps Buffers Components

[1112] 1 PBS 1x phosphate buffer solution pH 7.4

[1113] 2 A1 pH 8.0, 2M NaCI, 300 mM Tris, 5% Glycerol, 0.02% (w / v) sodium azide

[1114] 3 Buffer Acide pH 5.6, 40 mM citrate-phosphate, 0.02% (w / v) sodium azide 4 PBS 1x phosphate buffer solution pH 7.4

[1115] 5 Buffer Alkaline pH 10.0, 100 mM sodium carbonate, 0.02% (w / v) sodium azide 6 PBS 1x phosphate buffer solution pH 7.4

[1116] 7 Buffer Imidazole pH 8.0, 1 M NaCI, 0.02 M Tris, 5% Glycerol, 0.3 M Imidazole,

[1117] 0.02% (w / v) sodium azide

[1118] 8 A2 pH 9.0, 25 mM Tris, 10% isopropanol, 2M urea, 0.02% (w / v) sodium azide

[1119] 9 PBS 1x phosphate buffer solution pH 7.4

[1120] 10 A3 pH 8.0, 600 mM NaCI, 300 mM Tris ph 8, 0, 5% Glycerol

[1121] 11 Elution 3C pH 7.4, 3C-Protease, PBS + 300 mM NaCI

[1122]

[1123] To cleave off the bacterial-folding-tag and the CL7-tag from the N-terminal end of the Diabodies, 800 pL of Elution 3C Buffer, containing 1,25 pg / mL PreScission Protease (Trialtus Bioscience, 30-2030), was added on the column. The column was closed with a stopper, and rotated at 4°C for three hours. Afterward the column was eluted using gravity and the elution fraction with the Diabodies was saved (E1). Then, another 800 pL of Elution 3C Buffer without PreScission Protease was added to the column, incubated for three minutes, and the elution fractions were saved (E2-E7) for six more times. From the different washing and elution fractions, 21 pL was mixed with 7 pL of 4x protein loading buffer and incubated at 95°C for 5 minutes. Afterwards, the protein samples were loaded onto a 12% SDS-PAGE gel and run first at 90 volt for 20 minutes and then at 130 volt for the final 90 minutes. After the run, the gels were stained with Coomassie Blue solution for 10 minutes and afterwards washed in destaining solution for 10 minutes and then transferred into VE-water for imaging.

[1124] For each CL7-tag purified Diabody, the elution fractions with the highest purity (often excluding E1 fraction) were pooled, concentrated using Aquacide II ((Sigma Aldrich, 17851-M) tubes, and dialyzed against 1x PBS (pH 7,4) at 4°C over night. D-Trehalose was added to the Diabodies at a final concentration of 60 mM as a solvent and preservative. The Diabody solution (PBS pH 7,4 + 60 mM D-Trehalose) was sterilized through 0,2 pm filtration and kept at 4°C until further use.

[1125] After the CL7 tag purification with 3C protease digestion, the final purified Diabodies (according to any one of SEQ ID NOs: 228 to 239) have the amino acid sequence GPGT (SEQ ID NO: 277) attached at the N-terminal end. GP amino acids are left from the 3C protease cutting side (SEQ ID NO: 47) and GT amino acids are from the Kpnl restriction enzyme cloning side.

[1126] 5. Measuring PCC Diabody concentration in solution

[1127] To determine the final Diabody concentration, the Pierce™ BCA Protein Assay Kit (Thermo Scientific™, #23225) was used after the manufactures protocol. In short, protein-standard and Diabody samples were pipette into separate test tubes. Then working reagent was added to each tube, mixed and incubate at room temperature for 30-60 minutes in the dark. With a spectrophotometer set to 562 nm the absorbance of all samples was measured within 10 minutes. The average 562 nm absorbance measurement of the Blank standard was subtracted from the 562nm absorbance measurement of all other individual standards and Diabody samples, to generate a standard curve to determine the protein concentration of each Diabody sample.

[1128] 6. Testing in vitro activity of Diabodies against tumor cells in regular 2D cell culture Recombinant produced Diabodies (SEQ ID NOs: 228 to 239) were tested in cell culture against different human and murine tumor cell lines (Table 12). The tumor cells are positive for the luziferase fire fly protein, expressed by viable cells only. The tumor cells were seeded into 96-well cell culture plates (7500 cells in 50 pL per well for 150 cells per pL) in their respective media and incubated at 37°C and 5% CO2 for 24 hours. The next day, fresh human CD3+ / CD8+ T-cells were added to each well, with 50 lll / ml human IL-2 (# AF-200-02-100UG, ThermoFisher Scientific), for a final tumor cell to T cells ratio of 1 to 3 or 1 to 5. The Diabodies were added at their respective concentration in 20 pL per well. As positive control for target cell killing a DMSO solution (#472301, MERCK) with a final concentration of 5-10% (v / v) in the medium was used. As reference control, to control for unspecific donor T cell activation against the target cells, target cells only and target cells mixed with T cells was used. The plates were incubated at 37°C and 5% CO2 for 24 or 48 hours. For measuring cell viability 20 pL of 3.5 mM luciferin solution was added to each well (final concentration 0.5 mM) and the plates were incubate at 37°C for 30 minutes. Afterwards, the bioluminescence of the firefly-luciferase was measured, using a Tecan Spark plate reader (with an absorbance scan from 350 nm to 700 nm with an integration time interval of 1000 ms).

[1129] Table 12: Tumor cell lines

[1130] ATCC / Cell type

[1131] Cell Line DSMZ Medium

[1132] Number

[1133] RPMI1640 supplemented with 10% (v / v) FBS, 1% Pancreas

[1134] AsPC-1 CRL-1682 (v / v) GlutaMAX™, 1% (v / v) penicillin-streptomycin- Adenocarcinoma

[1135] neomycin antibiotic mixture

[1136] Macrophage

[1137] RPMI1640 supplemented with 10% (v / v) FBS, 1% Biphenotypic B

[1138] MV4-11 CRL-9591 (v / v) GlutaMAX™, 1% (v / v) penicillin-streptomycin- Myelomonocytic

[1139] neomycin antibiotic mixture

[1140] Leukemia

[1141] RPMI1640 supplemented with 10% (v / v) FBS, 1% B lymphoblast

[1142] MM.1S CRL-2974 (v / v) GlutaMAX™, 1% (v / v) penicillin-streptomycin- Multiple Myeloma

[1143] neomycin antibiotic mixture

[1144] DMEM / F12 supplemented with 10% (v / v) FBS, 1% Neuroendocrine

[1145] (v / v) GlutaMAX™, 1% (v / v) penicillin-streptomycin- MS18 - carcinoma NET G3 of

[1146] neomycin antibiotic mixture, 1% (v / v) Gibco™ the rectum

[1147] insulin-transferrin-selenium (ITS-G)

[1148] DMEM (4.5 g / L) supplemented with 10% (v / v) FBS, Pancreas Epithelioid

[1149] Panc-1 CRL-1469 1% (v / v) GlutaMAX™, 1% (v / v) penicillin- Carcinoma

[1150] streptomycin-neomycin antibiotic mixture RPMI1640 supplemented with 10% (v / v) FBS, 1% Promyelocytic (v / v) GlutaMAX™, 1% (v / v) penicillin-streptomycin- HL-60 CCL-240

[1151] leukemia neomycin antibiotic mixture, + 0.05 mM 2- mercaptoethanol

[1152] RPMI1640 supplemented with 10% (v / v) FBS, 1% Chronic myeloid

[1153] K562 CCL-243 (v / v) GlutaMAX™, 1% (v / v) penicillin-streptomycin- leukemia cells

[1154] neomycin antibiotic mixture

[1155] Cells from a female

[1156] ATCC: DMEM (Sigma-Aldrich # D6429) with 10% v / v FBS; C1498 mouse with acute

[1157] TIB-49™ 1% v / v PSN; 1% v / v GlutaMAX

[1158] myeloid leukemia

[1159]

[1160] RPMI1640 supplemented with 10% (v / v) FBS, 1% MOLM- Acute myeloid

[1161] ACC 544 (v / v) GlutaMAX™, 1% (v / v) penicillin-streptomycin- 13 leukemia

[1162] neomycin antibiotic mixture

[1163] RPMI1640 supplemented with 10% (v / v) FBS, 1% Jurkat ACC 282 T cell leukemia (v / v) GlutaMAX™, 1% (v / v) penicillin-streptomycin- neomycin antibiotic mixture

[1164]

[1165] 7. 3D co-culture assay

[1166] A mix of tumor cells and healthy human cells (Table 13) is seeded into 96-well cell culture plates. Per well a mix of 600 tumor cells, 600 HLIEC, 600 HDLEC, 600 NHDF and 1800 T cells in 100 pL medium. The medium consist of RPMI1640 supplemented with 10% (v / v) FBS, 1% (v / v) GlutaMAX™, 1% (v / v) penicillin-streptomycin-neomycin antibiotic mixture, 50 lll / ml human IL-2 (# AF-200-02-100UG, ThermoFisher Scientific), 10% (v / v) cultrex basement membrane extract (BME) containing laminin, collagen IV, entactin and heparin sulfate proteoglycan (#3434-050- RTU, R& D Systems®), 10% (v / v) methocult (#04434, StemCell™) and 1% lipid-mix (v / v) (Lipid Mixture 1, Chemically Defined, L0288, Sigma-Aldrich®). The coculture plates are incubated at 37°C and 5% CO2.

[1167] Table 13: Healthy human tissue cells

[1168] PromoCell Cell type

[1169] Cell Line Medium

[1170] Number

[1171] Endothelial Cell GrowthMedium MV 2 (PromoCell # C- Umbilical artery

[1172] HUAEC C- 12202 22022), Growth Medium MV 2 SupplementMix; 1% v / v Endothelial cells

[1173] PSN

[1174] Keratinocyte Growth Medium 2 (PromoCell # C-20011), HaCaT C- 12006 Keratinocytes

[1175] Growth Medium SupplementMix; 1% v / v PSN Fibroblast Growth Medium (PromoCell # C-23020), NHDF C- 12300 Fibroblast

[1176] Growth Medium SupplementMix; 1% v / v PSN Endothelial Cell GrowthMedium MV 2 (PromoCell # C- Lymphatic

[1177] HDLEC C-12217 22022), Growth Medium MV 2 SupplementMix; 1% v / v Endothelial Cells

[1178] PSN

[1179]

[1180] 8. Testing the in vitro activity of Diabodies against tumor cells in 3D co-culture At day 1 the luziferase positive tumor cells are seeded in the co-culture assay and incubated for 5 days at 37°C and 5% CO2. At day 5 Diabody PCC04D (SEQ ID NO: 231) was added (10 pL per well) at the respective concentration. Also 1800 fresh human T cells (in 10 pL pure RPMI medium per well) were added. As positive control for target cell killing a DMSO solution (#472301, MERCK) with a final concentration of 5-10% (v / v) in the medium was used. As reference control, to control for unspecific donor T cell activation against the target cells, target cells only and target cells mixed with T cells was used. The plates were incubated at 37°C and 5% CO2 for 3 more days. At day 8 the tumor cell viability was measured adding 20 pL of 3.5 mM luciferin solution to each well (final concentration 0.5 mM) and the plates were incubate at 37°C for 30 minutes. Afterwards, the bioluminescence of the firefly-luciferase was measured, using a Tecan Spark plate reader (with an absorbance scan from 350 nm to 700 nm with an integration time interval of 1000 ms).

[1181] 9. Testing in vitro the combination of PCC04D with different small molecule inhibitors

[1182] At day 1 the luziferase positive tumor cells are seeded in the co-culture assay and incubated for 5 days at 37°C and 5% CO2. At day 5 the Diabody PCC04D (SEQ ID NO: 231) was added (10 pL per well) at the respective concentration. Also 1800 fresh human T cells (in 10 pL per well) were added. For inhibitor treatment, the respective SMI were resuspended in pure RPMI medium and 20 pL was added per well. For combination of two or more SMI, the respective inhibitors were combined before in medium and added in 20 pL per well. Most SMI were ordered from commercial companies, and most SMI were resuspended in DMSO for stock concentrations and stored at -80°C. SMIs ordered from MedChem Express® are ATRA (HY-14649), Simvastatin (HY-17502), Fluvastatin (HY-14664), Lapaguistat (HY-14925), Bemfivastatin (HY-106281), Lenalidomide (HY-A0003), Iberdomide (HY-101291), HOMO-PROTAC cereblon degrader 1 (HY-111594), Eragidomide (HY-130800), Adagrasib (HY-130149), BI-2852 (HY-126247), MRTX-1133 (HY-134813), RMC-0331 (HY-134885), SOS1 activator 1 (HY-111671), Narciclasine (HY-16563), Ruxolitinib (HY-50856), Anagrelide hydrochloride (HY-B0523A), NSC-70220 (HY-101796), SAH-SOS1A TFA (HY-P2265A).. The SMI SAH-SOS1A TFA was dissolved in pure H2O. As positive control for target cell killing, DMSO (#472301, MERCK) at a final concentration of 5-10% (v / v) in the medium was used. As reference control, to control for unspecific donor T cell activation against the target cells, target cells only and target cells mixed with T cells was used. The plates were incubated at 37°C and 5% CO2 for 3 more days. At day 8 the tumor cell viability was measured adding 20 pL of 3.5 mM luciferin solution to each well (final concentration 0.5 mM) and the plates were incubated at 37°C for 30 minutes. Afterwards, the bioluminescence of the firefly-luciferase was measured, using a Tecan Spark plate reader (with an absorbance scan from 350 nm to 700 nm with an integration time interval of 1000 ms).

[1183] 10. Testing the expression of Ras protein on the cell surface with flow cytometry

[1184] The day before, the tumor cells in regular 2D-culture were washed 1x with PBS and resuspended in their respective medium without FBS (serum starving). The next day, 10-14 hours before the flow cytometry analysis (FACS) the cells are returned in regular medium with FBS. Two hundred thousand cells were used for each flow cytometry sample. For adherent cell lines, cell culture medium was first removed and the cells were washed one time with PBS. After washing, 1 ml of 0.5% Trypsin-EDTA (Gibco, #25300054) was added to trypsinise the cells for 3 min at 37°C. Then, 9 ml of complete medium was added to stop trypsinisation process. Suspension cells were directly taken from the cell culture medium. Cell number of each sample was determined via Trypan Blue staining and a total of 2 X 106cells were harvested and washed one time with ice-cold PBS. The cells were then resuspended into 1 ml of ice-cold FACS buffer (PBS supplemented with 2% FBS) and blocked with 10 pl of human FcR Blocking Reagent (MACS Miltenyi #130-059-901) for 10 min at 4°C. The cells were then aliquoted into a 96-well round bottom plate with 100 pl per well. For antibody staining, 1 pl of primary antibody (1 to 100 dilution) was added per sample. For detecting Ras protein on the cell surface the anti-Ras antibodies (Ras10 clone, Invitrogen #MA1-012) was used, or primary labeled PCC04D Diabody (10). For detecting human HLA-I and HLA-II antigens on the cell surface the HLA-I-APC (MACS Miltenyi #130-120-569) and the HLA-II-PE (MACS Miltenyi #130-120-784) antibodies. For detecting intra cellular myeloperoxidase (MPO), a direct labeled antibody was used (Beckman Coulter®, #B23132). To detect the dead cell population, 1 pl of cell viability stain was added per sample. Two viability stains were used, 7-AAD (Biolegend®, #420404) or Zombie NIR™ (BioLegend®, #423105). Cells were incubated for 30 minutes at 4°C in the dark. Then, cells were pelleted through centrifugation at 1500 rpm for 5 min at 4°c and washed with 5 ml of FACS buffer for 3 times. After last wash, cells were resuspended into 100 pl FACS buffer. For detecting the Ras10 antibody clone, 1 pl of the secondary antibodies (FITC anti-mouse IgG (Biolegend #406001) was added into their respective wells and incubated f...

Claims

CLAIMS1. Combination ofan antigen binding protein (ABP) or an isolated nucleic acid comprising a sequence encoding the ABP anda modulator compoundfor use as medicament, wherein the ABP comprises at least a first antigen binding domain capable of binding to an extracellular Ras antigen and wherein the modulator compound is selected from the group consisting ofa) an All-trans-retinoic acid (ATRA) based modulator,b) a modulator of the cholesterol and triglyceride synthesis or metabolism, preferably Simvastatin, Fluvastatin, Lapaquistat, or Bemfivastatinc) an immunomodulatory drug targeting cereblon, preferably Lenalidomide, Iberdomide, HOMO-Protac cereblon degrader 1, or Eragidomide,d) a modulator of monomeric GTP-binding proteins, preferably the Ras family, preferably the Ras signaling complex, preferably NSC-70220, SAH-SOS1A TFA, Adagrasib, BI-2852, MRTX-1133, RMC-0331 or SOS 1 -activator 1,e) a kinase modulator, preferably of the Rho kinase pathway, preferably Narciclasine, f) a tyrosine kinase modulator, preferably of the JAK signaling pathway, preferably Ruxolitinib,g) a modulator of esterases, preferably of the phosphodiesterase pathway, preferably Anagrelide,h) a lipid, preferably a lipid part of a cell membranei) a fatty acid, preferably a saturated fatty acid or unsaturated fatty acidj) a modulator of the gut microbiome, andk) an inhibitor of the naphthylisoquinoline alkaloid class.l) a combination of two or more of a) to k).

2. The combination for use according to claim 1, wherein said extracellular Ras antigen is not part of, or is not restricted to, a human leukocyte antigen (HLA)-peptide complex, or specific HLA alleles, presented on the cell surface by the major histocompatibility complex (MHC).

3. The combination for use according to claim 1 or 2, wherein said ABP comprises one or more additional antigen binding domain(s) that is capable of binding to antigen(s) present on a mammalian T-cell, preferably a human cluster of differentiation 3 (CD3) antigen or a human T cell receptor (TCR).

4. The combination for use according to any one of claims 1 to 3, wherein the first antigen binding domain comprises an amino acid sequence having a sequence identity of at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% sequence identity, to an amino acid sequence selected from SEQ ID NO: 20 to 31, 66 to 71, 134 to 178 and 290 to 396, or, in each case independently, optionally with no more than ten, nine, eight, seven, six, five, four, preferably no more than three, two or one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.

5. The combination for use according to any one of claims 1 to 4, wherein the isolated nucleic acid is comprised in an expression construct, preferably further comprising promoter and / or terminator sequences.

6. The combination for use according to claim 5, wherein the isolated nucleic acid and / or the expression construct is comprised in a recombinant host cell.

7. The combination for use according to any one of claims 1 to 6, wherein the ABP is PCC04D diabody (SEQ ID NOs 23 and 231).

8. The combination for use according to any one of claims 1 to 7, wherein the ABP enhances a cell-mediated immune response, such as the immune response mediated by an activated cytotoxic T-cell (CTL) to a mammalian cell expressing said extracellular Ras antigen.

9. The combination for use according to any one of claims 1 to 8, wherein the ABP is selected from the group consisting of an immunoglobulin molecule, such as an IgG, IgE, IgD, IgA, or IgM immunoglobulin, preferably an IgG immunoglobulin, a monoclonal antibody, a chimeric antibody, a bispecifc ABP, such as a bispecific antibody, a CDR- grafted antibody, a humanized antibody, a single domain antibody, such as a VHHsingle domain antibody, a hemibody antibody, a single-chain KeyLock-antibody, a diabody, a single chain diabody, a variable domain of the antibody heavy chain or antibody light chain, a multispecific antibody, a Chimeric Antigen Receptor (CAR), alternative protein binders including monobodies (derived from fibronectin type III), anticalins (derived from lipocalins), affibodies (derived from immunoglobulin-binding protein A), DARPins (Designed Ankyrin Repeat Proteins), proteins with repeating motifs like leucine-rich repeats (LRRs), ankyrin repeats (ARs), Armadillo repeats (Arms), tetratricopeptide repeats (TPRs), and / or a fragment of an antibody, such as a fragment of a monoclonal antibody, for example a single chain Fv (scFv), (scFv)2, a Fv, a disulfide linked Fv, Fab, Fab', F(ab')2 or a scFv-Fc, preferably wherein said ABP is a bispecific antibody or a diabody.

10. The combination for use according to any one of claims 1 to 9, wherein the ABP comprises at least one antigen binding domain capable of binding with one, two, three, four, or preferable more than five amino acids to a Ras antigen, wherein the at least one antigen binding domain is alone or in a bipartite complex with another Ras binding protein, or in a tripartite complex with another Ras binding protein and the membrane, or in a multipartite complex with one or more Ras binding proteins and / or the membrane, and wherein said amino acids in the ABP optionally comprise:(i) a domain comprising the RBD-CRD region (amino acids 52 to 188) of the human RAF1 protein as set forth in SEQ ID NO: 23,(ii) optionally wherein one or multiple amino acids as depicted in Tables A and B are in contact with KRAS residues, or wherein one or multiple amino acids as depicted in Table C are in contact with the membrane, or wherein one or multiple amino acids as depicted in Table D are in contact with KRAS residues in a tripartite complex comprised of RBD-CRD, KRAS and the membrane, or(iii) a domain comprising the CDC25H region (amino acids 780 to 1019) of the human SOS1 protein as set forth in SEQ ID NO: 24, optionally wherein one or multiple amino acids as depicted in Table E are in contact with KRAS residues, or(iv) a domain comprising the CDC25 region (amino acids 1038 to 1270) of the human RASGRF1 protein as set forth in SEQ ID NO: 29, optionally whereinone or multiple amino acids as depicted in Table F are in contact with Ras residues in a tripartite complex with Ras, Sos1 and RasGRF1, or(v) a domain comprising the RAS binding region (amino acids 274 to 364) of the human RASSF5 protein (UniProt Q8WWW0-1) as set forth in SEQ ID NO: 377, optionally wherein one or multiple amino acids are in contact with RAS residues, or(vi) a domain comprising the RAS binding region (amino acids 201 to 363) of a splice variant of the human RASSF5 protein (UniProt Q8WWW0-2) as set forth in SEQ ID NO: 378, optionally wherein one or multiple amino acids are in contact with RAS residues, or(vii)a domain comprising the RAS binding region (amino acids 201 to 363) of the human RASSF1 protein (UniProt Q9NS23-1) as set forth in SEQ ID NO: 379, optionally wherein one or multiple amino acids are in contact with RAS residues, or(viii) a domain comprising the RAS binding region (amino acids 176 to 264) of the human RASSF2 protein (UniProt P50749-1) as set forth in SEQ ID NO: 380, optionally wherein one or multiple amino acids are in contact with RAS residues, or(ix) a domain comprising the RAS binding region (amino acids 6 to 89) of the human RASSF7 protein (UniProt Q02833-1) as set forth in SEQ ID NO: 381, optionally wherein one or multiple amino acids are in contact with RAS residues, or(x) a domain comprising the RAS binding region (amino acids 19 to 91) of the human ARAF protein (UniProt P10398-1) as set forth in SEQ ID NO: 382, optionally wherein one or multiple amino acids are in contact with RAS residues, or(xi) a domain comprising the RAS binding region (amino acids 19 to 148) of the human ARAF protein (UniProt P10398-1) as set forth in SEQ ID NO: 383, optionally wherein one or multiple amino acids are in contact with RAS residues, or(xii)a domain comprising the RAS binding region (amino acids 151 to 232) of the human BRAF protein (UniProt P15056) as set forth in SEQ ID NO: 384, optionally wherein one or multiple amino acids are in contact with RAS residues, or(xiii) a domain comprising the RAS binding region (amino acids 151 to 320) of the human BRAF protein (UniProt P15056) as set forth in SEQ ID NO: 385, optionally wherein one or multiple amino acids are in contact with RAS residues, or(xiv) a domain comprising the RAS binding region (amino acids 56 to 131) of the human RAF1 protein (UniProt P04049-1) as set forth in SEQ ID NO: 386, optionally wherein one or multiple amino acids are in contact with RAS residues, or(xv) a domain comprising the RAS binding region (amino acids 1235 to 1451) of the human NF1 protein (UniProt P21359-1) as set forth in SEQ ID NO: 387, optionally wherein one or multiple amino acids are in contact with RAS residues, or(xvi) a domain comprising the RAS binding region (amino acids 748 to 942) of the human RASA1 protein (UniProt P20936-1) as set forth in SEQ ID NO: 388, optionally wherein one or multiple amino acids are in contact with RAS residues, or(xvii) a domain comprising the RAS binding region (amino acids 302 to 512) of the human RASA4 protein (UniProt 043374-1) as set forth in SEQ ID NO: 389, optionally wherein one or multiple amino acids are in contact with RAS residues, or(xviii) a domain comprising the RAS binding region (amino acids 938 to 1130) of the human RGS12 protein (UniProt 014924-1) as set forth in SEQ ID NO: 390, optionally wherein one or multiple amino acids are in contact with RAS residues.

11. The combination for use according to any one of claims 1 to 10, wherein the ABP comprises at least one antigen binding domain capable of binding to an extracellular Ras antigen, preferably provided that said extracellular Ras antigen is not part of, or is not restricted to, a HLA-peptide complex, or specific HLA alleles presented on the cell surface, and wherein said Ras binding ABP comprises a diabody format as set forth in SEQ ID NO: 228 to 239, wherein in each case independently comprise a sequence having no more than three or two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to SEQ ID NO: 224, and 228 to 239, respectively; or comprising a sequence having at least 75% sequence identity, preferably at least 80% sequence identity, more preferably at least 90% sequence identity, and most preferably at last 95% sequence identity with SEQ ID NO: 224, and 228 to 239.

12. The combination for use according to any one of claims 1 to 11 for use in a method of diagnosis, prevention and / or treatment of a proliferative disorder, such as cancer, and / or for use in adoptive, target-cell specific immunotherapy and / or for use in drug development and / or for use in a method of diagnosis, prevention and / or treatment of a non-malignant disease.

13. Pharmaceutical composition for use as medicament comprising the combination according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier, stabilizer and / or excipient.

14. Composition comprisingan antigen binding protein (ABP) or an isolated nucleic acid comprising a sequence encoding the ABP anda modulator compound,wherein the ABP comprises at least a first antigen binding domain capable of binding to an extracellular Ras antigen and wherein the modulator compound is selected from the group consisting ofa) an All-trans-retinoic acid (ATRA) based modulator,b) a modulator of the cholesterol and triglyceride synthesis or metabolism, preferably Simvastatin, Fluvastatin, Lapaquistat, or Bemfivastatinc) an immunomodulatory drug targeting cereblon, preferably Lenalidomide, Iberdomide, HOMO-Protac cereblon degrader 1, or Eragidomide,d) a modulator of monomeric GTP-binding proteins, preferably the Ras family, preferably the Ras signaling complex, preferably NSC-70220, SAH-SOS1A TFA, Adagrasib, BI-2852, MRTX-1133, RMC-0331 or SOS 1 -activator 1,e) a kinase modulator, preferably of the Rho kinase pathway, preferably Narciclasine, f) a tyrosine kinase modulator, preferably of the JAK signaling pathway, preferably Ruxolitinib,g) a modulator of esterases, preferably of the phosphodiesterase pathway, preferably Anagrelide,h) a lipid, preferably a lipid part of a cell membranei) a fatty acid, preferably a saturated fatty acid or unsaturated fatty acidj) a modulator of the gut microbiome, andk) an inhibitor of the naphthylisoquinoline alkaloid class.l) a combination of two or more of a) to k).

15. A kit comprisingan antigen binding protein (ABP) or an isolated nucleic acid comprising a sequence encoding the ABP anda modulator compound,wherein the ABP comprises at least a first antigen binding domain capable of binding to an extracellular Ras antigen and wherein the modulator compound is selected from the group consisting ofa) an All-trans-retinoic acid (ATRA) based modulator,b) a modulator of the cholesterol and triglyceride synthesis or metabolism, preferably Simvastatin, Fluvastatin, Lapaquistat, or Bemfivastatinc) an immunomodulatory drug targeting cereblon, preferably Lenalidomide, Iberdomide, HOMO-Protac cereblon degrader 1, or Eragidomide,d) a modulator of monomeric GTP-binding proteins, preferably the Ras family, preferably the Ras signaling complex, preferably NSC-70220, SAH-SOS1A TFA, Adagrasib, BI-2852, MRTX-1133, RMC-0331 or SOS 1 -activator 1,e) a kinase modulator, preferably of the Rho kinase pathway, preferably Narciclasine, f) a tyrosine kinase modulator, preferably of the JAK signaling pathway, preferably Ruxolitinib,g) a modulator of esterases, preferably of the phosphodiesterase pathway, preferably Anagrelide,h) a lipid, preferably a lipid part of a cell membranei) a fatty acid, preferably a saturated fatty acid or unsaturated fatty acidj) a modulator of the gut microbiome, andk) an inhibitor of the naphthylisoquinoline alkaloid class.l) a combination of two or more of a) to k).

16. An in vitro 3D cell culture model comprising tumor cells and at least one non-malignant cell selected from the group comprising or consisting of a fibroblast, an endothelial cell of an arterial blood vessel, an endothelial cell of a venous blood vessel, an endothelial cell of a lymphatic vessel, a tissue macrophage, a fat cell, an osteoblast, a chondrocyte, a smooth muscle cell, a preadipocyte, a pericyte, a mesenchymal stem cell, a melanocyte, a keratinocyte, hematopoietic progenitors, a dendritic cell, a skeletal muscle cell, a T cell and a B cell, preferably wherein the at least non-malignant cell is a T cell, a fibroblast and an endothelial cell.

17. The in vitro 3D cell culture model according to claim 16, further comprising the combination according to any one of claims 1 to 12 or the ABP as defined in any one of claims 1 to 12 for testing the influence of the combination on the viability and extracellular Ras expression of the at least one tumor cell.

18. An in vitro method for detecting the expression of predominantly extracellular Ras proteins on the surface of predominantly living cells selected from the group consisting ofa) a method using a Ras protein labeling compound with substantially no penetration inside the cell and / or predominantly binding to the extracellular Ras proteinb) a method using an agent blocking binding to intracellular Ras proteins and / or using a washing step to substantially remove binding to intracellular Ras proteinsc) a method using a Ras protein labeling compound as of a) and one or more labeling compounds binding to intracellular proteins not belonging to the Ras protein familyd) a method using a dual-antigen protein-protein interaction (PPI) reportere) a method using cells expressing an altered Ras protein, preferably a Ras-tag fusion protein, wherein the tag can be detected on the extracellular side of the cell, optionally wherein the tag requires a second tag to be detectable (split-tag PPI)f) a method using an ABP as defined in any one of claims 1 to 12g) a method using 2D, 3D cell culture and / or spheroid or organoid cultures and / or stem cell-based embryo-like structuresh) a method combining two or more of a) to g).

19. An in vitro method for detecting modulation of cell activity induced by binding of ABPs or compounds to extracellular Ras on the surface of predominantly living cells selected from the group consisting ofa) a method using an agent to detect a cytokine levelb) a method using at least one electrode to detect a change in cells’ electrical potentialc) a method using genetically modified cells with a reporter plasmid to detect and / or measure activation of a signaling cascaded) a method combining two or more of a) to c).

20. An in silico method for detecting modulation of extracellular Ras on the surface of artificial cells selected from the group consisting ofa) a method using artificial cell membranesb) a method using artificial intelligence for calculating the extracellular Ras interface with small molecule compounds, with other proteins, with the lipid cell membrane and / or with the glycocalyx.

21. An antigen binding compound (ABC) comprising a small molecule inhibitor (SMI) covalent connected directly or via a PEG linker to a carrier compound for targeting an extracellular antigen on human or animal cells, wherein the SMI is selected from the group consisting ofa) A KRas, or HRas, or NRas inhibitorb) A pan-Ras inhibitorc) A mutant specific KRas, or HRas, or NRas inhibitord) A mutant specific pan-Ras inhibitore) A small GTPase inhibitor, preferably of the Ras family, preferably Pan-KRAS- IN-2, MRTX-EX 185, ARS-1323, CP-609754, MRTX1133, KRAS_G12D_inhibitor 1, KRAS_G12D_inhibitor 3, KRAS_G12D_IN_1, KRAS_G12D_IN_2, YL-17231, RMC-6236, RSC-1255, LY4066434, QTX3034, PF-07934040, BI-3706674, QLC1101, TSN1611, LY3962673, QTX3046, GFH375, ASP3082, INCB161734, RMC-9805, HRS-4642, YL-15293, Sotorasib, RMC-6291, MK-1084, LY3537982, LY3499446, JNJ-74699157, JDQ443, JAB-21822, IBI351, HS-10370, HBI-2438, GH35, GEC255, FMC-376, Divarasib, D3S-001, Garsorasib, BPI-421286, BI-1823911, BEBT-607, BBO- 8520 or Adagrasibf) A GTPase inhibitorg) A Rho inhibitor, preferably Netarsudil, Y-27632, H 1152, Chroman 1, Fasudil hydrochloride, GSK 269962, SB 772077B, TC-S 7001, RKI 1447 or CHDI 00580985h) An Arf inhibitor, preferably QS11, Bragsinl, Ran-IN-1, CHNQD-01255, Arf1- GEFs-IN-1, CHNQD-01269, myristoylated ARF6i) A Ran inhibitor, preferably Ran-IN-1 or M36j) A Rab inhibitor, preferably CID-1067700, Nexinhib20, MLS-573151 (MLS000573151), ML141 (CID-2950007), SBI-581, Psoromic acid, Asnuciclib or BQZ-485,k) A Kinase inhibitor, preferably Abemaciclib, Abivertinib, Abrocitinib, Acalabrutinib, Acrizanib, Acumapimod, Afatinib, Agerafenib, AKI-001, Alectinib, Alisertib, Almonertinib, Alpelisib, Alvocidib, AMG-900, Asciminib, AT9283, AUM-302, AV-412, Avapritinib, Avutometinib, Axitinib, AZD-0424, AZD-7648,AZD-8330, Bafetinib, Barasertib, Belumosudil, Binimetinib, Bosutinib, Branebrutinib, Brepocitinib, Brigatinib, Cabozantinib, Capivasertib, Capmatinib, Cediranib, Ceritinib, Chiauranib, Cobimetinib, Conteltinib, Copanlisib, Crizotinib, CYC116, Dabrafenib, Dacomitinib, Danusertib, Dasatinib, Deucravacitinib, Doramapimod, Dorsomorphin, Duvelisib, Emodin, Encorafenib, ENMD-981693, Ensartinib, Entrectinib, Erdafitinib, Erlotinib, Everolimus, Fasudil, Fedratinib, FF-10101-01, Firmonertinib, Flumatinib, Fruquintinib, Futibatinib, Gedatolisib, Gefitinib, Genistein, Gilteritinib, GSK- 2636771, H-89, Ibrutinib, Icotinib, Idelalisib, llorasertib, Imatinib, Indirubin, Infigratinib, Ipatasertib, KX-2361, Lapatinib, Larotrectinib, Lazertinib, Lenvatinib, Lorlatinib, Losmapimod, Mangostin, Masitinib, Merestinib, Midostaurin, Mirdametinib, MK-5108, MKC-1, MLN8054, Mobocertinib, Momelotinib, Motesanib, Naporafenib, Naquotinib, Nedisertib, Nemiralisib, Neratinib, Nilotinib, Nintedanib, Olmutinib, ONO-7579, Onvansertib, Orantinib, Orelabrutinib, Osimertinib, Pacritinib, Palbociclib, Parsaclisib, Pazopanib, PD- 98059, Pelitinib, Pemigatinib, Pexidartinib, PF-03814735, PF-477736, Pimasertib, Pirtobrutinib, Ponatinib, Pralsetinib, Prexasertib, Quizartinib, Rabusertib, Ravoxertinib, Rebastinib, Regorafenib, Remibrutinib, Repotrectinib, Reversine, Ribociclib, Ridaforolimus, Rigosertib, Rimacalib, Ripretinib, Rivoceranib, Rociletinib, Ruxolitinib, Seliciclib, Selonsertib, Selpercatinib, Selumetinib, Semaxanib, Sirolimus, Sitravatinib, SNS-314, Sorafenib, Spebrutinib, Sunitinib, Surufatinib, TAK-901, Temsirolimus, Tepotinib, Tesevatinib, Tirabrutinib, Tivozanib, Tofacitinib, Tozasertib, TPCA-1, Trametinib, Trilaciclib, Tucatinib, Umbralisib, Upadacitinib, Vactosertib, Vandetanib, Vatalanib, Vemurafenib, Vistusertib, Vorolanib, Voruciclib, Wortmannin, X-376, Zabedosertib or Zanubrutinibfor use as a medicament, wherein the carrier compound is selected from the group consisting ofa) a serum protein, preferably albumin, preferably Albutein, or Plasbumin, or Buminate, or Alburx, or Flexbumin, or Plasbumin-25, or Albuked, or Kedbuminb) recombinantly produced albumin, preferably consisting of subdomain IA, or IB, or IIA, or IIB, or IIIA, or IIIB, preferably any combination of these subdomainsc) immunoglobulins, preferably I gG 1, or I gG2, or I gG3, or I gG4, or I g E, or IgD, or IgA, or IgMd) plasma protein fraction systemic, preferably Octaplase) hydroxyethyl starchf) hetastarch, preferably Hespan, or Hextendg) high molecular dextran, preferably Hyskon, or Dextran 70h) extracellular matrix proteinsi) fatty acid binding proteins or peptidesj) nanoparticles, preferably with a diameter between one and one hundred nanometers.

22. The antigen binding compound according to claim 21, wherein said PEG linker is selected from the group consisting ofa) linear PEK linkersb) branched and multi-arm PEG linkersc) monodisperse PEG linkersd) polydisperse PEG linkerse) cleavable PEG linkers.

23. The combination according to any one of claims 1 to 12 or the ABC according to claim 21 or 22, wherein said extracellular antigen is part of the large GTPase superfamily, for example as listed in Table 1, or part of the kinase family, and is a mutated or non-mutated antigen.

24. The combination according to any one of claims 1 to 12 and 23 or the ABC according to any one of claims 21 to 23, wherein said extracellular antigen is notpart of, or is not restricted to, a human leukocyte antigen (HLA)-peptide complex, or specific HLA alleles, presented on the cell surface by the major histocompatibility complex (MHC).

25. The ABC according to any one of claims 21 to 24, wherein said ABC is a single compound, not covalent linked to a second compound, instead it is a hydrophilic compound with high water solubility and low membrane permeability, therefore increasing its binding to extracellular antigen.

26. The ABC according to any one of claims 21 to 25 for use in a method of diagnosis, prevention and / or treatment of a malignant or non-malignant human or animal disease, or as supplement for stimulating healthy cells.

27. The ABC according to any one of claims 21 to 25 for use as fertilizer or pesticide to treat plant cells or algae.

28. The ABC according to any one of claims 21 to 27, wherein said ABC has a twofold, or fivefold, or tenfold, or hundredfold, or thousandfold or higher extracellular concentration compared to its cytoplasmic concentration, preferable measured in an in vitro cell model.

29. The ABP as defined in any one of claims 1 to 12, 23 and 24 or the ABC according to any one of claims 21 to 28 for use in diagnosis and / or therapy of human or animal disease or for agricultural or biotechnology purpose.

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