EP4694907A1 - Peptides as antagonists of ikaros zinc finger family (ikzf) proteins to activate the immune system against tumor cells - Google Patents

Abstract

The invention relates to a therapeutic peptide, comprising a section which inhibits the homo or hetero dimerization of members of the Ikaros family of zinc finger proteins (IKZF inhibiting section), wherein the length of the peptide is in the range of 9 amino acids to 100 amino acids and wherein the amino acid sequence of the IKZF inhibiting section comprises SEQ ID NO: 1 (FTIHM) or SEQ ID NO: 2 (YTIHM). The invention further relates to an IKZF dimerization inhibitor for use in the treatment of a cancer in a subject, wherein the use comprises administering the IKZF dimerization inhibitor to the subject wherein the inhibitor inhibits the homo or hetero dimerization of members of the IKZF family.

Description

PEPTIDES AS ANTAGONISTS OF IKAROS ZINC FINGER FAMILY (IKZF) PROTEINS TO ACTIVATE THE IMMUNE SYSTEM AGAINST TUMOR CELLS

FIELD OF THE INVENTION

The invention relates to inhibitors of the homo or hetero dimerization of members of the Ikaros of zinc finger (IKZF) family, in particular therapeutic peptides, and their use for immunomodulation.

BACKGROUND OF THE INVENTION

As the global population continues to age, particularly in western nations, cancer is becoming a significant cause of death. This disease has an outsized impact on those over 70 years old and constitutes a staggering 50% of all identified cases (Roser and Ritchie 2015). In the year 2017, the collective toll of diverse cancer types claimed the lives of nearly 10 million individuals, solidifying its position as the second most fatal disease globally. Notwithstanding the notable milestones made in the realm of cancer therapeutics, the United States, as projected by the American Cancer Society, anticipates a staggering figure of over 600,000 cancer-related mortalities in the year 2023, with Lung, Colorectal, Stomach and Liver cancer among the worst prognostic types (Siegel et al 2015).

The recent advances in cancer therapeutics and drug development have undoubtedly revolutionized the survival rate of cancer patients and provided novel non-invasive treatments (Jemal et al. 2017). The manifestation of such an effect may not be readily apparent, given the concurrent rise in the incidence of cancer cases. Next generation sequencing that allowed the introduction of personalized medicine (Carlson 2012; Gullapalli et al. 2012). Human papillomavirus vaccines that reduced cervical cancers (St Laurent et al. 2018) and cancer immunotherapy (Jiang and Zhou 2015; Shahid et al. 2019) are among the most significant advances.

The immune system, intricately intertwined with intercellular and intracellular pathways and networks, stands as one of the most complex systems within the human body. These networks are meticulously regulated, and even slight alterations in the timing and location of effector molecules can have paradoxical consequences, either thwarting or inadvertently bolstering the progression of tumors (Vesely and Schreiber 2011 ). The dynamic process encompassing the interaction between immune cells and tumor cells is known as "Immunoediting". Cancer immunoediting comprises three distinct phases: Elimination, Equilibrium, and Escape. During the Elimination phase, cancer cells are recognized by specific tumor antigens displayed on their surface and subsequently eliminated by immune cells. However, some cells within the tumor may acquire mutations that confer resistance to immune destruction. If a small population of cells manages to evade immune control, the second phase, termed "Equilibrium," ensues. In this phase, the tumor persists without spreading further. Subsequent mutations in the tumor cells, enabling uncontrolled growth and the ability to inhibit immune cells, ultimately lead to the dominance of tumor cells, marking the transition to the "Escape" phase (Vesely and Schreiber 2011 ; O'Donnell et al. 2019; Schreiber et al. 2011 ).

IMiDs (immunomodulatory substances) such as lenalidomide and pomalidomide have become established treatments for multiple myeloma and, to some extent, nonHodgkin's lymphoma. Besides their direct cytotoxic effects on tumor cells, these substances possess immunomodulatory properties that activate the immune system in order to target tumor cells. In 2014, the mechanism of action of these drugs was unraveled, revealing that they bind to cereblon, thereby expediting the subsequent degradation of cereblon substrates. Considering the numerous substrates associated with cereblon, these drugs effectively counteract multiple targets of cereblon. Prominently, within this group, the primary targets exhibiting significant functional relevance were members of the Ikaros zinc finger transcription factors family: IKZF1 and IKZF3 (Krdnke et al. 2014).

The IKZF transcription factors family (IKZF) is characterized by its possession of a highly conserved zinc finger (ZF) domain. The ZF domain is a small structural motif that exists in various transcription factors across different eukaryotic organisms. It is composed of two [3-sheets and one a-helix, which are stabilized by one or more zinc ions.

The Ikaros family contains 6 highly conserved C2-H2-type ZF domains, IKZFs translocate and bind to the DNA, and form homo- or heterodimers with other proteins (Cassandri et al., 2017). Previous reports have suggested that the 4 ZF domains, located at the N-terminus of IKZFs, are responsible for DNA recognition, while the other 2 ZF domains at the C-terminus are responsible for heterodimerization (McCarty et al., 2003). Many recent studies have also challenged this classical structure related function, as an example B lymphocyte-induced maturation protein-1 (Blimp-1 ) was found to form heterodimer with IKZF3 by the N-terminal 119 amino acid of IKZF3, moreover, RUNX1 did not interact with IKZF1 and IKZF3 at the C-terminal (see Figure 1 ) (Hung et al., 2016; Zhou et al., 2019).

Noteworthy, IKZF family control gene expression by forming different homo- and hetero-dimers that affect their DNA binding specificity. They modulate gene expression by:

• Binding directly to gene promoters and enhance or suppress target gene expression.

• By relocating and forming chromatin remodeling complex to regulate DNA accessibility.

• Gene expression regulation on RNA level.

(Georgopoulos et al., 1992; Hsi et al., 2008; John and Ward, 2011 ; Yoshida and Georgopoulos, 2014).

As mentioned above, Krdnke et al. demonstrated that the interaction between lenalidomide and the CRBN-CRL4 ubiquitin ligase complex enhances the binding affinity of CRBN to IKZF1/3. This specific binding event then triggers the ubiquitination and subsequent degradation of IKZF1/3, which plays a crucial role in the survival of multiple myeloma (MM) cells. In order to study the role of IKZF1/3 expression in T cells on cancer patients, the inventors Awwad et al., have analysed in a previous study the IKZF1/3 expression levels in T-cells from 45 MM stage I (MMI) and 50 newly diagnosed MM stage III (MM III) patients, according to Dune-Salmon staging system, by flow cytometry to examine their prognostic and predictive value. Awwad et al. also combined in vivo observations with in vitro assays to determine the effect of IKZF1/3 expression on the T-cell immunophenotype and anti-tumour T-cell response in 162 MMIII patients. Awwad et al. found that high IKZF3, but not IKZF1 , expression in T-cells correlates with superior overall survival in MMIII patients treated with immunomodulatory drugs (thalidomide, lenalidomide and pomalidomide). Moreover, Awwad et al. showed that higher IKZF3 expression in T-cells inhibits myeloma- specific T-cell response in vitro and that the immunophenotype of patients with high IKZF3 expression shows features that are contrary to the changes induced by immunomodulatory drugs. Although they observed higher IKZF3 expression levels in T-cells from patients with MMIII compared to MMI, IKZF3 expression was unaffected by the tumour microenvironment. (Awwad et al., 2018).

SUMMARY OF THE INVENTION

The present invention is, inter alia, based on the surprising finding that by inhibiting Aiolos (the gene product of IKZF3) and Ikaros (the gene product of IKZF1 ), the interleukin-2 (IL-2) secretion of T-cells could be significantly increased. Interleukin-2 is crucial for the activity of T cells against tumour cells.

Based on this knowledge specific inhibitors of Ikaros and Aiolos were developed. In the cell culture investigations with human T-cells, the inventors were able to show that the addition of peptide inhibitors strongly increased the interleukin-2 secretion of T-cells, even in comparison to the interleukin-2 secretion of pomalidomide. At the same time, there was no reduction in the vitality of the T cells at the selected dosage.

While other approaches of inhibiting Aiolos and Ikaros are also suitable, the invention is illustrated by therapeutic peptides that serve as inhibitors. The therapeutic peptides comprise a section, which inhibits the homo or hetero dimerization of Aiolos and Ikaros and also the other members of the Ikaros family of zinc finger proteins (IKZF inhibiting section).

Thus, according to a first aspect the invention relates to a therapeutic peptide, comprising a IKZF inhibiting section, which inhibits the homo or hetero dimerization of members of the Ikaros of zinc finger (IKZF) family, wherein the length of the therapeutic peptide is in the range of 9 amino acids to 100 amino acids and wherein the amino acid sequence of the IKZF inhibiting section comprises SEQ ID NO: 1 (FTIHM) or SEQ ID NO: 2 (YTIHM).

According to a second aspect, the invention provides a method for the treatment of a cancer in a subject, comprising administering to the subject an IKZF dimerization inhibitor, which inhibits the homo or hetero dimerization of members of the IKZF family.

According to a third aspect, the invention relates to a nucleic acid encoding the therapeutic peptide according to the first aspect.

According to a fourth aspect, the invention relates to a vector containing the nucleic acid according to the third aspect.

According to a fifth aspect, the invention relates to a host cell containing the nucleic acid according to the third aspect or the vector according to the fourth aspect.

FIGURES

Fig. 1 shows the domain organization of IKZF3 together with known binding partners and their position of binding.

Fig. 2 shows the results of a coimmunoprecipitation (co-IP) of IKZF3 protein combined mass spectrometry analysis of 18 sorted healthy donors CD8+ T cells induced to exhaustion with strong activation of CD3/CD28 dyna beads.

Fig. 3 shows the result of an assay in human embryonic kidney 293T (HEK 293T), in which cells were co-transfected with full length IKZF1 protein, and recombinant gluthathione S transferase (GST)-fused on the C-terminal of the full length IKZF3 protein (GST-IKZF3_V1 ) or different GST-tagged variants of IKZF3. By a pull-down co-IP assay with antiGST beads, the complex was isolated and IKZF1 expression was captured by immunoblot analysis.

Fig. 4 shows the results of an enzyme-linked immunosorbent assay (ELISA) for the secretion of IL-2 was then performed to accurately assess the secretion of IL2 from each condition, i.e. T cells cultured for 24 hours in serum-free T cell medium and either 10 pM of the C1 M peptide, scrambled peptide as a negative control, or 10 pM Lenalidomide or 1 pM of Pomalidomide as IMiDs control.

Fig. 5 shows the results of a cytokine screening assay. 119 different cytokines were screened in the supernatant from T cells cultured for 24 hours in serum-free T cell medium either 10 pM of the C1 M peptide showing a strong specific increase in IL-2, IL-6 and INFy secretion from T cells.

Fig. 6 shows an illustration of the process steps of the sandwich ELISA approach for determining the ability of the testing peptides to bind to IKZF1 or IKZF3.

Fig. 7 shows the results of the peptides PepM1 , PepM2 and PepM3 in IL-2 analyses (Figure 7A), IKZF3 binding assay (Figure 7B) and IKZF1 binding assay (Figure 7C) with C1 M peptide as reference.

Fig. 8 shows the results of the peptides PepAlal to PepAla15, in IL-2 analyses (Figure 8A), IKZF3 binding assay (Figure 8B) and IKZF1 binding assay (Figure 8C) with C1 M peptide as reference.

Fig. 9 shows a comparison of the sequences of the test peptides PepM1 to PepM3, PepM7 to PepM14 and PepAlal to PepAlal 5.

Fig. 10 shows the results of a sequence comparison of the core of the ZF5 domain of IKZF1 , IKZF2, IKZF3, IKZF4, and the ZF4 domain of IKZF5.

Fig. 11 shows the Fluorescence Lifetime Imaging Microscopy (FLIM) experiment results in the form of phasor plots for A) IKZF1 and B) IKZF3 using GFP lifetime signal in HEK cells. Each panel contains three plots: Top Plot: Average GFP lifetime signal in HEK cells expressing only GFP protein.

Middle Plot: Average GFP lifetime signal in HEK cells expressing IKZF1 or IKZF3 fused to GFP.

Bottom Plot: Average GFP lifetime signal in HEK cells co-expressing IKZF1 or IKZF3 fused to either GFP or mCherry. The data represents the average phasor of 15 measurements (n=15) for each condition.

Fig. 12 shows the results of FLIM experiments focusing on the heterodimerization of IKZF1 and IKZF3 using GFP lifetime signal in HEK cells. The results are shown as phasor plots. The figure contains three plots:

Top Plot: Average GFP lifetime signal in HEK cells expressing only GFP protein.

Middle Plot: Average GFP lifetime signal in HEK cells expressing IKZF1 fused to GFP.

Bottom Plot: Average GFP lifetime signal in HEK cells co-expressing IKZF1 fused to GFP and IKZF3 fused to mCherry.

Data represent the average phasor of 15 measurements (n=15) for each condition.

Fig. 13 shows the results of a FLIM experiment testing the effect of the C1 M peptide on the heterodimerization of IKZF1 and IKZF3 using GFP lifetime signal in HEK cells. The results are shown as phasor plots. The figure contains five plots:

First Plot: Average GFP lifetime signal in HEK cells expressing only GFP protein.

Second Plot: Average GFP lifetime signal in HEK cells expressing IKZF1 fused to GFP.

Third Plot: Average GFP lifetime signal in HEK cells expressing IKZF3 fused to GFP.

Fourth Plot: Average GFP lifetime signal in HEK cells co-expressing IKZF1 fused to GFP and IKZF3 fused to mCherry with scrambled peptide.

Fifth Plot: Average GFP lifetime signal in HEK cells co-expressing

IKZF1 fused to GFP and IKZF3 fused to mCherry with C1 M peptide.

C1 M peptide is tested for its ability to inhibit the heterodimerization of

IKZF1 and IKZF3. The data represents the average phasor of 15 measurements (n=8, 10, 12, 12, & 15 respectively) for each condition. Fig. 14 shows the results of a FLIM experiment results testing the effect of the C1 M peptide on the heterodimerization of IKZF1 and IKZF3 using GFP lifetime signal in HEK cells. The figure contains five plots:

First Plot: Average GFP lifetime signal in HEK cells co-expressing IKZF1 fused to GFP and IKZF3 fused to mCherry with scrambled peptide.

Second Plot: Average GFP lifetime signal in HEK cells co-expressing IKZF1 fused to GFP and IKZF3 fused to mCherry with C1 M peptide.

Figure 15 shows a dot plot illustrating the T cell activation ability of the C1 M peptide. T cells from healthy donors were incubated for 24 hours with either a scrambled peptide, C1 M peptide, or no peptide (control). Subsequently, these T cells were co-cultured with NCI-H929 tumor cells for either 24 hours (dots) or 48 hours (crosses). Flow cytometry was used to analyze the percentage of tumor cells among live cells (n=6). The results indicate that T cells pre-cultured with C1 M peptide killed significantly more tumor cells than those treated with scrambled peptide or the control, demonstrating the potent activation effect of the C1 M peptide on T cells.

DETAILED DESCRIPTION OF THE INVENTION

In order to provide a clear and consistent understanding of the specification and claims, and the scope to be given such terms, the following definitions are provided.

Definitions

A "peptide" as used herein relates to a chain of amino acids connected by peptide bonds. A peptide may be composed of any number of amino acids of any type, preferably naturally occurring amino acids, which, preferably, are linked by peptide bonds. In particular, a peptide comprises at least 3 amino acids, preferably at least 5, at least 7, at least 9, at least 12, or at least 15 amino acids. Furthermore, there is no upper limit for the length of a peptide. However, preferably, a peptide according to the invention does not exceed a length of 500 amino acids, more preferably it does not exceed a length of 300 amino acids; even more preferably it is not longer than 250 amino acids. Thus, the term “peptide” includes “oligopeptides”, which usually refer to peptides with a length of 2 to 20 amino acids and “polypeptides” with at least 60, at least 80, preferably at least 100 amino acids.

The term “polypeptide” as used herein refers to a peptide. The terms “polypeptide” and “protein” are used interchangeably. The polypeptides and proteins as used herein include chemically synthesized proteins as well as naturally synthesized proteins, which are encoded by genes. The polypeptides or proteins may be obtained from a natural source, such as human blood or produced in cell culture as recombinant proteins.

The term “protein” as used herein may contain one or more polypeptide chains. Proteins with more than one polypeptide chain are often expressed on one polypeptide chain from one gene and cleaved post-translationally. Thus, the terms “protein” and “polypeptide” are used interchangeably.

The term "protein domain" or “domain” as used herein refers to a region of a protein that can fold into a stable three-dimensional structure independently of the rest of the protein. This structure may maintain a specific function associated with the domain's function within the intact protein, including enzymatic activity, creation of a recognition motif for another molecule, or provision of the necessary structural components for a protein to exist in a particular environment. Protein domains are usually evolutionarily conserved regions of proteins, both within a protein family and within other protein superfamilies that require similar functions.

The term “fusion protein” according to the invention relates to proteins created through the joining of two or more genes, cDNAs or sequences that originally coded for separate proteins/peptides. The genes may be naturally occurring in the same organism or different organisms or may synthetic polynucleotides.

The term “therapeutic peptide” as used herein relates to peptides with a therapeutic effect, i.e. peptides used as active pharmaceutical ingredient. The term “patient” as used herein denotes a mammal, in particular a human with a condition or disease who is to be treated.

As used herein, the term “mammal” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits, Carnivora, including Felines (cats) and Canines (dogs), Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses), Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes).

The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity". For purposes of the present invention, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et a/., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the no brief option) is used as the percent identity and is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment).

For purposes of the present invention, the degree of sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et a/., 2000, supra), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -no brief option) is used as the percent identity and is calculated as follows: (Identical Desoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

For example an identity of at least 90 % includes 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or even 99% identity to the subject sequence. Typically, similar sequences will include the same residues in positions that are relevant for the function of the peptide or polynucleotide, such as active site residues or glycosylated amino acids, however though may include any number of conservative amino acid substitutions.

The term “carrier” according to the invention relates to molecules useful for delivery of a cargo into cells of a patient, in particular an active pharmaceutical ingredient, e.g. a polynucleotide or protein. Carriers may covalently or non-covalently bind to the cargo or may encapsulate the cargo.

The term "recombinant" when used in reference to a cell, nucleic acid, protein or vector, indicates that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (nonrecombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature.

An "isolated" polynucleotide (e.g., an RNA, DNA or a mixed polymer) or peptide according to the invention is one, which is substantially separated from other cellular components which naturally accompany a native human sequence or protein, e.g., ribosomes, polymerases, the chromosome, other RNA molecules and proteins. The term embraces a nucleic acid sequence or protein, which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.

As used herein, the terms "transformed", "stably transformed", and "transgenic", used with reference to a cell, means that the cell contains a non-native (e.g. heterologous) nucleic acid sequence integrated into its genome or carried as an episome that is maintained through multiple generations.

The term “fragment” as used herein refers to a polypeptide that has an aminoterminal and/or carboxy terminal deletion of one or more amino acids as compared to the native or wild-type protein, but where the remaining amino acid sequence is identical to the corresponding positions in the amino acid sequence deduced from a full-length cDNA.

The terms “binding affinity” or “affinity” as used herein indicate the strength of the binding between two molecules, in particular a ligand and a protein target. Binding affinities are influenced by non-covalent intermolecular interactions between the two molecules such as hydrogen bonding, electrostatic interactions, hydrophobic interactions, and van der Waals forces.

“Dimerization” is a biological process in which two proteins (or other molecules) join together to form a complex known as a dimer. The individual proteins in these complexes are known as subunits, and they can either be identical (in a homodimer) or different (in a heterodimer). The dimerization process can be reversible or irreversible, and it is regulated by a variety of factors, including the concentration of the individual proteins, their spatial distribution within the cell, and the presence of specific chemical signals.

“Heterodimerization” refers to the interaction between two different protein subunits to form a dimer. In this case, the two subunits are distinct and may have different amino acid sequences and functions. “Homodimerization”, on the other hand, involves the interaction between two identical protein subunits to form a dimer.

The transitional term “comprising”, which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim, except for impurities ordinarily associated therewith. When the phrase “consists of’ appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. “A ‘consisting essentially of claim occupies a middle ground between closed claims that are written in a ‘consisting of’ format and fully open claims that are drafted in a ‘comprising’ format.”

A “Zinc Finger (ZF) domain” is a type of protein structural motif that is capable of binding to specific DNA sequences, RNA, proteins, and small molecules. These domains are typically small, functionally independent units within a protein. The name "zinc finger" comes from the presence of one or more zinc ions in the protein structure, which helps stabilize the fold.

“Proteinaceous amino acid” as used herein refers to any one of Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic acid (Asp, D), Cysteine (Cys, C), Glutamic acid (Glu, E), Glutamine (Gin, Q), Glycine (Gly, G), Histidine (His, H), Isoleucine (lie, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Vai, V).

An “IKZF inhibiting section” as used herein is a section of a peptide, which inhibits the homo or hetero dimerization of members of the IKZF family.

An “IKZF dimerization inhibitor” as used herein is any molecule, which inhibits the homo and/or hetero dimerization of members of the IKZF family.

“Fluorescence-lifetime imaging microscopy” or “FLIM” is an imaging technique based on the differences in the exponential decay rate of the photon emission of a fluorophore from a sample. It can be used as an imaging technique in confocal microscopy, two-photon excitation microscopy, and multiphoton tomography. The fluorescence lifetime (FLT) of the fluorophore, rather than its intensity, is used to create the image in FLIM. Fluorescence lifetime depends on the local microenvironment of the fluorophore, thus precluding any erroneous measurements in fluorescence intensity due to change in brightness of the light source, background light intensity or limited photo-bleaching. This technique also has the advantage of minimizing the effect of photon scattering in thick layers of sample.

“Phasor” approach as used herein refers to a method, which is used for vectorial representation of sinusoidal waves like alternative currents and voltages or electromagnetic waves. The amplitude and the phase of the waveform is transformed into a vector where the phase is translated to the angle between the phasor vector and X axis and the amplitude is translated to vector length or magnitude. In this concept the representation and the analysis become very simple and the addition of two wave forms is realized by their vectorial summation. The representation of the data is also referred to as “phasor plot”.

“Scrambled peptide” is a well-established concept in the literature and ubiquitously used where biological or functional activity of peptides is studied. It is regularly created via random shuffling of the original sequence and normally used as the synonym of homologous negative control peptide.

According to a first aspect, the invention relates to therapeutic peptide, comprising a IKZF inhibiting section, which inhibits the homo or hetero dimerization of members of the Ikaros of zinc finger (IKZF) family. The length of the peptide is in the range of 9 amino acids to 100 amino acids. Moreover, the amino acid sequence of the IKZF inhibiting section comprises SEQ ID NO: 1 (FTIHM) or SEQ ID NO: 2 (YTIHM).

The IKZF family is a group of transcription factors that play vital roles in hematopoiesis, the process by which blood cells are produced. These proteins have both activating and repressive functions, and they control the expression of genes involved in cell fate decisions, proliferation, and differentiation. The family is named after Ikaros, the first member discovered and characterized.

Zinc finger domains are characterized by the coordination of one or more zinc ions in order to stabilize the fold. The most common type of zinc finger, and the one found in the IKZF family, is the C2H2-type zinc finger. This type of zinc finger has a conserved sequence with two cysteine residues and two histidine residues that coordinate a single zinc ion.

The C2H2 zinc finger typically folds into a structure with a beta-hairpin followed by an alpha helix. The side chains of the cysteine and histidine residues bind to the zinc ion, allowing the protein to fold into its correct three-dimensional shape. In terms of structure, IKZF proteins contain several C2H2-type zinc finger domains, which allow them to bind to DNA and regulate gene expression. The number and arrangement of these domains vary between family members, providing each protein with unique DNA binding properties. Moreover, these proteins can form homo- or heterodimers, enhancing their functional diversity.

The IKZF proteins interact with a wide range of other proteins, including other transcription factors, chromatin modifiers, and signaling proteins. These interactions enable the IKZF proteins to integrate signals from various cellular pathways and coordinate complex gene expression programs.

The IKZF family generally contains 6 highly-conserved C2-H2-type ZF domains, IKZFs translocate and bind to the DNA, and form homo- or heterodimers with other proteins (Cassandri et al., 2017). Previous reports have suggested that the 4 ZF domains, located at the N-terminus of IKZFs, are responsible for DNA recognition, while the other 2 ZF domains at the C-terminus are responsible for heterodimerization (McCarty et al., 2003). The six ZF domains are numbered ZF1 , ZF2, ZF3, ZF4, ZF5 and ZF6.

There are five known members of the IKZF family: IKZF1 (Ikaros). The names IKZF1 and Ikaros are used interchangeably. Ikaros is the most studied member of the IKZF family. It is essential for the development of all lymphoid cells and has been shown to have tumor suppressor activity. Ikaros is a key regulator of B cell and T cell development, and it also plays a role in the development and function of natural killer cells and dendritic cells. Mutations in the Ikaros gene have been linked to leukemia, specifically acute lymphoblastic leukemia (ALL). The amino acid sequence of Ikaros is identified by Uniprot entry Q13422 (SEQ ID NO: 11 ). IKZF2 (Helios): The names IKZF2 and Helios are used interchangeably. Like Ikaros, Helios is involved in T cell development, but it's also associated with the regulation of regulatory T cells (Tregs), which play a key role in maintaining immune tolerance. Dysregulation of Helios expression can contribute to autoimmune disorders. The amino acid sequence of Helios is identified by Uniprot entry Q9UKS7 (SEQ ID NO: 12).

IKZF3 (Aiolos): The names IKZF3 and Aiolos are used interchangeably. Aiolos shares many functions with Ikaros and is also involved in B cell and T cell development. Aiolos can cooperate with Ikaros to regulate gene expression and control lymphocyte development. Dysregulation of Aiolos has been linked to systemic lupus erythematosus (SLE), an autoimmune disease. The amino acid sequence of Aiolos is identified by Uniprot entry Q9UKT9 (SEQ ID NO: 13).

IKZF4 (Eos): The names IKZF4 and Eos are used interchangeably. Eos functions primarily as a transcriptional repressor and is involved in regulating the differentiation and function of T cells, particularly T regs. Eos can interact with the transcription factor FOXP3 to suppress the expression of pro-inflammatory genes in Tregs. Dysregulation of Eos can lead to inflammation and autoimmunity. The amino acid sequence of Eos is identified by Uniprot entry Q9H2S9 (SEQ ID NO: 14).

IKZF5 (Pegasus): The names IKZF5 and Pegasus are used interchangeably. The least characterized member of the IKZF family, Pegasus is thought to be involved in lymphocyte development like the other family members, but its specific functions are not well understood. The amino acid sequence of Pegasus is identified by Uniprot entry Q9H5V7 (SEQ ID NO: 15). Pegasus differs from the other IKZF members such that it only contains three N-terminal ZF domains. Thus, ZF4 of IKZF5 corresponds to ZF5 of the other members and ZF5 of IKZF5 corresponds to ZF6 of the other members.

As shown in the examples, the minimal core structure of the binding domains of the IKZF family members necessary for homo or hetero dimerization is the SEQ ID NO: 1 found in the fifth ZF domain (ZF5) of IKZF3 and IKZF4. A sequence comparison of the core of the ZF5 domains (ZF4 in case of IKZF5) is shown in Figure 9. Peptides with this sequence are able to bind IKZF1 and IKZF3 and are shown to cause the effect of disrupting the homo and hetero dimerization of IKZF1 and IKZF3 and to cause an increase in IL-2 expression in T cells. It is expected that therapeutic peptides consensus sequence SEQ ID NO: 2 of IKZF1 , IKZF2, IKZF5 shows the same effective binding.

Specifically, the IKZF inhibiting section exerts an inhibitory effect on the dimerization process of any of the IKZF family members, either homodimerization of any of IKZF1 , IKZF2, IKZF3, IKZF4, and IKZF5 or the heterodimerization between any two of IKZF1 , IKZF2, IKZF3, IKZF4, and IKZF5.

In accordance with an embodiment of the therapeutic peptide, the IKZF inhibiting section has the capability to inhibit the homo or heterodimerization of Ikaros and Aiolos. Specifically, the IKZF inhibiting section exerts an inhibitory effect on the dimerization process of these two zinc finger proteins. This inhibition could be observed in the homodimerization of IKZF1 or the heterodimerization between IKZF1 and IKZF3. The inhibitory action of the IKZF section on the homo or heterodimerization of IKZF1 and IKZF3 holds significant therapeutic potential as it can modulate the biochemical activities of these proteins and as shown in the examples has a major impact on the IL-2 production of T cells.

Interleukin-2 (IL-2) is a critical cytokine in the immune system, essential for the activation and proliferation of T cells.

The increase in IL-2 has the following beneficial effects on cancer treatment: Activation of T cells: T cells are an essential component of the immune response to cancer. They can recognize and destroy cancer cells. IL-2 is crucial for the growth, differentiation, and survival of these cells. IL-2 can induce the proliferation of cytotoxic T cells and enhance their ability to kill cancer cells.

Induction of immune cell-mediated tumor killing: In addition to activating T cells, IL-2 also stimulates natural killer (NK) cells, which are part of the innate immune system and have the ability to kill tumor cells without prior sensitization.

Promotion of anti-tumor immune responses: By enhancing the activity of T cells and NK cells, IL-2 promotes a robust immune response against the tumor. Additionally, IL-2 may support the development and maintenance of memory T cells, which can provide long-term immunity against the tumor.

IL-2 promotes the growth, differentiation, and survival of antigen-selected cytotoxic T cells (CTLs), thereby enhancing the body's ability to mount an effective immune response. It acts by binding to the IL-2 receptor on the surface of T cells, triggering a cascade of signaling events that lead to T cell expansion and increased cytotoxic activity. An increase in IL-2 secretion is a strong indicator of T cell activation and enhanced immune response.

Increased production of other essential cytokines such as INF-y: IL-2 can stimulate the production of other key cytokines, like interferon-gamma (INF-y). INF-y can directly inhibit tumor growth and works synergistically with other components of the immune system to enhance the anti-tumor response.

Enhancement of immune checkpoint inhibitors: Checkpoint inhibitors are a type of immunotherapy that blocks proteins (like PD-1 , PD-L1 , CTLA-4) from putting the brakes on T cells, thus boosting their ability to kill cancer cells. IL-2 can enhance the efficacy of these inhibitors by promoting T cell proliferation and activation.

In the examples, the therapeutic peptide have been shown to significantly increase IL-2 production in T cells, which suggests that it effectively enhances T cell activation.

As shown in Example 7, the therapeutic peptides have a cytotoxic effect on cancer cells. This effect is likely caused by the activation of T cells against the tumor via IL- 2.

According to one embodiment, the amino acid sequence of the IKZF inhibiting section comprises SEQ ID NO: 3 (XXXTIHM). The Xaa at position 1 is preferably Vai or lie, because these are residues found in the position in the IKZF family members. More preferably, the Xaa at position 1 is Vai as it is Vai in IKZF3 and as shown in the Examples, peptides with the Vai at position 1 show a strong binding to IKZF1 and IKZF3 and an increase in IL-2 production. The Xaa at position 3 is preferably Phe or Tyr, because these are residues found in the position in the IKZF family members. More preferably, Xaa at position 3 is Phe as it is Phe in IKZF3 and as shown in the Examples, peptides with the Phe at position 3 show a strong binding to IKZF1 and IKZF3 and an increase in IL-2 production. The Xaa at position 2 may be any proteinaceous amino acid. The Xaa at position 2 is preferably Met or Leu, because these are residues found in the position in the IKZF family members. More preferably, Xaa at position 2 is Met as it is Met in IKZF3 and as shown in the Examples, peptides with the Met at position 2 show a strong binding to IKZF1 and IKZF3 and an increase in IL-2 production.

According to one embodiment, the amino acid sequence of the IKZF inhibiting section comprises SEQ ID NO: 4 (XXXTIHMXX). The Xaa at position 1 is preferably Vai or lie, because these are residues found in the position in the IKZF family members. More preferably, the Xaa at position 1 is Vai as it is Vai in IKZF3 and as shown in the Examples, peptides with the Vai at position 1 show a strong binding to IKZF1 and IKZF3 and an increase in IL-2 production. The Xaa at position 3 is preferably Phe or Tyr, because these are residues found in the position in the IKZF family members. More preferably, Xaa at position 3 is Phe as it is Phe in IKZF3 and as shown in the Examples, peptides with the Phe at position 3 show a strong binding to IKZF1 and IKZF3 and an increase in IL-2 production. The Xaa at position 9 is preferably Cys, because this is the residue found in IKZF3 and as shown in the Examples, peptides with the Cys at position 9 show a strong binding to IKZF1 and IKZF3 and an increase in IL-2 production. The Xaa at position 2 is preferably Met or Leu, because these are residues found in the position in the IKZF family members. More preferably, Xaa at position 2 is Met as it is Met in IKZF3 and as shown in the Examples, peptides with the Met at position 2 show a strong binding to IKZF1 and IKZF3 and an increase in IL-2 production. The Xaa at position 8 is preferably Gly, because this is the residue found in IKZF3 and as shown in the Examples, peptides with the Gly at position 8 show a strong binding to IKZF1 and IKZF3 and an increase in IL-2 production.

According to one embodiment, the amino acid sequence of the IKZF inhibiting section comprises a sequence selected from SEQ ID NO: 5 (VMFTIHM), SEQ ID NO: 6 (VMFTIHMGC), SEQ ID NO: 7 (YVMFTIHMGCH), SEQ ID NO: 8 (RVLFLDYVMFTIHMG, PepMI O), SEQ ID NO: 9 (LFLDYVMFTIHMGCH, PepM11 ), or SEQ ID NO: 10 (YRCDHCRVLFLDYVMFTIHMGCH, C1 M). As shown in the Examples, peptides with these sequences show a strong binding to IKZF1 and IKZF3 and an increase in IL-2 production. While the experiments shown in the examples have identified specific IKZF inhibiting sections or core sequences thereof, it is understood that any peptide that is similar to the domain ZF5 of any one of IKZF1 , IKZF2, IKZF3, or IKZF4, or of ZF4 of IKZF5 may inhibit the homodimerization of any of IKZF1 , IKZF2, IKZF3, IKZF4, and IKZF5 or the heterodimerization between any two of IKZF1 , IKZF2, IKZF3, IKZF4, and IKZF5. ZF5 of IKZF3 consists of AA 452 to 474 of SEQ ID NO: 13. ZF5 of IKZF1 consists of AA 462 to 484 of SEQ ID NO: 11 . ZF5 of IKZF2 consists of AA 471 to 493 of SEQ ID NO: 12. ZF5 of IKZF4 consists of AA 530 to 552 of SEQ ID NO: 14. ZF4 of IKZF5 consists of AA 364 to 386 of SEQ ID NO: 15.

Moreover, some experiments suggest that the C-terminal ZF6 domain (ZF5 in case of IKZF5) is also involved in the homo and heterodimerization processes. Thus, IKZF inhibiting sections additionally comprising the domain ZF6 of IKZF1 , IKZF2, IKZF3, or IKZF4, or ZF5 of IKZF5 are expected to be highly effective in the inhibition of homo and hetero dimerization. ZF6 of IKZF3 consists of AA 480 to 504 of SEQ ID NO: 13. ZF6 of IKZF1 consists of AA 490 to 514 of SEQ ID NO: 11. ZF6 of IKZF2 consists of AA 499 to 523 of SEQ ID NO: 12. ZF6 of IKZF4 consists of AA 558 to 582 of SEQ ID NO: 14. ZF5 of IKZF5 consists of AA 364 to 386 of SEQ ID NO: 15.

Thus, according to one embodiment, the IKZF inhibiting section comprises a) an amino acid sequence that is at least 90 % identical to AA 452 to 474 of SEQ ID NO: 13 and optionally an amino acid sequence that is at least 90 % identical to AA 480 to 504 of SEQ ID NO: 13; b) an amino acid sequence that is at least 90 % identical to AA 452 to 504 of SEQ ID NO: 13; c) an amino acid sequence that is at least 90 % identical to AA 462 to 484 of SEQ ID NO: 11 and optionally an amino acid sequence that is at least 90 % identical to AA 490 to 514 of SEQ ID NO: 11 ; d) an amino acid sequence that is at least 90 % identical to AA 462 to 514 of SEQ ID NO: 11 ; e) an amino acid sequence that is at least 90 % identical to AA 471 to 493 of SEQ ID NO: 12 and optionally an amino acid sequence that is at least 90 % identical to AA 499 to 523 of SEQ ID NO: 12 f) an amino acid sequence that is at least 90 % identical to AA 471 to 523 of SEQ ID NO: 12; g) an amino acid sequence that is at least 90 % identical to AA 530 to 552 of SEQ ID NO: 14 and optionally an amino acid sequence that is at least 90 % identical to AA 558 to 582 of SEQ ID NO: 14; h) an amino acid sequence that is at least 90 % identical to AA 530 to 582 of SEQ ID NO: 14; i) an amino acid sequence that is at least 90 % identical to AA 364 to 386 of SEQ ID NO: 15 and optionally an amino acid sequence that is at least 90 % identical to AA 392 to 416 of SEQ ID NO: 15; and j) an amino acid sequence that is at least 90 % identical to AA 364 to 416 of SEQ ID NO: 15.

According to one embodiment of the therapeutic peptide, the amino acid identity of the IKZF inhibiting section is at least 95%. The level of amino acid identity can be, for example, 95%, 96%, 97%, 98%, 99%, or even 100%. In another embodiment, the amino acid identity of the therapeutic peptide is more preferably at least 98%. In yet another embodiment, the amino acid identity of the therapeutic peptide is most preferably 100%.

According to one embodiment of the therapeutic peptide, the length of the IKZF inhibiting section is less than 80 amino acids. The length of the IKZF inhibiting section may be for example 80 amino acids, 78 amino acids, 76 amino acids, 74 amino acids, 72 amino acids, 70 amino acids, 68 amino acids, 66 amino acids, 64 amino acids, 62 amino acids, 60 amino acids, 58 amino acids, 56 amino acids, 54 amino acids, 52 amino acids, 50 amino acids, 48 amino acids, 46 amino acids, 44 amino acids, 42 amino acids, 40 amino acids, 38 amino acids, 36 amino acids, 34 amino acids, 32 amino acids, 30 amino acids, 28 amino acids, 26 amino acids, 24 amino acids, 22 amino acids, 20 amino acids, 18 amino acids, 16 amino acids, 14 amino acids, 12 amino acids, 10 amino acids, 8 amino acids. A length of the more than 80 has the disadvantage it leads to a very long total length of the peptide, which in turn is the cause of a very low internalization rate into the cells and a fast degradation in the serum. According to one embodiment, the length of the IKZF inhibiting section is less than 60 amino acids. A length of less than 60 amino acids facilitates the peptide’s internalization and significantly decreases the degradation chance compared to longer peptides. According to one embodiment, the length of the IKZF inhibiting section is less than 40 amino acids. A length of less than 40 amino acids further facilitates the therapeutic peptide’s internalization and further decreases the degradation chance. According to one embodiment, the length of the IKZF inhibiting section is less than 30 amino acids. A length of less than 30 amino acids further facilitates the therapeutic peptide’s internalization and further decreases the degradation chance.

According to one embodiment of the therapeutic peptide, the length of the IKZF inhibiting section is more than 12 amino acids. The length of the IKZF inhibiting section may be for example 14 amino acids, 16 amino acids, 18 amino acids, 20 amino acids, 22 amino acids, 24 amino acids, 26 amino acids, 28 amino acids, 30 amino acids, 32 amino acids, 34 amino acids, 36 amino acids, 38 amino acids, 40 amino acids, 42 amino acids, 44 amino acids, 46 amino acids, 48 amino acids, 50 amino acids, 52 amino acids, 54 amino acids, 56 amino acids, 58 amino acids, 60 amino acids, 62 amino acids, 64 amino acids, 66 amino acids, 68 amino acids, 70 amino acids, 72 amino acids, 74 amino acids, 76 amino acids, 78 amino acids, 80 amino acids. A length of less than 12 amino acids has the disadvantage that [Please insert disadvantages of a decreased stability, a lower bioavailability and fast renal clearance.

According to one embodiment of the therapeutic peptide, the total length of the therapeutic peptide is less than 110 amino acids. The total length of the therapeutic peptide may be for example 110 amino acids, 108 amino acids, 106 amino acids, 104 amino acids, 102 amino acids, 100 amino acids, 98 amino acids, 96 amino acids, 94 amino acids, 92 amino acids, 90 amino acids, 88 amino acids, 86 amino acids, 84 amino acids, 82 amino acids, 80 amino acids, 78 amino acids, 76 amino acids, 74 amino acids, 72 amino acids, 70 amino acids, 68 amino acids, 66 amino acids, 64 amino acids, 62 amino acids, 60 amino acids, 58 amino acids, 56 amino acids, 54 amino acids, 52 amino acids, 50 amino acids, 48 amino acids, 46 amino acids, 44 amino acids, 42 amino acids, 40 amino acids, 38 amino acids, 36 amino acids, 34 amino acids, 32 amino acids, 30 amino acids, 28 amino acids, 26 amino acids, 24 amino acids, 22 amino acids, 20 amino acids, 18 amino acids, 16 amino acids, 14 amino acids, 12 amino acids, 10 amino acids, 8 amino acids. According to one embodiment, the total length of the therapeutic peptide is less than 70 amino acids. According to one embodiment, the total length of the therapeutic peptide is less than 50 amino acids. According to one embodiment, the total length of the therapeutic peptide is less than 30 amino acids.

According to one embodiment, the length of the IKZF inhibiting section is more than 15 amino acids. According to one embodiment, the length of the IKZF inhibiting section is more than 20 amino acids. According to one embodiment, the length of the IKZF inhibiting section is more than 25 amino acids.

According to one embodiment, the therapeutic peptide contains in addition to the IKZF inhibiting section at least one part or section that is not derived from any of IKZF1 , IKZF2, IKZF3, IKZF4, and IKZF5. According to one embodiment, the therapeutic peptide is not only a fragment of any of IKZF1 , IKZF2, IKZF3, IKZF4, and IKZF5. According to one embodiment the therapeutic peptide does not only contain amino acid sequence of IKZF1 , IKZF2, IKZF3, IKZF4, and/or IKZF5.

According to one embodiment, the therapeutic peptide further comprises one or more elements selected from a group consisting of charge modulating elements, half-life prolonging moieties, stability enhancing elements, and chemical staples.

Particularly, the charge modulating elements may be selected from arginine residues, histidine residues, or lysine residues. These elements influence the charge on the therapeutic peptide, which can impact its interaction with other molecules or structures. For example, the inclusion of charge modulating elements may help to enhance peptide solubility, improve cellular uptake, and facilitate binding to target proteins.

Furthermore, the half-life prolonging elements in this embodiment can include albumin, Polyethylene Glycol (PEG), or Proline, Alanine, and Serine (PAS) sequences. By incorporating these elements, the therapeutic peptide's half-life, or the time it remains active within the body, can be extended. This results in a significant advantage that it allows for less frequent dosing and improved patient compliance.

Stability enhancing elements such as methylation or acetylation can also be included in the therapeutic peptide structure. These elements can enhance the stability of the therapeutic peptide, thus increasing its resistance to various degrading forces or conditions that can enhance the peptide’s therapeutic potential and longevity in the body.

In some cases, the therapeutic peptide may also contain chemical staples, specifically a hydrocarbon chain that forms a bridge between two amino acids on the peptide. The presence of this chemical staple can enhance the structure's rigidity and stability, thereby improving the therapeutic peptide's resistance to conformational changes, pharmacokinetic properties, binding affinity, and proteolytic stability.

In accordance with an embodiment of the therapeutic peptide, at least one arginine residue, preferably at least two arginine residues are situated at the N-terminus of the therapeutic peptide. This specific location of arginine residues at the N-terminus can confer certain advantages, such as enhanced peptide stability and improved cellular uptake.

Alternatively or in addition, at least two arginine residues, and more preferably at least three arginine residues are situated at the C-terminus of the therapeutic peptide. This arrangement can offer its own benefits such as an enhanced peptide stability and improved cellular uptake.

It is important to note that the specific benefits or impacts of arginine residues' location in the therapeutic peptide can vary based on the overall structure and properties of the peptide.

According to one embodiment, the sequence of the therapeutic peptide is RRYRCDHCRVLFLDYVMFTIHMGCHRRRRRRRRR (SEQ ID NO: 16).

According to one embodiment, the therapeutic peptide is for use in medical treatment. According to one embodiment, the therapeutic peptide is for use in the treatment of a cancer in a subject.

The method of treatment The inventors have designed peptides that successfully bind to the ZF5 domain of the members of the IKZF family (ZF4 in case of IKZF5) and prevent homo or heterodimerization. These peptides successfully prevent dimerization of IKZF3 and IKZF1 , thus leading to an increase in IL-2 production. As the function of the therapeutic peptides is in particular the prevention of dimerization of IKZF3 and IKZF1 , the function can be transferred to any inhibitor, which inhibits the homo or hetero dimerization of members of the IKZF family. Such inhibitor is referred to as IKZF dimerization inhibitor.

According to a second aspect, the invention relates to a method for the treatment of a cancer in a subject, comprising administering to the subject an IKZF dimerization inhibitor.

In other words, the second aspect relates to an IKZF dimerization inhibitor for use in the treatment of a cancer in a subject, wherein the use comprises administering to the subject an IKZF dimerization inhibitor.

According to one embodiment of the method, the IKZF dimerization inhibitor has a binding affinity to the ZF5 and optionally the ZF6 domain of a member of the IKZF family. The member is preferably selected from IKZF1 , IKZF2, IKZF3, IKZF4. According to one embodiment of the method, the IKZF dimerization inhibitor has a binding affinity to the ZF4 and optionally the ZF5 domain of IKZF5.

The IKZF dimerization inhibitor may be any molecule that binds to the ZF5 domain IKZF3 and/or IKZF1. In particular, the IKZF dimerization inhibitor binds to the ZF5 domain of IKZF1 , IKZF2, IKZF3, and IKZF4, and to ZF4 of IKZF5.

The IKZF dimerization inhibitor may be selected from the group consisting of an antibody, an antibody derivative, a peptide, a nucleic acid encoding the antibody or the peptide, or a small molecule.

If an antibody is engineered to target the ZF5 domain (or the ZF4 domain of IKZF5), it could bind to this site and physically obstruct the interaction surface, preventing the formation of homo or heterodimers, similar to the therapeutic peptide according to the first aspect. The sequence and structure of the ZF5 domain of IKZF3 and IKZF1 are known. Based on this, the skilled person knows how to prepare polyclonal or monoclonal antibodies binding to the ZF5 domain. The creation of antibodies against a specific domain like ZF5 of IKZF3 or IKZF1 involves the following steps:

The first step is antigen preparation. First, the ZF5 domain must be prepared as an antigen. This could involve expressing and purifying the domain itself, or a larger part of IKZF3 that includes the ZF5 domain. If the ZF5 domain cannot be expressed and purified on its own, peptides mimicking the specific regions (epitopes) of the ZF5 domain can also be synthesized and used as the antigen.

The next step is immunization. The antigen is then injected into an animal (often a mouse, rabbit, or goat) to stimulate an immune response. The immune system of the animal will produce antibodies against the antigen. This is followed by hybridoma generation (for monoclonal antibodies): For monoclonal antibodies, the B-cells producing antibodies in the immunized animal are harvested and fused with immortal myeloma cells to generate hybridomas, which are cells that can continuously produce the desired antibody. The next step is screening and selection. The hybridomas are screened for those producing antibodies that recognize and bind to the ZF5 domain. This is often done through techniques like ELISA (Enzyme-Linked Immunosorbent Assay). The hybridoma cell lines that produce antibodies recognizing the ZF5 domain are selected and cloned. The final step is production and purification: The selected hybridoma cells are grown in culture and the antibodies are purified from the culture medium for further use.

According to one embodiment, the binding epitope of the antibody or antibody derivative is a section of the amino acid sequence AA 452 to 474 of SEQ ID NO: 13. According to one embodiment, the binding epitope of the antibody or antibody derivative is a section of the amino acid sequence AA 452 to 504 of SEQ ID NO: 13. According to one embodiment, the binding epitope of the antibody or antibody derivative is a section of the amino acid sequence AA 462 to 484 of SEQ ID NO: 11 . According to one embodiment, the binding epitope of the antibody or antibody derivative is a section of the amino acid sequence. AA 462 to 514 of SEQ ID NO: 11 .

According to one embodiment, the antibody derivative is an antibody fragment.

According to one embodiment, the antibody fragment is selected from the group consisting of Fab fragments, F(ab’)2 fragments and Fab’ fragments. Fab fragments are antibody fragments that consist of the variable regions of the heavy and light chains of an antibody, as well as the first constant region of the heavy chain. Fab fragments can be produced by enzymatic digestion of full-length antibodies with papain. F(ab')2 fragments are antibody fragments that consist of two Fab fragments linked together by a disulfide bond. F(ab')2 fragments can be produced by enzymatic digestion of full-length antibodies with pepsin. Fab’ fragments are antibody fragments that consist of the variable regions of the heavy and light chains of an antibody, as well as a portion of the constant region of the heavy chain. Fab’ fragments can be produced by enzymatic digestion of full-length antibodies with papain followed by reduction of the disulfide bonds connecting the heavy chains. These fragments are commonly used in research and diagnostic applications and methods for the generation of antibody fragments are not particularly limited and are known in the art.

According to one embodiment, the antibody derivative is an antibody mimetic. The antibody mimetic according to the invention may be selected from the group consisting of single-chain variable fragments (scFv), single-domain antibodies, affibodies, affilins, affimers, affitins, anticalins, DARPins, monobodies, and peptide aptamers.

Single-chain variable fragments (scFv) are a type of antibody fragment that consist of the variable domains of the heavy and light chains of an antibody linked together by a short peptide linker. They can be produced in bacteria, yeast, or mammalian cells. Single-domain antibodies, also known as nanobodies, are antibody fragments that consist of a single variable domain from either the heavy or light chain of an antibody. They are smaller and more stable than traditional antibodies. Affibodies are small protein scaffolds that are engineered to bind to specific targets with high affinity and specificity. They are based on the B-domain of protein A, which is a natural ligand for the Fc region of antibodies. Affilins are a type of small protein scaffold that are engineered to bind to specific targets with high affinity and specificity. They are based on the cystatin protein family, which are natural protease inhibitors. Affimers are a type of protein scaffold that are engineered to bind to specific targets with high affinity and specificity. They are based on a protein called staphylococcal nuclease. Affitins are a type of protein scaffold that are engineered to bind to specific targets with high affinity and specificity. They are based on a protein called ExbB, which is involved in iron transport in bacteria, and can be used in research, diagnostic, and therapeutic applications. Anticalins are a type of protein scaffold that are engineered to bind to specific targets with high affinity and specificity. They are based on a protein called lipocalin, which is involved in the transport of small hydrophobic molecules. DARPins, or designed ankyrin repeat proteins, are a type of protein scaffold that are engineered to bind to specific targets with high affinity and specificity. They are based on the ankyrin repeat protein family, and can be used in research, diagnostic, and therapeutic applications. Monobodies are a type of antibody mimetic that consist of a single protein domain engineered to bind to specific targets with high affinity and specificity. They are based on the fibronectin type III domain, and can be used in research, diagnostic, and therapeutic applications. Peptide aptamers are a type of protein scaffold that consist of a short peptide sequence that is engineered to bind to specific targets with high affinity and specificity. They are often generated using phage display or other selection methods, and can be used in research, diagnostic, and therapeutic applications. Respective antibody mimetics and methods for producing the same are not particularly limited and are known in the art.

With the known sequence and structural information for the ZF5 domain (see AlphaFoldDB Q9UKT9 for the predicted structure of IKZF3 and AlphaFoldDB Q13422 for the predicted structure of IKZF1 ) and the identified therapeutic peptides that bind to the ZF5 domain and inhibit its dimerization, and therefore the dimerization of the IKZF3 and/or IKZF1 , the skilled person can design or identify small molecules that can do the same. The skilled person would, for example use the following approach to identify small molecules as IKZF dimerization inhibitors.

The skilled person will, for example, start with an in-depth understanding of the ZF5 domain, particularly its structure and the region that mediates dimerization, i.e. the peptides' binding site. The skilled person may then use in silico methods to screen large libraries of small molecules for potential binders. This often involves molecular docking, where the small molecules are computationally 'docked' into the binding pocket to predict their binding mode and affinity. The next step may be in vitro high- throughput screening: This is an experimental method where a large library of small molecules is tested for binding to the ZF5 domain or for their ability to inhibit ZF5- mediated dimerization. This process can identify potential 'hit' compounds. This is followed by hit validation and optimization: Once potential small molecules have been identified, these hits are validated in secondary assays to confirm their activity. Biophysical methods like surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) can be used to confirm binding. Hits are then optimized through a cycle of medicinal chemistry, where their structures are systematically modified to improve their potency, selectivity, and drug-like properties. The optimized molecules are then tested again for activity. The small molecules are then tested in cell-based assays to confirm their activity i.e. the impact on IL-2 production. This may be followed by in vivo testing.

The peptide may be a peptide derived from the IKZF3-binding domain of IKZF1 or from the IKZF1 -binding domain of IKZF3. Specifically, the peptide may be a therapeutic peptide according to the first aspect.

According to one embodiment, the IKZF dimerization inhibitor a nucleic acid encoding the antibody or the peptide. According to one embodiment, the nucleic acid encoding is an mRNA molecule. According to one embodiment, the mRNA molecule encodes an antibody. According to one embodiment, the mRNA molecule encodes a therapeutic peptide according to the first aspect as described above. Administering an IKZF dimerization inhibitor using an mRNA-based approach would involve a slightly different process than traditional mRNA vaccines. Instead of encoding an antigen to stimulate an immune response, the mRNA would encode the antibody or therapeutic itself.

In the following, the process is briefly outlined. A) Design and production: The mRNA sequence encoding the desired antibody or therapeutic peptide is synthesized. This sequence would typically consist of both the heavy and light chains of the antibody, which are optimized for stability, efficient translation, and reduced immunogenicity. B) Delivery system: Similar to the mRNA vaccines, the mRNA encoding the antibody may be encapsulated in lipid nanoparticles (LNPs) to protect it from degradation and facilitate its entry into host cells. C) Administration: The mRNA-antibody construct is injected into the recipient, usually via intramuscular or subcutaneous routes, in the present case using one of the administration routes listed below, e.g. by intraocular application. The mRNA is taken up by cells at the injection site. D) Translation and secretion: Once inside the host cells, the mRNA is released from the LNPs and translated by cellular machinery to produce the antibody. The antibody's heavy and light chains assemble into a functional antibody, which is then secreted from the cell into the extracellular space and subsequently the bloodstream. E) Binding and neutralization: The synthesized IKZF dimerization inhibitor specifically binds IKZF1 and/or IKZF3, which leads to increasing the IL-2 secretion.

According to this invention, the IKZF dimerization inhibitor may be administered by any appropriate route, but preferably by a route, which transmits the peptide directly into the blood, e.g., intravenous injection. Subcutaneous injection is also a useful mode of administration. Other routes of administration include, without limitation, oral, intradermal, transdermal, intraperitoneal, intramuscular, intrathecal, mucosal (e.g., intranasal), and by inhalation.

The amount of the IKZF dimerization inhibitor, i.e. therapeutic peptide, antibody, antibody derivative, nucleic acid or small molecule of the invention present in each effective dose is selected with regard to consideration to the half-life of the compound, the identity and/or stage of the cancer, the patient's age, weight, sex, general physical condition and the like.

The amount of active component required to induce an effective immunomodulatory effect on cancer cells without significant adverse side effects varies depending upon the pharmaceutical composition employed and the optional presence of other components. Generally, for the compositions containing protein/peptide, or fusion protein, each dose will comprise between about 5 pg peptide/kg patient body weight to about 10 mg/kg. Generally, a useful therapeutic dosage is between 1 to 5 mg peptide/kg body weight. Another embodiment of a useful dosage may be about 500 pg/kg of peptide. Other dosage ranges may also be contemplated by one of skill in the art. For example, dosages of the therapeutic peptides of this invention may be similar to the dosages discussed for other peptide cancer therapeutics and dosages of the IKZF dimerization inhibitor of this invention may be similar to the dosages discussed for other antibody cancer therapeutics.

In accordance with an embodiment of the method, the IKZF dimerization inhibitor is administered in conjunction with a carrier. This carrier can assist in the delivery of the inhibitor to the desired location in the body and can potentially enhance the inhibitor's therapeutic efficacy. In a preferred embodiment, the carrier is selected from the group consisting of liposomes and nanoparticles. The use of these carriers can provide various advantages. For example, liposomes, which are small spherical vesicles, can protect the IKZF dimerization inhibitor from degradation in the body, facilitate its passage across biological barriers, and direct its delivery to specific cells or tissues. Similarly, nanoparticles, which are particles of a very small size typically in the nanometer range, can offer enhanced delivery and targeting capabilities.

However, the carrier selection depends on several factors including the specific properties of the IKZF dimerization inhibitor, the desired delivery route, the target cells or tissues, and the patient's overall condition. Therefore, a carrier that is most suitable for a given application may be selected from liposomes, nanoparticles, or other suitable materials based on these considerations.

According to one embodiment, the method further involves administering a therapeutic agent. This therapeutic agent may be selected from a group consisting of immune checkpoint inhibitors, which may include but are not limited to anti-PD-1 , PD- L1 , CTLA-4, TIGIT, LAG-3, TIM-3 antibodies, or other cancer immunomodulation agents.

Specifically, in one such embodiment, the therapeutic agent can be an anti-PD-1 antibody, which functions by blocking the programmed death-1 (PD-1 ) pathway, a mechanism often exploited by cancers to evade the immune system. The use of this agent in combination with the IKZF dimerization inhibitor could bring about the advantage of 1 ) a synergistic effect with immune checkpoint inhibitors, 2) augmented T-cell proliferation and survival which should help achieve better response in the patients, and/or 3) overcoming immunosuppression which is a major problem for many tumors.

Alternatively or in addition, the therapeutic agent could be an anti-PD-L1 , anti-CTLA- 4, anti-TIGIT, anti-LAG-3, or anti-TIM-3 antibody. Each of these agents inhibits a specific immune checkpoint pathway, thereby enhancing the immune system's ability to attack cancer cells. The therapeutic agent could also be any other cancer immunomodulation agents, which modulate the immune system in a way that improves its ability to fight against cancer.

It should be noted that the choice of the therapeutic agent will depend on various factors, including the specific type of cancer, the stage of the disease, the patient's overall health, and other treatments the patient may be receiving. Therefore, the therapeutic agent that is most suitable for a given application can be chosen based on these considerations.

In one embodiment, the method further comprises employing cancer cell therapy approaches. These include the adoptive transfer of allogenic or autologous cells.

In one such embodiment, the adoptive transfer involves allogenic cells. These are cells that originate from a donor other than the patient. The use of the IKZF dimerization inhibitor together with allogenic cells in cancer cell therapy can have certain advantages, such as 1 ) enhanced cellular expansion and persistence, 2) improved immune cell functionality, 3) overcoming immunosuppression, and 4) achieving a synergistic effect.

In another embodiment, the adoptive transfer involves autologous cells. These are cells that are harvested from the patient, possibly modified or expanded ex vivo, and then re-introduced back into the patient. The use of the IKZF dimerization inhibitor together with autologous cells can have distinct advantages, such as 1 ) enhanced cellular expansion and persistence, 2) improved immune cell functionality, 3) overcoming immunosuppression, 4) achieving a synergistic effect.

The choice between allogenic or autologous cells will depend on various factors, including the specific type of cancer, the stage of the disease, the patient's overall health, and the availability of a suitable donor in the case of allogenic cells. Therefore, the most appropriate type of cell for adoptive transfer can be selected based on these considerations.

According to one embodiment of the method, the cancer is selected from the group consisting of lymphatic neoplasia and immunogenic solid tumors, preferably selected from myeloma, renal cancers, melanoma, lung cancer, breast cancers, head and neck cancers, pancreatic cancer, more preferably myeloma.

In the method of the second aspect, the IKZF dimerization inhibitor may be a therapeutic peptide defined according to first aspect. All the embodiments described above in connection with the first aspect of the invention apply to the method of the second aspect of the invention.

The

According to a third aspect, the invention provides an isolated polynucleotide that comprises a nucleic acid sequence encoding a therapeutic peptide according to the first aspect of the invention.

The isolated polynucleotide may be a DNA molecule or an RNA molecule. The isolated polynucleotide is preferably a DNA molecule, in particular a cDNA molecule. The techniques used to isolate or clone a polynucleotide encoding a peptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the polynucleotides from such genomic DNA can be effected, e.g., by using the well-known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features (see, e.g., Innis et al, 1990). Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligation activated transcription (LAT) and polynucleotide-based amplification (NASBA) may be used.

vector

In a fourth aspect, the invention also relates to expression vectors comprising a polynucleotide according to the third aspect of the invention.

The expression vector further preferably comprises control elements such as a promoter, and transcriptional and translational stop signals. The polynucleotide according to the second aspect and the control elements may be joined together to produce a recombinant expression vector that may include one or more restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites. The polynucleotide may be inserted into an appropriate expression vector for expression. In creating the expression vector, the coding sequence is located in the expression vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or a virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide of the fourth aspect of the invention. The choice of the expression vector will typically depend on the compatibility of the expression vector with the host cell into which the expression vector is to be introduced. The expression vectors may be a linear or closed circular plasmid.

The expression vector is preferably adapted to expression in mammalian cells. The expression vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicator" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

The vector is preferably one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. For integration into the host cell genome, the expression vector may rely on any other element of the expression vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location in the chromosome.

The vectors of the present invention preferably contain one or more (e.g., several) selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

The procedures used to ligate the elements described above to build the recombinant expression vectors of the present invention are well known to one skilled in the art (see Green and Sambrook 2012; Chapter 3)).

According to one embodiment the vector backbone of the vector according to the third aspect is selected from pCDNA3, pCDNA3.1 , pCDNA4, pCDNA5, pCDNA6, pCEP4, pCEP-puro, pCET1019, pCMV, pEF1 , pEF4, pEF5, pEF6, pExchange, pEXPR, pIRES, and pSCAS.

Host Cell

According to a fifth aspect, the invention relates to a host cell containing the nucleic acid according to the third aspect or the vector according to the fourth aspect. The expression vector according to the third aspect is introduced into a host cell so that the expression vector is maintained as a chromosomal integrant or as a selfreplicating extra-chromosomal vector as described earlier. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.

According to one embodiment the fusion protein is produced by expression in a mammalian host cell line. The fusion protein is preferably produced in a human host cell line. Generally, any human host cell line is suitable for expression of the fusion protein. The host cell is preferably of human origin in order to ensure that the fusion protein is properly processed during folding and receives the proper post-translational modifications (e.g. glycosylation, hydroxylation, phosphorylation and sulfation). A favourable glycosylation profile of the fusion protein is particularly obtained with human kidney cell lines. Preferred human kidney cell lines are HEK cell-lines, in particular HEK 293 cell lines.

Examples of HEK cell-lines for production of the glycosylated polypeptide are HEK 293 F, Flp-ln ^TM-293 (Invitrogen, R75007), 293 (ATCC® CRL-1573), 293 EBNA, 293 H (Thermo Scientific 11631017), 293S, 293T (ATCC® CRL-3216™), 293T/17 (ATCC® CRL11268™), 293T/17 SF (ATCC® ACS4500™), HEK 293 STF (ATCC® CRL 3249™), HEK-293.2sus (ATCC® CRL-1573™). A preferred cell line for production of the polypeptide is the HEK 293 F cell line.

Other human cell lines suitable as host cells for expression include, without limitation, cell lines derived from myeloid leukemia cells. Specific examples of host cells are K562, NM-F9, NM-D4, NM-H9D8, NM-H9D8-E6, NM H9D8-E6Q12, GT-2X, GT-5s and cells derived from anyone of said host cells. K562 is a human myeloid leukemia cell line present in the American Type Culture Collection (ATCC CCL-243). The remaining cell lines are derived from K562 cells and have been selected for specific glycosylation features.

Further mammalian host cell lines suitable for producing fusion proteins according to the invention include cell lines of hamster, mouse, and monkey origin. Suitable host cells include Chinese hamster ovary cells (CHO cells, e.g., DG44, DXB11 , and K1 [ATCC CCL-61 , including its glutamine auxotroph derivative CHOZn, SAFC CHOGS]) and baby hamster kidney (BHK) cells.

EXAMPLES

Example 1 - The Identification of binding partners of IKZF3

Coimmunoprecipitation (co-IP) combined mass spectrometry analysis of 4 sorted healthy donors CD8+ T cells was used and exhaustion with strong activation of CD3/CD28 dynabeads was induced. Cells were only used in a pull-down co-IP assay capturing the full length IKZF3 protein. Mass spectrometry analysis to identify binding proteins were performed and analysed at the Proteomics Core Facility at EMBL Heidelberg. Benjamini Hochberg (BH) adjusted p-value threshold <0.05 and Iog2 Fold Change (FC) threshold of 1 were set to determine potential interaction partners of IKZF3. We found that IKZF1 was highly enriched as a heterodimer of IKZF3 (Figure 2). Further, mass spectrometry analysis showed that not only IKZF1 , but also ribosomal proteins, proteins for splice variant expression and proteins of posttranslational modification were increased in the samples as well.

Example 2 - Identification of binding site of heterodimerization domain in IKZF3

As a next step, the binding domain of IKZF1 to IKZF3 was sought for in a proteinprotein binding assay. An assay in human embryonic kidney 293T (HEK 293T) cells was established as they do not express members of the IKZF protein family.

Cells were co-transfected with full length IKZF1 protein, and recombinant gluthathione S transferase (GST)-fused on the C-terminal of the full length IKZF3 protein (GST- IKZF3_V1 ) or different GST-tagged variants of IKZF3 (GST-IKZF3_V1 to GST- IKZF3_V5). For IKZF3 variant expression and determination of the binding site, we designed GST fused variants, which only lacked one functional domain in each variant. GST-IKZF3_V1 contains aa1 - aa519, GST-IKZF3_V2 aa1 - aa240, GST- IKZF3_V3 aa1 - aa119, GST-IKZF3_V4 aa118 - aa240 and GST-IKZF3_V5 aa240 - aa519. By a pull-down co-IP assay with anti-GST beads, the complex was isolated and IKZF1 expression was captured by immunoblot analysis.

The results showed that IKZF1 binds to full-length IKZF3 protein, but also to GST- IKZF3 variant 5, which comprises the two C-terminal zinc finger domains of IKZF3 for protein interaction (Figure 3). Taken together, the data determined that IKZF1 and IKZF3 heterodimerized in exhausted CD8+ T cells, potentially binding to one or both two zinc finger domains at the C-terminal of the IKZF3 transcription factor. Moreover, ribosomal and proteins for posttranslational modification were also associated to the heterodimer complex.

Example 3 - Designing Blocking Peptides

As the results showed that the amino acid from 240 to 519 are responsible for the binding, the binding site was expected to be one of the highly conserved ZF domains at the C terminal.

• C1 : represents the 5th ZF domain of the IKZF3 protein and consists of the amino acids from 452 to 474: (YRCDHCRVLFLDYVMFTIHMGCH);

To narrow down the binding site two peptide constructs were: designed mimicking the natural Zinc finger domains at the C-terminus in order to block the formation of IKZF1 - IKZF3 heterodimers. The natural peptide from IKZF3 exhibited a very low net charge at pH of 7. This was expected to hinder its internalization inside the cells. Therefore, an arginine-rich derivative peptide was designed which exhibited a net charge of about 6 at pH 7. The peptide was labelled Fluorescein isothiocyanate (FITC) in order to monitor the peptide internalization with flow cytometry. After confirming the internalization of the peptide, the peptides were tested on cellular models.

Table 1

C1 M = SEQ ID NO: 32

C2M = SEQ ID NO: 33

Ctrl = SEQ ID NO: 34

Example 4 - Testing blocking peptide mimicking the natural domain

C1 IKZF3 Domain Modified peptide (C1 M) (Table 1 ) was tested in cell culture model to examine its ability to abrogate the IKZF1 -IKZF3-induced IL-2 suppression. 18 different healthy donors were used to isolate the T cells using magnetic cell sorting approach. Afterwards, T cells were cultured for 24 hours in serum-free T cell medium and either 10pM of the C1 M peptide or Scrambled as a negative control or 10pM Lenalidomide or 1 pM of Pomalidomide (as an IMiD control which have been previously described).

After 24 hours, the supernatant was collected and centrifuged to remove any cells and stored in -20 degrees. An enzyme-linked immunosorbent assay (ELISA) for the secretion of IL-2 was then performed to accurately assess the secretion of IL2 from each condition.

Interestingly, in all cases, IL-2 secretion increased when treating the cells with C1 M peptide, we performed 3 replicated of the experiment with each 18 different healthy donors’ cells and confirmed the findings (Figure 4A and 4B). To confirm that the increase of IL-2 is a specific increase of IL-2 and not due to random increase in the cellular secretion, we performed a cytokines screening assay at Sciomics GMBH. 119 different cytokines were screened in the supernatant from the same previous experiment and showed strong specific increase in IL-2, IL-6 and INFy secretion from T cells (Figure 5).

Example 5 - The Ala Scanning Peptides Approach

Ala scanning, also known as alanine scanning mutagenesis, is a commonly used approach in peptide or protein engineering to identify which amino acid residues are essential for the function of the molecule. This approach involves systematically substituting each amino acid residue in the molecule with alanine, a nonpolar amino acid with minimal steric hindrance. The basic principle of Ala scanning is to identify which amino acid residues contribute to the overall function of the molecule by testing the activity of each variant against the original molecule. By comparing the activity of each alanine-substituted variant to the original molecule, it is possible to identify, which amino acid residues are essential for the overall function of the molecule. The Ala scanning approach typically involves generating a series of mutant peptides or proteins, in which each amino acid residue in the original molecule is replaced with alanine, one at a time. The resulting variants are then tested in a functional assay, such as measuring the ability of the peptide to bind to a target molecule, or its ability to induce a specific cellular response.

Therefore, we evaluated the peptides using two different approaches:

A) ELISA IL-2:

In this assay, T cells were isolated from the peripheral blood of healthy donors and cultured in serum-free medium. The cells were then activated with anti-CD3/CD28 beads and cultured in the presence of either 10 pM of the testing peptide or a 10 pM scrambled control peptide. After 24 hours, the supernatants from the cells were collected, and the levels of IL-2 were measured using a sensitive ELISA kit according to the manufacturer's instructions. This approach was previously used in our analyses and has been shown to provide reliable and reproducible results. By comparing the levels of IL-2 secretion in cells treated with the testing peptide versus the control peptide, we were able to determine the peptide's ability to increase IL-2 secretion in T cells. This assay provided a quantitative readout of the peptide's activity and allowed us to accurately assess the effectiveness of the peptide in promoting IL-2 secretion.

B) IKZF1/IKZF3-Peptide Binding Assay

To overcome the challenge of testing peptides that are not soluble in appropriate solvents, we utilized a method in which we coated polystyrene plates with the peptides. This allowed us to test the functionality of the peptides, even if they were not soluble in solvents that are typically used for cell culture approaches. We used a sandwich ELISA approach, briefly, we coated polystyrene plates with the testing peptides and incubated them overnight to allow the peptides to bind to the plate. Next, we used these coated plates to test the ability of the peptides to capture IKZF1 or IKZF3 from lysates of HEK293 cells that had been transiently transfected with the respective IKZF protein. We chose to use HEK293T cells for this purpose, as they do not normally express IKZF1 and IKZF3. To detect the captured IKZF proteins, we incubated the coated plates with antibodies specific to IKZF1 or IKZF3. We then used a secondary antibody conjugated to horseradish peroxidase (HRP) to detect the primary antibody and quantified the proteins using a chemiluminescence detection substrate (Figure 6). This sandwich ELISA approach allowed us to determine the ability of the testing peptides to bind to IKZF1 or IKZF3. By comparing the signal obtained from the peptides to that obtained from a scrambled control, we were able to determine whether the peptides had specific binding activity for the target proteins. This approach provided a quantitative readout of the peptides' binding activity and allowed us to accurately assess their effectiveness in capturing IKZF1 or IKZF3, which is hypothesized as a mechanism of action.

Before the Ala scanning approach, we designed and synthesized three peptides with overlapping sequences that spanned 8 amino acids each, namely Pep M1 (SEQ ID NO: 35), Pep M2 (SEQ ID NO: 36), and Pep M3 (SEQ ID NO: 37), as detailed in the table in Figure 9. To enhance their internalization ability in cells, all three peptides were modified with arginine-rich domains. To assess the solubility of the peptides, we conducted solubility tests. We found that Pep M2 and Pep M3 exhibited very good solubility, while Pep M1 showed no solubility in an appropriate solvent. So, all peptides were analysed for their IKZF1/IKZF3 binding, and only Pep M2 and Pep M3 were tested for IL-2 on T cells as well.

The results are shown in Figure 7 A and B. Compared to the scrambled control peptides, only Pep M1 and Pep M2, but not Pep M3 exhibited an activity similar to that observed with C1 M peptide the binding assay. (Figure 7A, 7B). The result was confirmed for Pep M2 in the IL-2 ELISA (in which Pep M1 could not be tested for lack of solubility). These results indicate a possible importance of amino acids L9 to M20 of C1 M.

In order to identify the functional peptides, we considered a threshold representing 50% of the activity of the original peptide C1 M. So, a peptide isoform that exhibits almost 50% of the original C1 M was considered a functional peptide. The threshold was added to all figures as dotted line. The Cut-off was set the following values based on our previous experiments:

An Ala-scanning group was designed in which substitute each amino acid from L9 to H23 (15 peptides, see table in Figure 9: Ala Scanning Group). All of the peptides were modified with arginine-rich domains to increase their internalization. Based on a solubility test showed that PepAla 3, PepAla 4 and PepAla 11 were not soluble in the appropriate solvent for IL-2 analyses. All peptides were analysed for their IKZF1/IKZF3 binding, and on T cells for IL-2 increase (if soluble).

The analyses showed that:

- The removal of V14 slightly decreases the peptide function (represented by Ala 6).

- The removal of F16 to M20 strongly decreases/abolish the peptide function (represented by Ala 8 to Ala 12).

- The removal of C22 also showed decrease in the IL-2 function, despite no evidence being observed in terms of IKZF1/IKZF3 binding (represented by A14). (See Figure 8A, 8B & 8C)

There is a discrepancy in the reported binding data and the effectiveness of the C1 M peptide in promoting IL-2 secretion between Figures 7B and 8B. It must be recognized that variability in in vitro assays is a standard and expected part of experimental work. This variability is largely due to differences in human donor cells and their responses, which is why rigorous statistical tests are applied to determine significant differences.

Human donor cells exhibit a wide range of responses to experimental conditions due to genetic, epigenetic, and environmental differences. This natural variability is particularly pronounced in immunological assays.

For instance, when assessing IL-2 secretion upon IKZF blocking and T cell activation, different donors' cells can behave very differently. It is not uncommon to observe variations in outcomes by as much as 20 times between different donors. This variability reflects the complex nature of the human immune system, where individual responses to the same stimulus can vary significantly.

To address this variability and draw reliable conclusions, we apply rigorous statistical tests to our data. These tests help identify significant differences and ensure that our findings are not due to random variation. By using appropriate statistical methods, we can confidently determine the effectiveness of the C1 M peptide in inhibiting IKZF dimerization and promoting IL-2 secretion, despite the natural variability in donor cell responses.

Example 6 - FLIM assay for testing the Inhibition of dimerization of IKZFs

To confirm that the C1 M peptide inhibits the homo or heterodimerization of IKZF proteins the inventors used a fluorescence lifetime imaging microscopy (FLIM) assay, which is a powerful technique to study protein-protein interactions within live cells by detecting energy transfer between two closely positioned fluorophores.

FLIM occurs when two fluorescent proteins are in close proximity (typically 1 -10 nm), resulting in energy transfer from one fluorophore to another, leading to a measurable shift in the lifetime of the fluorescence signal of one fluorophore compared to the lifetime of the flurorophore alone. In this application, FLIM is used to determine the dimerization state of IKZF proteins.

Experimental Design 1. Fluorescent Tagging

IKZF1 and IKZF3 were fused with a fluorescent protein, such as Green Fluorescent Protein (GFP) or mCherry (a red fluorescent protein). Methods for tagging protein mit fluorescent tags are known to the skilled person.

2. Validation of Homo and Heterodimerization:

Homo-dimerization: To validate the homo-dimerization of IKZF1 and IKZF3 IKZF1 -

GFP was co-expressed with IKZF1 -mCherry and IKZF3-mCherry with IKZF3-GFP in cells. When these proteins dimerize, the close proximity of the fluorophores results in a detectable change in the lifetime of the fluorescence signals compared to the lifetime of the flurorophores in the unbound proteins.

Hetero-dimerization: For heterodimerization, IKZF1 -GFP was co-expressed with

IKZF3-mCherry. An interaction between IKZF1 and IKZF3 brings GFP and mCherry into proximity, which would result in a detectable change a detectable change in the lifetime of the fluorescence signals compared to the lifetime of the flurorophores in the unbound proteins.

3. Peptide Intervention:

Upon introducing the C1 M peptide, a reduction of the FLIM signal shift should be observed if the peptide inhibits the homo- or hetero-dimerization. This would indicate that the C1 M peptide disrupts the interaction between IKZF1 and IKZF3, validating its function in inhibiting heterodimerization.

Results:

The results are shown with the phasor approach in phasor plots. In FLIM, phasor can be used to visualize the spectra and decay curves. In this method the Fourier transformation of the spectrum or decay curve is calculated and the resulted complex number is plotted on a 2D plot where the X axis represents the Real component and the Y axis represents the Imaginary component. This facilitates the analysis since each spectrum and decay is transformed into a unique position on the phasor plot, which depends on its spectral width or emission maximum or to its average lifetime. The most important feature of this analysis is that it is fast and it provides a graphical representation of the measured curve.

1. Homo-dimerizatioir.

As shown in Figure 11 , the FLIM experiment confirms the homo dimerization of both IKZF1 and IKZF3.

Specifically, the signal of GFP (upper phasor plot in Figure 11 A) and IKZF1 -GFP (middle phasor plot in Figure 11 A ) is at a comparable position (close to the left vertical dotted line). In the experiment co-expressing IKZF1 -GFP with IKZF1 -Cherry (lower phasor plot in Figure 11 A), the GFP signal is significantly shifted to right, i.e. into the middle of the two vertical dotted lines. This shift is a proof for the interaction of IKZF1 -GFP with IKZF1 -Cherry, i.e. a homodimerization.

The results of the same experiment using IKZF3 are shown in the lower panel of Figure 11. The signals of GFP (upper phasor plot in Figure 11 B) and IKZF3-GFP (middle phasor plot in Figure 11 B) are at an identical position (close to the left vertical dotted line). In the experiment co-expressing IKZF3-GFP with IKZF3-Cherry (lower phasor plot in Figure 11 B), the GFP signal is significantly shifted to right, i.e. into the middle of the two vertical dotted lines. This shift is a proof for the interaction of IKZF3- GFP with IKZF3-Cherry and therefore a homodimerization of IKZF3.

2. Hetero-dimerization:

Moreover, as shown in Figure 12, also the heterodimerization of IKZF1 and IKZF3 is confirmed by FLIM. Specifically, the results for GFP and IKZF1 -GFP are confirmed, which means that the signals of GFP (upper phasor plot of Figure 12) and IKZF1 -GFP (middle phasor plot of Figure 12) are at an identical position (close to the left vertical dotted line). In the experiment co-expressing IKZF1 -GFP with IKZF3-Cherry (lower phasor plot in Figure 12), the GFP signal is significantly shifted to right, i.e. smeared out towards the middle of the two vertical dotted lines. This shift is a proof for the interaction of IKZF1 -GFP with IKZF3-Cherry, i.e. a heterodimerization of IKZF1 and IKZF3. Co-expression of IKZF1 -GFP and IKZF3-mCherry resulted in a clear FLIM signal shift as well, indicating successful heterodimerization (shorter lifetime signal indicates clear interaction, Figure 12).

3. Peptide Intervention:

It was then confirmed that C1 M inhibits the hetrerodimerization of IKZF1 and IKZF3. As shown Figure 13, the results for GFP, IKZF1 -GFP and IKZF3-GFP are confirmed, which means that the signals of GFP (first phasor plot from the top of Figure 13), IKZF1 -GFP (second phasor plot from the top of Figure 13) and IKZF3-GFP (third phasor plot from the top of Figure 13) and are at an identical position (close to the left vertical dotted line). In the experiment co-expressing IKZF1 -GFP with IKZF3- Cherry and scrambled peptide (fourth phasor plot from the top of Figure 13), the GFP signal is significantly shifted to right, i.e. smeared out towards the middle of the two vertical dotted lines. This shift confirmed the interaction of IKZF1 -GFP with IKZF3- Cherry, i.e. a heterodimerization of IKZF1 and IKZF3. As the signal is comparable to the one in the lower panel of Figure 12, the peptides have no influence on the signal, which means that they not interfere with the dimerization.

In contrast, addition of the C1 M peptide to the co-expression of IKZF1 -GFP with IKZF3-Cherry led to decrease in the shift caused by heterodimer of IKZF1/IKZF3. This clearly shows the blocking of the dimerization.

We then measured the effect directly on the nuclei to prove the heterodimerization inhibition in the functional core. In contrast to the previous approach, in which the whole signal from the cell (signal coming from cytosol and nuclei) was measured (see Figure 13), this experimental set up, we only measured the signal from the nuclei. As shown in Figure 14, the measurement in the nuclei confirmed the previous measurement in the whole cell: The phasor plots in Figure 14 show a similar result as seen in the two bottom plots of Figure 13. Accordingly, also when measuring in the nucleus, the samples with IKZF1 -GFP/IKZF3-mCherry and scrambled peptide have a higher concentration of shorter lifetimes (similar to the Ctrl, not shown) than the samples with IKZF1 -GFP/IKZF3-mCherry and C1 M.

The results showed that C1 M interfered with (i.e. prevents more) the interaction of IKZF1 with IKZF3, resulting in less shorter lifetimes than with scrambled (Figure 14). Thus, C1 M has access to the nucleus, where the IKZF1/IKZF3 heterodimer functions by binding to the DNA, and blocks of the dimerization also in the nucleus.

Example 7 - Cytotoxicity of the therapeutic peptides

To illustrate the therapeutic relevance of the previous results, i.e. the inhibition of IKZF1/3 homo and hetero dimerization and the consequent IL-2 activation, we provide the additional experimental evidence.

Method

T cells from healthy donors were incubated for 24 hours with either a scrambled peptide, C1 M peptide, or no peptide. Subsequently, these T cells were co-cultured with NCI-H929 tumor cells for either 24 hours or 48 hours.

Flow cytometry was used to analyze the percentage of tumor cells among live cells (n=6).

Results

The results are shown in Figure 15 and indicate that T cells pre-cultured with C1 M peptide killed significantly more tumor cells than those treated with scrambled peptide or the control, demonstrating the potent activation effect of the C1 M peptide on T cells.

This show that treatment of T cells with the C1 M peptide strongly enhanced their killing of tumor cells. Specifically, we have observed increased cytotoxic activity in T cells treated with C1 M peptide compared to controls, demonstrating that the peptide not only activates T cells but also boosts their ability to eliminate tumor cells (Figure 15)

The enhanced killing of tumor cells is likely due to the increased activation and proliferation of cytotoxic T cells driven by IL-2 upregulation.

By inhibiting the homo and heterodimerization of IKZF proteins, the C1 M peptide prevents the exhaustion of T cells, maintaining their cytotoxic potential and improving their efficacy in targeting tumor cells. Conclusion

The increase in IL-2 production is a well-established marker of T cell activation and has significant implications for enhancing immune responses against tumors. The resulting cytotoxicity of the therapeutic peptides according to the invention is confirmed experimentally.

Many modifications and other embodiments of the invention set forth herein will come to mind to the one skilled in the art to which the invention pertains having the benefit of the teachings presented in the foregoing description and the associated drawings. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

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Claims

C l a i m s

1. A therapeutic peptide, comprising a section which inhibits the homo or hetero dimerization of members of the Ikaros family of zinc finger proteins (IKZF inhibiting section), wherein the length of the peptide is in the range of 9 amino acids to 100 amino acids and wherein the amino acid sequence of the IKZF inhibiting section comprises SEQ ID NO: 1 (FTIHM) or SEQ ID NO: 2 (YTIHM).

2. The therapeutic peptide according to claim 1 , wherein the IKZF inhibiting section inhibits the homo or heterodimerization of Ikaros (IKZF1 ) and Aiolos (IKZF3).

3. The therapeutic peptide according to claim 1 or 2, wherein the amino acid sequence of the IKZF inhibiting section comprises

SEQ ID NO: 3 (XXXTIHM), wherein Xaa at position 1 is Vai or lie, preferably Vai, wherein Xaa at position 3 is Phe or Tyr, preferably Phe, and wherein preferably Xaa at position 2 is Met or Leu, more preferably Met; or

SEQ ID NO: 4 (XXXTIHMXX), wherein Xaa at position 1 is V or I, preferably V, wherein Xaa at position 3 is Phe or Tyr, preferably Phe, and Xaa at position 9 is Cys, wherein preferably Xaa at position 2 is Met or Leu, more preferably Met and wherein preferably Xaa at position 8 is Gly.

4. The therapeutic peptide according to any one of the preceding claims, wherein the IKZF inhibiting section comprises a sequence selected from SEQ ID NO: 5 (VMFTIHM), SEQ ID NO: 6 (VMFTIHMGC), SEQ ID NO: 7 (YVMFTIHMGCH), SEQ ID NO: 8 (RVLFLDYVMFTIHMG), SEQ ID NO: 9 (LFLDYVMFTIHMGCH,), or SEQ ID NO: 10 (YRCDHCRVLFLDYVMFTIHMGCH).

5. The therapeutic peptide according to any one of the preceding claims, wherein IKZF inhibiting section comprises a) an amino acid sequence that is at least 90 % identical to AA 452 to 474 of SEQ ID NO: 13 and optionally an amino acid sequence that is at least 90 % identical to AA 480 to 504 of SEQ ID NO: 13; b) an amino acid sequence that is at least 90 % identical to AA 452 to 504 of SEQ ID NO: 13; c) an amino acid sequence that is at least 90 % identical to AA 462 to 484 of SEQ ID NO: 11 and optionally an amino acid sequence that is at least 90 % identical to AA 490 to 514 of SEQ ID NO: 11 ; d) an amino acid sequence that is at least 90 % identical to AA 462 to 514 of SEQ ID NO: 11 ; e) an amino acid sequence that is at least 90 % identical to AA 471 to 493 of SEQ ID NO: 12 and optionally an amino acid sequence that is at least 90 % identical to AA 499 to 523 of SEQ ID NO: 12 f) an amino acid sequence that is at least 90 % identical to AA 471 to 523 of SEQ ID NO: 12; g) an amino acid sequence that is at least 90 % identical to AA 530 to 552 of SEQ ID NO: 14 and optionally an amino acid sequence that is at least 90 % identical to AA 558 to 582 of SEQ ID NO: 14; h) an amino acid sequence that is at least 90 % identical to AA 530 to 582 of SEQ ID NO: 14; i) an amino acid sequence that is at least 90 % identical to AA 364 to 386 of SEQ ID NO: 15 and optionally an amino acid sequence that is at least 90 % identical to AA 392 to 416 of SEQ ID NO: 15; and j) an amino acid sequence that is at least 90 % identical to AA 364 to 416 of SEQ ID NO: 15.

6. The therapeutic peptide according to claim 5, wherein the amino acid identity is at least 95 %, more preferably at least 98 %, most preferably 100 %.

7. The therapeutic peptide according to any one of the preceding claims, wherein the length of the IKZF inhibiting section is less than 80 amino acids, preferably less than 60 amino acids, more preferably less than 40 amino acids, most preferably less than 30 amino acids and/or wherein the length of the IKZF inhibiting section is more than 12 amino acids, preferably more than 15 amino acids, more preferably more than 20 amino acids, most preferably more than 25 amino acids.

8. The therapeutic peptide according to any one of the preceding claims, wherein the total length of the therapeutic peptide is less than 90 amino acids, preferably less than 70 amino acids, more preferably less than 50 amino acids, most preferably less than 30 amino acids.

9. The therapeutic peptide according to any one of the preceding claims, further comprising one or more elements selected from charge modulating elements, charge modulating elements, half-life prolonging moieties, stability enhancing elements, and chemical staples, wherein the charge modulating elements are preferably selected from the group consisting of arginine residues, histidine residues or lysine residues and/or wherein the half-life prolonging elements are preferably selected from albumin, PEG, and PAS and/or wherein the stability enhancing elements are preferably selected from methylation or acetylation, wherein the chemical staple is a hydrocarbon chain that forms a bridge between two amino acids on the p a hydrocarbon chain that forms a bridge between two amino acids on the peptide therapeutic peptide.

10. The therapeutic peptide according to claim 9, wherein at least one arginine residues is, preferably at least two arginine residues are located the N-terminus of the therapeutic peptide and/or at least two arginine residues, preferably at least three arginine residues are located at the C-terminus.

11. A nucleic acid encoding the therapeutic peptide according to any of the preceding claims.

12. An IKZF dimerization inhibitor for use in the treatment of a cancer in a subject, wherein the use comprises administering the IKZF dimerization inhibitor to the subject wherein the inhibitor inhibits the homo or hetero dimerization of members of the IKZF family.

13. The IKZF dimerization inhibitor for use of claim 12, wherein the IKZF dimerization inhibitor has a binding affinity to the ZF5 and optionally the ZF6 domain of a member of the Ikaros family of zinc finger proteins, preferably selected from IKZF1 , IKZF2, IKZF3, IKZF4, and IKZF5 and is selected from the group consisting of an antibody, or antibody derivative, a peptide derived from the IKZF3-binding domain of IKZF1 or from the IKZF1 -binding domain of IKZF3, a nucleic acid encoding the antibody or the peptide, or a small molecule.

14. The IKZF dimerization inhibitor for use according to claim 12 or 13, wherein the method further comprises administering a therapeutic agent selected from the group consisting of immune check point inhibitors such as anti- PD-1 , PD-L1 , CTLA-4, TIG IT, LAG-3, TIM-3 antibodies, or any cancer immunomodulation agents.

15. The IKZF dimerization inhibitor for use according to any of claims 12 to 14, wherein the cancer is selected from the group consisting of lymphatic neoplasia and immunogenic solid tumors, preferably selected from myeloma, renal cancers, melanoma, lung cancer, breast cancers, head and neck cancers, pancreatic cancer, more preferably myeloma.