Peptides acting as antagonists for Ikaros zinc finger family (IKZF) proteins to activate the immune system against tumor cells.

Abstract

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

Description

Technical Field

[0001] The present invention relates to inhibitors of homo- or hetero-dimerization of members of the zinc finger Ikaros (IKZF) family, particularly therapeutic peptides, and their use for immunomodulation.

Background Art

[0002] As the world's population ages, cancer has become the leading cause of death, particularly in Western countries. This disease disproportionately affects people over 70 years old, accounting for an astonishing 50% of all identified cases (Roser and Ritchie 2015). In 2017, the overall damage of various cancer types claimed the lives of approximately 10 million individuals, solidifying its position as the second most lethal disease worldwide. Despite notable milestones in cancer therapeutics and drug development, the American Cancer Society predicts that in the United States, deaths related to cancer, particularly lung, colorectal, gastric, and liver cancers, the most prognostically unfavorable types, will exceed 600,000 in 2023 (Siegel et al 2015).

[0003] Recent advancements in cancer therapeutics and drug development have undoubtedly revolutionized the survival rates of cancer patients and provided new non-invasive treatment methods (Jemal et al.2017). Considering that the incidence of cancer cases has simultaneously increased, the manifestation of such effects may not be easily apparent. Next-generation sequencing that enables personalized medicine (Carlson 2012;Gullapalli et al.2012). The human papillomavirus vaccine that has reduced cervical cancer (St Laurent et al.2018) and cancer immunotherapy (Jiang and Zhou 2015;Shahid et al.2019) are among the most notably advanced.

[0004] The immune system is one of the most complex systems in the human body, intricately intertwined with intercellular and intracellular pathways and networks. These networks are so precisely controlled that even slight changes in the timing and position of effector molecules can have paradoxical consequences, either inhibiting or unintentionally enhancing tumor progression (Vesely and Schreiber 2011). The dynamic process encompassing the interaction between immune cells and tumor cells is known as "immunoediting." Cancer immunoediting involves three distinct phases: elimination, equilibrium, and evasion. During the elimination phase, cancer cells are recognized by specific tumor antigens presented 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, a second phase called "equilibrium" occurs. In this phase, the tumor persists without further spread. Subsequent mutations in tumor cells, enabling uncontrolled proliferation, and their ability to inhibit immune cells, ultimately lead to a dominance of tumor cells and a transition to the "escape" phase (Vesely and Schreiber 2011; O'Donnell et al. 2019; Schreiber et al. 2011).

[0005] Immunomodulatory drugs (IMiDs) such as lenalidomide and pomalidomide are established treatments for multiple myeloma and (to some extent) non-Hodgkin lymphoma. In addition to their direct cytotoxic effects on tumor cells, these substances possess immunomodulatory properties that activate the immune system to target tumor cells. In 2014, the mechanism of action of these drugs was elucidated, revealing that they bind to cereblon, thereby promoting the subsequent degradation of cereblon substrates. Given the numerous substrates associated with cereblon, these drugs effectively target multiple targets of cereblon. In particular, the main targets showing significant functional relevance within this group were IKZF1 and IKZF3, members of the Ikaros zinc finger transcription factor family (Kroenke et al. 2014).

[0006] The IKZF transcription factor family (IKZF) is characterized by the presence of a highly conserved zinc finger (ZF) domain. The ZF domain is a small structural motif found in various transcription factors across different eukaryotes. This domain consists of two β-sheets and one α-helix and is stabilized by one or more zinc ions.

[0007] The Ikaros family contains six highly conserved C2-H2 type ZF domains, and IKZF migrates to and binds to DNA, forming homodimers or heterodimers with other proteins (Cassandri et al., 2017). Previous reports have suggested that the four ZF domains located at the N-terminus of IKZF are involved in DNA recognition, while the other two ZF domains at the C-terminus are involved in heterodimerization (McCarty et al., 2003). Furthermore, many recent studies have challenged this classical structure related to function. For example, it was found that B lymphocyte-induced maturation protein-1 (Blimp-1) heterodimerizes with IKZF3 via the 119 amino acids at the N-terminus, and that RUNX1 did not interact with the C-terminal IKZF1 and IKZF3 (see Figure 1) (Hung et al., 2016; Zhou et al., 2019).

[0008] Notably, the IKZF family regulates gene expression by forming different homodimers and heterodimers that affect their DNA binding specificity. The IKZF family regulates gene expression as follows: • It directly binds to the gene promoter to enhance or suppress the expression of the target gene. • It controls the reachability of DNA by rearranging and forming chromatin reconfiguration complexes. • Regulation of gene expression at the RNA level. (Georgopoulos et al., 1992; Hsi et al., 2008; John and Ward, 2011; Yoshida and Georgopoulos, 2014).

[0009] As mentioned above, Kroenke et al. demonstrated that the interaction between lenalidomide and the CRBN-CRL4 ubiquitin ligase complex enhances the binding affinity of CRBN to IKZF1 / 3. Subsequently, this specific binding event triggers the ubiquitination and subsequent degradation of IKZF1 / 3, which plays a crucial role in the survival of multiple myeloma (MM) cells. To study the role of IKZF1 / 3 expression in T cells of cancer patients, our inventors, Awwad et al., previously analyzed the expression levels of IKZF1 / 3 in T cells derived from 45 MM stage I (MMI) patients and 50 newly diagnosed MM stage III (MMIII) patients according to the Durie-Salmon staging system using flow cytometry, and investigated their prognostic and predictive values. Awwad et al. also combined in vivo observation with in vitro assays to determine the effects of IKZF1 / 3 expression on T cell immunophenotype and antitumor T cell response in 162 MMIII patients. Awwad et al. found that in MMIII patients treated with immunomodulatory drugs (thalidomide, lenalidomide, and pomalidomide), high expression of IKZF3 rather than IKZF1 on T cells correlated with superior overall survival. Furthermore, Awwad et al. demonstrated that higher IKZF3 expression on T cells inhibited myeloma-specific T cell responses in vitro, and that the immunophenotype of IKZF3-high-expressing patients exhibited characteristics opposite to those induced by immunomodulatory drugs. Awwad et al. observed higher IKZF3 expression levels in T cells derived from MMIII patients compared to MMI, but IKZF3 expression was not affected by the tumor microenvironment (Awwad et al., 2018). [Overview of the project] [Means for solving the problem]

[0010] This invention is based on the remarkable discovery that inhibiting Aiolos (the gene product of IKZF3) and Ikaros (the gene product of IKZF1) can significantly increase the secretion of interleukin-2 (IL-2) by T cells. Interleukin-2 is extremely important for the activity of T cells against tumor cells.

[0011] Based on these findings, we developed specific inhibitors of Ikaros and Aiolos. In cell culture studies using human T cells, we demonstrated that the addition of the peptide inhibitors significantly increased interleukin-2 secretion from T cells, even compared to the interleukin-2 secretion from pomalidomide. Simultaneously, T cell vitality did not decrease at the selected dosage.

[0012] While other methods of inhibiting Aiolos and Ikaros are also suitable, the present invention is described by therapeutic peptides that function as inhibitors. These therapeutic peptides contain sites (IKZF inhibitory sites) that inhibit the homodimerization of Aiolos and Ikaros, as well as other members of the Ikaros family of zinc finger proteins.

[0013] Accordingly, according to the first aspect, the present invention relates to a therapeutic peptide comprising an IKZF inhibitory site that inhibits homodimerization or heterodimerization of members of the Ikaros (IKZF) family of zinc fingers, wherein the length of the therapeutic peptide is in the range of 9 to 100 amino acids, and the amino acid sequence of the IKZF inhibitory site comprises SEQ ID NO: 1 (FTIHM) or SEQ ID NO: 2 (YTIHM).

[0014] According to a second aspect, the present invention provides a method for treating a target cancer, comprising administering an IKZF dimerization inhibitor that inhibits homodimerization or heterodimerization of a member of the IKZF family to the target.

[0015] According to a third aspect, the present invention relates to a nucleic acid encoding a therapeutic peptide according to the first aspect.

[0016] According to a fourth aspect, the present invention relates to a vector containing nucleic acid according to a third aspect.

[0017] According to a fifth aspect, the present invention relates to a host cell containing a nucleic acid according to a third aspect or a vector according to a fourth aspect. [Brief explanation of the drawing]

[0018] [Figure 1] The domain structure of IKZF3 is shown along with its known binding partners and their binding locations. [Figure 2] This image shows the results of co-IP (co-IP) of IKZF3 protein combined with mass spectrometry of CD8+ T cells from 18 selected healthy donors, whose exhaustion was induced by potent activation of CD3 / CD28 dynabeads. [Figure 3] The results of an assay in human fetal kidney 293T (HEK293T) cells co-transfected with full-length IKZF1 protein and recombinant glutathione S-transferase (GST) fused to the C-terminus of full-length IKZF3 protein (GST-IKZF3_V1) or different variants of GST-tagged IKZF3 are shown. The complex was isolated by a pull-down co-IP assay using anti-GST beads, and IKZF1 expression was captured by immunoblotting. [Figure 4] Next, enzyme-linked immunosorbent assay (ELISA) was performed to evaluate IL-2 secretion, and the results of accurately evaluating IL-2 secretion under each condition are shown. Specifically, T cells were cultured for 24 hours in serum-free T cell medium, and either 10 μM C1M peptide or scrambled peptide as a negative control, or 10 μM lenalidomide or 1 μM pomalidomide as an IMiD control. [Figure 5]The results of the cytokine screening assay are shown. When 119 different cytokines were screened in the supernatant of T cells cultured for 24 hours in serum-free T cell medium with either 10 μM of the C1M peptide, a strong specific increase in the secretion of IL-2, IL-6, and INFγ from T cells was observed. [Figure 6] The process steps of the sandwich ELISA technique for determining the ability of the test peptide to bind to IKZF1 or IKZF3 are shown. [Figure 7] The results of peptides PepM1, PepM2, and PepM3 in the analysis of IL-2 (Figure 7A), IKZF3 binding assay (Figure 7B), and IKZF1 binding assay (Figure 7C) with C1M peptide as the standard are shown. [Figure 8] The results of peptides PepAla1 - PepAla15 in the analysis of IL-2 (Figure 8A), IKZF3 binding assay (Figure 8B), and IKZF1 binding assay (Figure 8C) with C1M peptide as the standard are shown. [Figure 9] The sequence comparison of test peptides PepM1 - PepM3, PepM7 - PepM14, and PepAla1 - PepAla15 is shown. [Figure 10] The results of the core sequence comparison of the ZF5 domain of IKZF1, IKZF2, IKZF3, IKZF4, and the ZF4 domain of IKZF5 are shown. [Figure 11] The results of fluorescence lifetime imaging microscopy (FLIM) experiments in the form of phase vector plots of A) IKZF1 and B) IKZF3 using the GFP lifetime signal in HEK cells are shown. Each panel contains three plots: Upper plot: The average GFP lifetime signal in HEK cells expressing only the GFP protein. Middle plot: The average GFP lifetime signal in HEK cells expressing IKZF1 or IKZF3 fused to GFP. Lower plot: The average GFP lifetime signal in HEK cells co-expressing IKZF1 or IKZF3 fused to either GFP or mCherry. The data represent the average phase vector of 15 measurements (n = 15) for each condition. [Figure 12]This report presents the results of FLIM experiments focusing on the heterodimerization of IKZF1 and IKZF3 using GFP lifetime signaling in HEK cells. The results are presented as phase vector plots. The figure includes three plots: Top plot: Mean GFP lifetime signal in HEK cells expressing only GFP protein. Middle plot: Mean GFP lifetime signal in HEK cells expressing GFP-fused IKZF1. Bottom plot: Mean GFP lifetime signal in HEK cells co-expressing GFP-fused IKZF1 and mCherry-fused IKZF3. The data represent the mean phase vectors from 15 measurements (n=15) for each condition. [Figure 13] This report presents the results of a FLIM experiment testing the effect of the C1M peptide on the heterodimerization of IKZF1 and IKZF3 using the GFP lifetime signal in HEK cells. The results are presented as phase vector plots. The figure includes five plots: Plot 1: Mean GFP lifetime signal in HEK cells expressing only GFP protein. Plot 2: Mean GFP lifetime signal in HEK cells expressing GFP-fused IKZF1. Plot 3: Mean GFP lifetime signal in HEK cells expressing GFP-fused IKZF3. Plot 4: Mean GFP lifetime signal in HEK cells co-expressing GFP-fused IKZF1 and mCherry-fused IKZF3 with a scrambled peptide. Plot 5: Mean GFP lifetime signal in HEK cells co-expressing GFP-fused IKZF1 and mCherry-fused IKZF3 with the C1M peptide. The C1M peptide was tested for its ability to inhibit the heterodimerization of IKZF1 and IKZF3. The data represents the average phase vector of 15 measurements for each condition (n=8, 10, 12, 12, and 15, respectively). [Figure 14]Results of a FLIM experiment testing the effect of the C1M peptide on the heterodimerization of IKZF1 and IKZF3 using the GFP lifetime signal in HEK cells are shown. The figure includes five plots: The first plot: The average GFP lifetime signal in HEK cells co-expressing IKZF1 fused to GFP and IKZF3 fused to mCherry with a scrambled peptide. The second plot: The average GFP lifetime signal in HEK cells co-expressing IKZF1 fused to GFP and IKZF3 fused to mCherry with the C1M peptide. [Figure 15] Dot plots illustrating the ability of the C1M peptide to activate T cells are shown. T cells from healthy donors were incubated for 24 hours with either a scrambled peptide, the C1M peptide, or no peptide (control). These T cells were then 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 the live cells (n = 6). The results showed that T cells pre-cultured with the C1M peptide killed significantly more tumor cells than T cells treated with the scrambled peptide or the control, demonstrating a strong activating effect of the C1M peptide on T cells.

Best Mode for Carrying Out the Invention

[0019] The following definitions are provided to facilitate a clear and consistent understanding of the specification and claims, and the scope given to such terms.

[0020] definition As used herein, “peptide” refers to a chain of amino acids linked by peptide bonds. A peptide may consist of any number of any type of amino acids, preferably naturally occurring amino acids, which are preferably linked by peptide bonds. In particular, a peptide contains 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 to the length of a peptide. However, preferably, the peptides according to the present invention do not exceed 500 amino acid lengths, more preferably 300 amino acid lengths, and even more preferably 250 amino acid lengths. Thus, the term “peptide” includes “oligopeptides,” which usually refer to peptides containing 2 to 20 amino acid lengths, and “polypeptides,” which contain at least 60, at least 80, preferably at least 100 amino acids.

[0021] As used herein, the term "polypeptide" refers to a peptide. The terms "polypeptide" and "protein" are interchangeable. As used herein, polypeptides and proteins include not only chemically synthesized proteins but also naturally synthesized proteins encoded by genes. Polypeptides or proteins may be obtained from natural sources such as human blood, or they may be produced in cell culture media as recombinant proteins.

[0022] As used herein, the term "protein" may contain one or more polypeptide chains. Proteins containing two or more polypeptide chains are often expressed on one polypeptide chain from one gene and cleaved post-translation. Therefore, the terms "protein" and "polypeptide" are used interchangeably.

[0023] As used herein, the terms “protein domain” or “domain” refer to a region of a protein that can fold into a stable three-dimensional structure independent of the rest of the protein. This structure allows for the maintenance of specific functions associated with the domain's function within an intact protein, including enzymatic activity, the generation of recognition motifs for other molecules, or the provision of structural components necessary for the protein to exist in a particular environment. Protein domains are typically evolutionarily conserved regions of a protein, both within the protein family and within other protein superfamilies that require similar functions.

[0024] The term "fusion protein" according to the present invention originally refers to a protein produced by joining two or more genes, cDNAs, or sequences that encode separate proteins / peptides. These genes may be naturally occurring in the same or different organisms, or they may be synthetic polynucleotides.

[0025] As used herein, the term “therapeutic peptide” refers to a peptide that has therapeutic effects, i.e., a peptide used as a pharmaceutical active ingredient.

[0026] As used herein, the term “patient” means a mammal having a condition or disease that requires treatment, in particular a human being.

[0027] As used herein, the term “mammal” means all mammals, including but not limited to rodents such as mice and hamsters, mammals of the order Logomorpha such as rabbits, Carnivora including cats and dogs, Artiodactyla including cats and pigs, Persodactyla including horses, primates, ceboids or simoids (monkeys), or apes (humans and apes).

[0028] The correlation between two amino acid sequences or two nucleotide sequences is described by a parameter called "identity." For the purposes of this invention, the degree of sequence identity between two amino acid sequences is preferably 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 al., 2000, Trends Genet. 16:276-277) version 3.0.0 or later. The optional parameters used are a gap-open penalty of 10, a gap-extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output, labeled "Longest Identity" (obtained using the no brief option), is used as an identity percentage and is calculated as follows: (Identical residues × 100) / (Length of alignment - Total number of gaps in alignment).

[0029] For the purposes of this invention, the degree of sequence identity between two nucleotide sequences is determined, preferably using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, cited above), as implemented in the Needle program of the EMBOSS package (EMBOSS; The European Molecular Biology Open Software Suite, Rice et al., 2000, cited above) version 3.0.0 or later. The optional parameters used are a gap-open penalty of 10, a gap-extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Needle output, labeled "Longest Identity" (obtained using the -no brief option), is used as the identity percentage and is calculated as follows: (100 identical desoxyribonucleotides) / (length of alignment - total number of gaps in alignment).

[0030] For example, at least 90% identity includes 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% identity with respect to the sequence in question. Typically, similar sequences contain identical residues at functionally relevant positions for peptides or polynucleotides, such as active site residues or glycosylated amino acids, but may also contain any number of conserved amino acid substitutions.

[0031] The term "carrier" in this invention relates to molecules useful for delivering cargo to a patient's cells, particularly pharmaceutical active ingredients, such as polynucleotides or proteins. The carrier may be covalently or noncovalently bonded to the cargo, or it may encapsulate the cargo.

[0032] When the term "recombinant" is used in relation to cells, nucleic acids, proteins, or vectors, it indicates that the cells, nucleic acids, proteins, or vectors have been modified by introducing heterologous nucleic acids or proteins, or by altering native nucleic acids or proteins, or that the cells are derived from such modified cells. For example, recombinant cells express genes not found in the cell's natural (non-recombinant) form, or express native genes at different levels or under different conditions than those found in nature.

[0033] The “isolated” polynucleotides (e.g., RNA, DNA, or mixed polymers) or peptides according to the present invention are polynucleotides substantially isolated from other cellular components naturally associated with a natural human sequence or protein, such as ribosomes, polymerases, chromosomes, other RNA molecules, and proteins. This term encompasses nucleic acid sequences or proteins isolated from their natural environment, recombinant or cloned DNA isolates, and chemically synthesized analogs or biologically synthesized analogs by heterologous systems.

[0034] As used herein, the terms “transformed,” “stable transformed,” and “transgenic” as used with respect to cells mean that the cell contains non-natural (e.g., heterologous) nucleic acid sequences incorporated into its genome or that are transmitted as episomes maintained over several generations.

[0035] As used herein, the term “fragment” refers to a polypeptide that, compared to a natural or wild-type protein, lacks one or more amino acid terminals and / or carboxyl terminals, but whose remaining amino acid sequence is identical to the corresponding positions of the amino acid sequence inferred from the full-length cDNA.

[0036] As used herein, the terms “binding affinity” or “affinity” refer to the strength of binding between two molecules, particularly a ligand and a protein target. Binding affinity is influenced by non-covalent intermolecular interactions between the two molecules, such as hydrogen bonds, electrostatic interactions, hydrophobic interactions, and van der Waals forces.

[0037] Dimerization is a biological process in which two proteins (or other molecules) bind together to form a complex known as a dimer. The individual proteins in these complexes are known as subunits and can be either identical (homodimers) or distinct (heterodimers). The dimerization process can be reversible or irreversible and is controlled by various factors, including the concentrations of the individual proteins, their spatial distribution within the cell, and the presence of specific chemical signals.

[0038] "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. On the other hand, "homodimerization" refers to the interaction between two identical protein subunits to form a dimer.

[0039] The transitional phrase “comprising” is synonymous with “including,” “contains,” or “characterized by,” and is comprehensive or non-restrictive, not excluding additional unlisted elements or process steps. The transitional phrase “consisting of” excludes any elements, processes, or components not expressed in the claim, except for impurities normally associated with them. If the phrase “consisting of” appears in a claim body section rather than immediately following the preamble, it limits the scope to the elements described in that section only, and does not exclude other elements as a whole from the claim. The transitional phrase “essentially consisting of” limits the scope of the claim to the expressed material or process, and is “not substantially affecting the basic and novel features” of the claimed invention. A claim “essentially consisting of” occupies an intermediate position between a closed claim written in the form of “consisting” and a fully open claim written in the form of “comprising.”

[0040] A "zinc finger (ZF) domain" is a type of protein structural motif that can bind to specific DNA sequences, RNA, proteins, and small molecules. These domains are typically small, functionally independent units within proteins. The name "zinc finger" comes from the presence of one or more zinc ions in the protein structure that help stabilize its folding.

[0041] As used herein, “proteinogenic amino acid” refers to any one of the following: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G), histidine (His, H), isoleucine (Ile, 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 (Val, V).

[0042] As used herein, "IKZF inhibition site" refers to a site in a peptide that inhibits homodimerization or heterodimerization of a member of the IKZF family.

[0043] As used herein, “IKZF dimerization inhibitor” is any molecule that inhibits homodimerization and / or heterodimerization of members of the IKZF family.

[0044] Fluorescence lifetime microscopy, or FLIM, is an imaging technique based on the exponential decay rate of photon emission from fluorophores in a sample. FLIM can be used as an imaging technique in confocal microscopy, two-photon excitation microscopy, and multiphoton tomography. FLIM generates images using the fluorescence lifetime (FLT) of fluorophores, rather than their intensity. Because fluorescence lifetime depends on the local microenvironment of the fluorophores, any measurement errors in fluorescence intensity caused by changes in light source brightness, background light intensity, or limited photobleaching are eliminated. This technique also has the advantage of minimizing the effects of photon scattering in thick layers of the sample.

[0045] As used herein, the “phase vector” method refers to a method used for the vector representation of alternating currents and voltages, or sinusoidal waves such as electromagnetic waves. The amplitude and phase of a waveform are converted into vectors, but the phase is converted into the angle between the phase vector and the X-axis, and the amplitude is converted into the length or magnitude of the vector. This concept simplifies representation and analysis, as the addition of two waveforms is achieved by their vector sum. The data representation is also referred to as a “phase vector plot.”

[0046] A "scrambled peptide" is a well-established concept in the literature and is universally used when studying the biological or functional activity of peptides. It is generally created by randomly shuffling the original sequence and is typically used as a synonym for a homologous negative control peptide.

[0047] Therapeutic peptides According to a first aspect, the present invention relates to a therapeutic peptide comprising an IKZF inhibitory site that inhibits homodimerization or heterodimerization of members of the Ikaros (IKZF) family of zinc fingers. The length of the peptide is in the range of 9 to 100 amino acids. Furthermore, the amino acid sequence of the IKZF inhibitory site comprises SEQ ID NO: 1 (FTIHM) or SEQ ID NO: 2 (YTIHM).

[0048] The IKZF family is a group of transcription factors that play a crucial role in hematopoiesis, the process by which blood cells are produced. These proteins possess both activating and repressive functions, regulating the expression of genes involved in cell fate determination, proliferation, and differentiation. The family is named after Ikaros, the first member discovered and characterized.

[0049] Zinc finger domains are characterized by the coordination of one or more zinc ions to stabilize folding. The most common type of zinc finger, found in the IKZF family, is the C2H2 type zinc finger. This type of zinc finger has a conserved sequence containing two cysteine ​​residues and two histidine residues that coordinate a single zinc ion.

[0050] C2H2 zinc fingers typically fold into a structure with a β-hairpin followed by an α-helix. The side chains of cysteine ​​and histidine residues bind to zinc ions, enabling the protein to fold into its correct three-dimensional shape. Structurally, the presence of several C2H2-type zinc finger domains in IKZF proteins allows them to bind to DNA and regulate gene expression. The number and arrangement of these domains differ among family members, giving each protein unique DNA-binding properties. Furthermore, the ability of these proteins to form homodimers or heterodimers enhances their functional diversity.

[0051] IKZF proteins interact with a wide range of other proteins, including other transcription factors, chromatin modifiers, and signaling proteins. These interactions allow IKZF proteins to integrate signals from various cellular pathways and modulate complex gene expression programs.

[0052] The IKZF family generally contains six highly conserved C2-H2 type ZF domains. IKZFs migrate to and bind to DNA and form homodimers or heterodimers with other proteins (Cassandri et al., 2017). Previous reports have suggested that the four N-terminal ZF domains of IKZF are involved in DNA recognition, while the other two C-terminal ZF domains are involved in heterodimerization (McCarty et al., 2003). The six ZF domains are numbered ZF1, ZF2, ZF3, ZF4, ZF5, and ZF6.

[0053] The IKZF family consists of five known members: IKZF(Ikaros). The names IKZF1 and Ikaros are used interchangeably. Ikaros is the most studied member of the IKZF family. Ikaros is essential for the development of all lymphoid cells and has been shown to have tumor suppressor activity. Ikaros is an important regulator for the development of B cells and T cells and also plays an important role in the development and function of natural killer cells and dendritic cells. Mutations in the Ikaros gene have been associated with leukemia, specifically acute lymphoblastic leukemia (ALL). The amino acid sequence of Ikaros has been identified by Uniprot entry Q13422 (SEQ ID NO: 11).

[0054] IKZF2 (Helios): The names IKZF2 and Helios are used interchangeably. Like Ikaros, Helios is involved in T cell development, but it is also involved in the regulation of regulatory T cells (Tregs), which play a crucial role in maintaining immune tolerance. Dysregulation of Helios expression may contribute to autoimmune diseases. The amino acid sequence of Helios has been identified by Uniprot entry Q9UKS7 (SEQ ID NO: 12).

[0055] IKZF3 (Aiolos): The names IKZF3 and Aiolos are used interchangeably. Aiolos shares many functions with Ikaros and is involved in the development of B cells and T cells. Aiolos can cooperate with Ikaros to regulate gene expression and control lymphocyte development. Dysregulation of Aiolos is associated with systemic lupus erythematosus (SLE), an autoimmune disease. The amino acid sequence of Aiolos has been identified by Uniprot entry Q9UKT9 (SEQ ID NO: 13).

[0056] IKZF4 (Eos): The names IKZF4 and Eos are used interchangeably. Eos primarily functions as a transcriptional repressor and is involved in regulating the differentiation and function of T cells, particularly Tregs. 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 has been identified by Uniprot entry Q9H2S9 (SEQ ID NO: 14).

[0057] IKZF5 (Pegasus): The names IKZF5 and Pegasus are used interchangeably. Pegasus, the least characterized member of the IKZF family, is thought to be involved in lymphocyte development like other family members, but its specific function is not well understood. The amino acid sequence of Pegasus has been identified by Uniprot entry Q9H5V7 (SEQ ID NO: 15). Pegasus differs from other IKZF members in that it contains only three N-terminal ZF domains. Therefore, ZF4 in IKZF5 corresponds to ZF5 in other members, and ZF5 in IKZF5 corresponds to ZF6 in other members.

[0058] As shown in the examples, the minimal core structure of the binding domain of IKZF family members required for homodimerization or heterodimerization is Sequence ID No. 1, found in the fifth ZF domain (ZF5) of IKZF3 and IKZF4. A sequence comparison of the core of the ZF5 domain (ZF4 in the case of IKZF5) is shown in Figure 9. Peptides containing this sequence have been shown to be able to bind to IKZF1 and IKZF3, causing an effect that inhibits homodimerization and heterodimerization of IKZF1 and IKZF3 and increasing IL-2 expression in T cells. Sequence ID No. 2, the consensus sequence for therapeutic peptides of IKZF1, IKZF2, and IKZF5, is expected to show the same effective binding.

[0059] Specifically, the IKZF inhibitory site exerts an inhibitory effect on the dimerization process of any of the IKZF family members, either homodimerization of IKZF1, IKZF2, IKZF3, IKZF4, and IKZF5, or heterodimerization between any two of IKZF1, IKZF2, IKZF3, IKZF4, and IKZF5.

[0060] According to one embodiment of the therapeutic peptide, the IKZF inhibitory site has the ability to inhibit the homodimerization or heterodimerization of Ikaros and Aiolos. Specifically, the IKZF inhibitory site 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 in the homodimerization between IKZF1 and IKZF3. The inhibitory effect of the IKZF moiety on the homodimerization or heterodimerization of IKZF1 and IKZF3 can modulate the biochemical activity of these proteins and has a significant impact on T cell IL-2 production, as shown in the examples, thus possessing great therapeutic potential.

[0061] Interleukin-2 (IL-2) is an extremely important cytokine in the immune system and is essential for the activation and proliferation of T cells.

[0062] Increased IL-2 levels have the following beneficial effects on cancer treatment: T cell activation: T cells are essential components of the immune response against cancer. They can recognize and destroy cancer cells. IL-2 is crucial for the proliferation, differentiation, and survival of these cells. IL-2 can induce the proliferation of cytotoxic T cells and enhance their ability to kill cancer cells.

[0063] Induction of immune cell-mediated tumor killing: In addition to activating T cells, IL-2 also stimulates natural killer (NK) cells. NK cells are part of the innate immune system and have the ability to kill tumor cells without prior sensitization.

[0064] Enhancement of anti-tumor immune response: IL-2 promotes a robust immune response against tumors by enhancing the activity of T cells and NK cells. In addition, IL-2 may support the development and maintenance of memory T cells, which can provide long-term immunity against tumors.

[0065] IL-2 promotes the proliferation, differentiation, and survival of antigen-selected cytotoxic T cells (CTLs), thereby enhancing the body's ability to initiate an effective immune response. IL-2 acts by binding to IL-2 receptors on the surface of T cells, triggering a cascade of signaling events that lead to T cell proliferation and increased cytotoxic activity. Increased IL-2 secretion is a strong indicator of T cell activation and enhanced immune response.

[0066] Increased production of other essential cytokines such as INF-γ: IL-2 can stimulate the production of other important cytokines such as interferon-γ (INF-γ). INF-γ can directly inhibit tumor growth and can work synergistically with other components of the immune system to enhance the antitumor response.

[0067] Enhancement of immune checkpoint inhibitors: Checkpoint inhibitors are a type of immunotherapy that enhances the ability of T cells to kill cancer cells by inhibiting proteins that act as brakes on T cells (such as PD-1, PD-L1, and CTLA-4). IL-2 can enhance the efficacy of these inhibitors by promoting T cell proliferation and activation.

[0068] In the examples, it has been shown that the therapeutic peptide significantly increases IL-2 production in T cells, which suggests that it effectively enhances T cell activation.

[0069] As shown in Example 7, the therapeutic peptide has a cytotoxic effect on cancer cells. This effect is thought to be caused by the activation of T cells against tumors via IL-2.

[0070] According to one embodiment, the amino acid sequence of the IKZF inhibition site includes SEQ ID NO: 3 (XXXTIHM). The Xaa at position 1 is preferably Val or Ile, because these are residues found at that position in IKZF family members. More preferably, the Xaa at position 1 is Val, which is Val in IKZF3, because, as shown in the examples, peptides containing Val at position 1 show strong binding to IKZF1 and IKZF3 and increased IL-2 production. The Xaa at position 3 is preferably Phe or Tyr, because these are residues found at that position in IKZF family members. More preferably, the Xaa at position 3 is Phe, which is Phe in IKZF3, because, as shown in the examples, peptides containing Phe at position 3 show strong binding to IKZF1 and IKZF3 and increased IL-2 production. The Xaa at position 2 may be any proteinogenic amino acid. The Xaa at position 2 is preferably Met or Leu, because these are residues found at that position in IKZF family members. More preferably, the Xaa at position 2 is Met, because in IKZF3 it is Met, and as shown in the examples, peptides containing Met at position 2 show strong binding to IKZF1 and IKZF3 and increased IL-2 production.

[0071] According to one embodiment, the amino acid sequence of the IKZF inhibition site includes SEQ ID NO: 4 (XXXTIHMXX). The Xaa at position 1 is preferably Val or Ile, because these are residues found at that position in IKZF family members. More preferably, the Xaa at position 1 is Val, which is Val in IKZF3, because, as shown in the examples, peptides containing Val at position 1 show strong binding to IKZF1 and IKZF3 and increased IL-2 production. The Xaa at position 3 is preferably Phe or Tyr, because these are residues found at that position in IKZF family members. More preferably, the Xaa at position 3 is Phe, which is Phe in IKZF3, because, as shown in the examples, peptides containing Phe at position 3 show strong binding to IKZF1 and IKZF3 and increased IL-2 production. The Xaa at position 9 is preferably Cys, because Cys is a residue found in IKZF3, and as shown in the examples, peptides containing Cys at position 9 show strong binding to IKZF1 and IKZF3 and increased IL-2 production. The Xaa at position 2 is preferably Met or Leu, because these are residues found at that position in IKZF family members. More preferably, the Xaa at position 2 is Met, because it is Met in IKZF3, and as shown in the examples, peptides containing Met at position 2 show strong binding to IKZF1 and IKZF3 and increased IL-2 production. The Xaa at position 8 is preferably Gly, because Gly is a residue found in IKZF3, and as shown in the examples, peptides containing Gly at position 8 show strong binding to IKZF1 and IKZF3 and increased IL-2 production.

[0072] According to one embodiment, the amino acid sequence of the IKZF inhibition site includes a sequence selected from SEQ ID NO: 5 (VMFTIHM), SEQ ID NO: 6 (VMFTIHMGC), SEQ ID NO: 7 (YVMFTIHMGCH), SEQ ID NO: 8 (RVLFLDYVMFTIHMG, PepM10), SEQ ID NO: 9 (LFLDYVMFTIHMGCH, PepM11), or SEQ ID NO: 10 (YRCDHCRVLFLDYVMFTIHMGCH, C1M). As shown in the examples, peptides containing these sequences exhibit strong binding to IKZF1 and IKZF3 and increased IL-2 production.

[0073] Experiments shown in the examples identified specific IKZF inhibition sites or their core sequences. It is understood that any single domain ZF5 of IKZF1, IKZF2, IKZF3, or IKZF4, or any peptide similar to the domain ZF4 of IKZF5, can inhibit homodimerization of any of IKZF1, IKZF2, IKZF3, IKZF4, and IKZF5, or heterodimerization between any two of IKZF1, IKZF2, IKZF3, IKZF4, and IKZF5. ZF5 of IKZF3 consists of AA 452-474 of SEQ ID NO: 13. ZF5 of IKZF1 consists of AA 462-484 of SEQ ID NO: 11. ZF5 of IKZF2 consists of AA 471-493 of SEQ ID NO: 12. The ZF5 of IKZF4 consists of AA 530-552 of sequence number 14. The ZF4 of IKZF5 consists of AA 364-386 of sequence number 15.

[0074] Furthermore, several experiments have suggested that the C-terminal ZF6 domain (ZF5 in the case of IKZF5) is also involved in homodimerization processes. Therefore, IKZF inhibitory sites containing the ZF6 domain of IKZF1, IKZF2, IKZF3, or IKZF4, or the ZF5 domain of IKZF5, are expected to be highly effective in inhibiting homodimerization and heterodimerization. The ZF6 of IKZF3 consists of AA 480-504 of SEQ ID NO: 13. The ZF6 of IKZF1 consists of AA 490-514 of SEQ ID NO: 11. The ZF6 of IKZF2 consists of AA 499-523 of SEQ ID NO: 12. The ZF6 of IKZF4 consists of AA 558-582 of SEQ ID NO: 14. The ZF5 of IKZF5 consists of AA 364-386 of SEQ ID NO: 15.

[0075] Therefore, according to one embodiment, the IKZF inhibition site is a) Amino acid sequences that are at least 90% identical to AA 452-474 of SEQ ID NO: 13, and optionally, amino acid sequences that are at least 90% identical to AA 480-504 of SEQ ID NO: 13; b) An amino acid sequence that is at least 90% identical to AA 452-504 of SEQ ID NO: 13; c) Amino acid sequences that are at least 90% identical to AA 462-484 of SEQ ID NO: 11, and optionally, amino acid sequences that are at least 90% identical to AA 490-514 of SEQ ID NO: 11; d) An amino acid sequence that is at least 90% identical to AA 462-514 of SEQ ID NO: 11; e) Amino acid sequences that are at least 90% identical to AA 471-493 of SEQ ID NO: 12, and optionally, amino acid sequences that are at least 90% identical to AA 499-523 of SEQ ID NO: 12; f) An amino acid sequence that is at least 90% identical to AA 471-523 of SEQ ID NO: 12; g) Amino acid sequences that are at least 90% identical to AA 530-552 of SEQ ID NO: 14, and optionally, amino acid sequences that are at least 90% identical to AA 558-582 of SEQ ID NO: 14; h) An amino acid sequence that is at least 90% identical to AA 530-582 of SEQ ID NO: 14; i) an amino acid sequence that is at least 90% identical to AA 364-386 of SEQ ID NO: 15, and an amino acid sequence that is at least 90% identical to AA 392-416 of SEQ ID NO: 15; and j) Contains an amino acid sequence that is at least 90% identical to AA 364-416 of SEQ ID NO: 15.

[0076] In one embodiment of the therapeutic peptide, the amino acid identity of the IKZF inhibition site is at least 95%. The level of amino acid identity may 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%.

[0077] According to one embodiment of the therapeutic peptide, the length of the IKZF inhibition site is less than 80 amino acids. The length of the IKZF inhibition site 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, or 8 amino acids. A length greater than 80 has the disadvantage of making the total length of the peptide very long, resulting in an extremely low rate of uptake into cells and rapid degradation in serum. According to one embodiment, the length of the IKZF inhibition site is less than 60 amino acids. For peptides with a length of less than 60 amino acids, internal translocation of the peptide is promoted, and the opportunity for degradation is significantly reduced compared to longer peptides. In one embodiment, the length of the IKZF inhibition site is less than 40 amino acids. For peptides with a length of less than 40 amino acids, internal translocation of the therapeutic peptide is further promoted, and the opportunity for degradation is further reduced. In another embodiment, the length of the IKZF inhibition site is less than 30 amino acids. For peptides with a length of less than 30 amino acids, internal translocation of the therapeutic peptide is further promoted, and the opportunity for degradation is further reduced.

[0078] According to one embodiment of the therapeutic peptide, the length of the IKZF inhibition site is greater than 12 amino acids. The length of the IKZF inhibition site may be, for example, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, or 80 amino acids. Lengths of less than 12 amino acids have the disadvantages of reduced stability, decreased bioavailability, and faster renal clearance.

[0079] According to one embodiment of a 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, or 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 another embodiment, the total length of the therapeutic peptide is less than 30 amino acids.

[0080] According to one embodiment, the length of the IKZF inhibition site is greater than 15 amino acids. According to one embodiment, the length of the IKZF inhibition site is greater than 20 amino acids. According to one embodiment, the length of the IKZF inhibition site is greater than 25 amino acids.

[0081] According to one embodiment, the therapeutic peptide includes, in addition to the IKZF inhibition site, at least one portion or site that does not originate from any of IKZF1, IKZF2, IKZF3, IKZF4, and IKZF5. According to one embodiment, the therapeutic peptide is not just a fragment of any of IKZF1, IKZF2, IKZF3, IKZF4, and IKZF5. According to one embodiment, the therapeutic peptide is not just the amino acid sequence of IKZF1, IKZF2, IKZF3, IKZF4, and / or IKZF5.

[0082] According to one embodiment, the therapeutic peptide further comprises one or more elements selected from the group consisting of charge modulating elements, half-life extending portions, stability enhancing elements, and chemical staples.

[0083] In particular, the charge-modulating element may be selected from arginine, histidine, or lysine residues. These elements can influence the charge of the therapeutic peptide, thereby affecting its interactions with other molecules or structures. For example, including a charge-modulating element may help increase the peptide's solubility, improve its intracellular uptake, and facilitate its binding to target proteins.

[0084] Furthermore, the half-life extension element in this embodiment may include albumin, polyethylene glycol (PEG), or a proline, alanine, and serine (PAS) sequence. Incorporating these elements makes it possible to extend the half-life of the therapeutic peptide, i.e., the time it maintains its activity in the body. This results in the significant advantage of reducing the frequency of administration and improving patient compliance.

[0085] Stability-enhancing elements such as methylation or acetylation can also be included in the structure of therapeutic peptides. These elements can increase the stability of therapeutic peptides, thereby improving their resistance to various degradation forces, and thus enhancing the therapeutic potential and durability of the peptides in the body.

[0086] In some cases, therapeutic peptides may also contain chemical staples, specifically hydrocarbon chains that form crosslinks between two amino acids on the peptide. The presence of these chemical staples can increase the rigidity and stability of the structure, thereby improving the therapeutic peptide's resistance to conformational changes, pharmacokinetic properties, binding affinity, and proteolytic stability.

[0087] According to one embodiment of the therapeutic peptide, at least one arginine residue, preferably at least two arginine residues, are positioned at the N-terminus of the therapeutic peptide. This specific position of the arginine residue at the N-terminus can provide certain advantages, such as increased peptide stability and improved intracellular uptake.

[0088] Alternatively or additionally, at least two arginine residues, more preferably at least three arginine residues, are positioned at the C-terminus of the therapeutic peptide. This arrangement can provide its own benefits, such as increased peptide stability and improved intracellular uptake.

[0089] It is important to note that the specific benefits or effects of the position of arginine residues in therapeutic peptides may vary based on the overall structure and properties of the peptide.

[0090] According to one embodiment, the sequence of the therapeutic peptide is RRYRCDHCRVLFLDYVMFTIHMGCHRRRRRRRRR (SEQ ID NO: 16).

[0091] 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 target cancer.

[0092] Treatment method The inventors have designed peptides that successfully bind to the ZF5 domain (ZF4 in the case of IKZF5) of members of the IKZF family, preventing homodimerization or heterodimerization. These peptides successfully prevent dimerization of IKZF3 and IKZF1, thereby increasing IL-2 production. Since the function of the therapeutic peptides is to prevent dimerization of IKZF3 and IKZF1 in particular, this function can be transferred to any inhibitor that inhibits homodimerization or heterodimerization of members of the IKZF family. Such inhibitors are referred to as IKZF dimerization inhibitors.

[0093] According to a second aspect, the present invention relates to a method for treating a target cancer, comprising administering an IKZF dimerization inhibitor to the target.

[0094] In other words, the second aspect relates to an IKZF dimerizing inhibitor for use in the treatment of a target cancer, wherein the use includes administering the IKZF dimerizing inhibitor to the target.

[0095] According to one embodiment of the method, the IKZF dimerization inhibitor has binding affinity to the ZF5 domain and optionally to the ZF6 domain of a member of the IKZF family. This member is preferably selected from IKZF1, IKZF2, IKZF3, and IKZF4. According to one embodiment of the method, the IKZF dimerization inhibitor has binding affinity to the ZF4 domain and optionally to the ZF5 domain of IKZF5.

[0096] IKZF dimerization inhibitors may be any molecule that binds to the ZF5 domains of IKZF3 and / or IKZF1. In particular, IKZF dimerization inhibitors bind to the ZF5 domains of IKZF1, IKZF2, IKZF3, and IKZF4, as well as the ZF4 domain of IKZF5.

[0097] IKZF dimerization inhibitors may be selected from the group consisting of antibodies, antibody derivatives, peptides, nucleic acids encoding antibodies or peptides, or small molecules.

[0098] If an antibody is genetically engineered to target the ZF5 domain (or the ZF4 domain of IKZF5), it can bind to this site and physically interfere with the interaction surface, preventing the formation of homodimers or heterodimers, similar to the therapeutic peptide according to the first embodiment. The sequences and structures of the ZF5 domains of IKZF3 and IKZF1 are known. Based on this, those skilled in the art know how to prepare polyclonal or monoclonal antibodies that bind to the ZF5 domain. The preparation of antibodies against specific domains such as ZF5 of IKZF3 or IKZF1 involves the following steps:

[0099] The first step is antigen preparation. First, the ZF5 domain must be prepared as an antigen. This preparation may involve expressing and purifying the domain itself, or a larger portion of IKZF3 containing the ZF5 domain. If the ZF5 domain cannot be expressed and purified directly, a peptide that mimics the specific region (epitope) of the ZF5 domain can be synthesized and used as an antigen.

[0100] 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 animal's immune system produces antibodies against the antigen. Following this, hybridoma formation takes place (in the case of monoclonal antibodies). For monoclonal antibodies, B cells producing the antibody are collected from the immunized animal and fused with immortalized myeloma cells to generate hybridomas (cells capable of continuously producing the desired antibody). The next step is screening and selection. Hybridomas are screened to produce antibodies that recognize and bind to the ZF5 domain. This screening is often performed using techniques such as ELISA (enzyme-linked immunosorbent assay). 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 medium, and the antibodies are purified from the culture medium for further use.

[0101] According to one embodiment, the binding epitope of the antibody or antibody derivative is the amino acid sequence AA 452-474 of SEQ ID NO: 13. According to one embodiment, the binding epitope of the antibody or antibody derivative is the amino acid sequence AA 452-504 of SEQ ID NO: 13. According to one embodiment, the binding epitope of the antibody or antibody derivative is the amino acid sequence AA 462-484 of SEQ ID NO: 11. According to one embodiment, the binding epitope of the antibody or antibody derivative is the amino acid sequence AA 462-514 of SEQ ID NO: 11.

[0102] 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. A Fab fragment is an antibody fragment consisting of the variable regions of the heavy and light chains of an antibody, and a first constant region of the heavy chain. Fab fragments can be produced by enzymatic digestion of a full-length antibody with papain. An F(ab')2 fragment is an antibody fragment consisting of two Fab fragments linked together by disulfide bonds. An F(ab')2 fragment can be produced by enzymatic digestion of a full-length antibody with pepsin. A Fab' fragment is an antibody fragment consisting of the variable regions of the heavy and light chains of an antibody, and a portion of the constant region of the heavy chain. A Fab' fragment can be produced by enzymatic digestion of a full-length antibody with papain, followed by reduction of the disulfide bonds connecting the heavy chains. These fragments are commonly used for research and diagnostic purposes, and the methods for producing antibody fragments are not particularly limited and are known in the art.

[0103] According to one embodiment, the antibody derivative is an antibody mimetic. The antibody mimetic according to the present invention may be selected from the group consisting of a single-chain variable fragment (scFv), a single-domain antibody, an affibody, an affilin, an affimer, an afitin, an antikalin, a DARPin, a monobody, and a peptide aptamer.

[0104] Single-chain variable fragments (scFv) are a type of antibody fragment consisting of variable domains from the heavy and light chains of an antibody, linked together by a short peptide linker. These can be produced in bacterial, yeast, or mammalian cells. Single-domain antibodies, also known as nanobodies, are antibody fragments consisting of a single variable domain derived from either the heavy or light chain of an antibody. They are smaller and more stable than conventional antibodies. Affibodies are scaffolds of small proteins genetically engineered to bind to specific targets with high affinity and specificity. These are based on the B domain of protein A, a natural ligand for the Fc region of antibodies. Affilins are a type of scaffold of small proteins genetically engineered to bind to specific targets with high affinity and specificity. These are based on the cystatin protein family, which are natural protease inhibitors. Affimers are a type of protein scaffolds genetically engineered to bind to specific targets with high affinity and specificity. These are based on proteins called Staphylococcal nucleases. Affitins are a type of protein scaffold that has been genetically engineered to bind to specific targets with high affinity and specificity. These are based on a protein called ExbB, which is involved in iron transport in bacteria, and are available for research, diagnostic, and therapeutic applications. Anticarins are a type of protein scaffold that has been genetically engineered to bind to specific targets with high affinity and specificity. These are based on a protein called lipocalin, which is involved in the transport of small hydrophobic molecules. DARPin, or engineered ankyrin repeat protein, is a type of protein scaffold that has been genetically engineered to bind to specific targets with high affinity and specificity. These are based on the ankyrin repeat protein family and are available for research, diagnostic, and therapeutic applications. Monobodies are a type of antibody mimetic consisting of a single protein domain that has been genetically engineered to bind to a specific target with high affinity and specificity. These are based on the fibronectin type III domain and are available for research, diagnostic, and therapeutic applications.Peptide aptamers are a type of protein scaffold consisting of short peptide sequences genetically engineered to bind to specific targets with high affinity and specificity. These are often produced using phage display or other preferred methods and are available for research, diagnostic, and therapeutic applications. The various antibody mimetic compounds and methods for producing them are not particularly limited and are publicly known in the art.

[0105] Those skilled in the art can design or identify small molecules capable of doing so, based on known sequence and structural information of the ZF5 domain (see AlphaFoldDB Q9UKT9 for the predicted structure of IKZF3 and AlphaFoldDB Q13422 for the predicted structure of IKZF1), as well as identified therapeutic peptides that bind to the ZF5 domain and inhibit its dimerization, thereby inhibiting the dimerization of IKZF3 and / or IKZF1. Those skilled in the art may use, for example, the following methods to identify small molecules as IKZF dimerization inhibitors.

[0106] Those skilled in the art would begin by gaining a deep understanding of the ZF5 domain, particularly its structure and region mediating dimerization, i.e., the peptide binding site. They could then use in silico methods to screen a large library of small molecules for potential binders. In silico methods often involve molecular docking, where small molecules are "docked" into binding pockets on a computer to predict binding modes and affinities. The next step might be in vitro high-throughput screening, an experimental method for testing a large library of small molecules for their ability to bind to the ZF5 domain or inhibit ZF5-mediated dimerization. This process can identify potential "hit" compounds. Subsequently, the hits are validated and optimized: once potential small molecules are identified, these hits are validated in secondary assays to confirm their activity. Biophysical methods such as surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) can be used to confirm binding. The hits are then optimized through a cycle of medicinal chemistry, with their structures systematically modified to improve potency, selectivity, and drug-like properties. The optimized molecules are then tested again for activity. Next, the small molecules are tested in cell-based assays to confirm their activity, i.e., their effect on IL-2 production. In vivo testing may then be performed.

[0107] The peptide may be a peptide derived from the IKZF3 binding domain of IKZF1 or the IKZF1 binding domain of IKZF3. Specifically, the peptide may be a therapeutic peptide according to the first embodiment.

[0108] According to one embodiment, the IKZF dimerization inhibitor is a nucleic acid encoding an antibody or a peptide. According to one embodiment, the encoding nucleic acid 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 embodiment described above. Administering an IKZF dimerization inhibitor using an mRNA-based method requires a process that differs slightly from conventional mRNA vaccines. Instead of encoding an antigen that stimulates an immune response, the mRNA encodes the antibody or the therapeutic agent itself.

[0109] The process is briefly outlined below. A) Design and Production: An mRNA sequence encoding the desired antibody or therapeutic peptide is synthesized. This sequence typically consists of both the heavy and light chains of the antibody and is optimized for stability, efficient translation, and reduced immunogenicity. B) Delivery System: Similar to mRNA vaccines, the mRNA encoding the antibody may be encapsulated in lipid nanoparticles (LNPs) to protect it from degradation and facilitate entry into host cells. C) Administration: The mRNA-antibody construct is injected into the recipient, usually via intramuscular or subcutaneous pathways, using one of the administration routes listed below, for example, intraocular application. The mRNA is taken up by cells at the injection site. D) Translation and Secretion: Once inside the host cell, the mRNA is released from the LNPs and translated by cellular mechanisms to produce antibodies. The heavy and light chains of the antibody associate to form a functional antibody, which is then secreted from the cell into the extracellular space and subsequently into the bloodstream. E) Binding and neutralization: The synthesized IKZF dimerization inhibitors specifically bind to IKZF1 and / or IKZF3, thereby increasing IL-2 secretion.

[0110] According to the present invention, the IKZF dimerization inhibitor may be administered by any suitable route, but it is preferable to administer it by a route that directly delivers the peptide into the bloodstream, such as intravenous injection. Subcutaneous injection is also a useful method of administration. Other routes of administration, but are not limited to, include oral, intradermal, transdermal, intraperitoneal, intramuscular, subarachnoid, mucosal (e.g., intranasal), and inhalation.

[0111] The amount of the IKZF dimerization inhibitor of the present invention, i.e., therapeutic peptide, antibody, antibody derivative, nucleic acid, or small molecule present in each effective dose is selected considering the half-life of the compound, the identification and / or stage of cancer, the patient's age, weight, sex, and overall physical condition.

[0112] The amount of active ingredient required to induce an effective immunomodulatory effect in cancer cells without serious and harmful side effects varies depending on the pharmaceutical composition used and the presence of other optional components. Generally, for compositions containing proteins / peptides or fusion proteins, each dose contains approximately 5 μg (peptide) / kg (patient body weight) to approximately 10 mg / kg. Generally, a useful therapeutic dose is 1 to 5 mg (peptide) / kg (body weight). Another embodiment of a useful dose may be approximately 500 μg / kg of peptide. Other dose ranges may be conceivable by those skilled in the art. For example, the dose of the therapeutic peptide of the present invention may be the same as the doses described for other peptide cancer therapies, and the dose of the IKZF dimerization inhibitor of the present invention may be the same as the doses described for other antibody cancer therapies.

[0113] According to one embodiment of the method, an IKZF dimerization inhibitor is administered together with a carrier. This carrier can help deliver the inhibitor to a desired location in the body and can potentially enhance the therapeutic effect of the inhibitor.

[0114] In preferred embodiments, the carrier is selected from the group consisting of liposomes and nanoparticles. Using these carriers can offer various advantages. For example, liposomes are small, spherical vesicles that can protect IKZF dimerization inhibitors from degradation in the body, facilitate their passage through biological barriers, and guide delivery to specific cells or tissues. Similarly, nanoparticles are very small particles, typically in the nanometer range, and can provide enhanced delivery and targeting capabilities.

[0115] However, the choice of carrier 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, based on these considerations, the most suitable carrier for a given application can be selected from liposomes, nanoparticles, or other suitable materials.

[0116] According to one embodiment, the method further comprises administering a therapeutic agent. This therapeutic agent can be selected from the group consisting of immune checkpoint inhibitors, including but not limited to anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-TIGIT antibodies, anti-LAG-3 antibodies, anti-TIM-3 antibodies, or other cancer immunomodulators.

[0117] Specifically, in one such embodiment, the therapeutic agent may be an anti-PD-1 antibody that functions by inhibiting the programmed death-1 (PD-1) pathway (a mechanism often used by cancer to evade the immune system). When used in combination with an IKZF dimerization inhibitor, this agent may offer benefits such as 1) synergistic effects with immune checkpoint inhibitors, 2) increased T cell proliferation and survival rates which should help achieve a better response in patients, and / or 3) overcoming immunosuppression, which is a significant problem for many tumors.

[0118] Alternatively or additionally, the therapeutic agent may be an anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-TIGIT antibody, anti-LAG-3 antibody, 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.

[0119] The therapeutic agent could also be any other cancer immunomodulator that modulates the immune system in a way that improves its ability to fight cancer.

[0120] It should be noted that the selection of a therapeutic agent depends on various factors, including the specific type of cancer, the stage of the disease, the patient's overall health, and any other treatments the patient may be receiving. Therefore, based on these considerations, the most suitable therapeutic agent for a given purpose can be selected.

[0121] In one embodiment, the method further includes utilizing techniques of cancer cell therapy. These techniques include adoptive transfer of allogeneic cells or autologous cells.

[0122] In one such embodiment, the adoptive transfer includes allogeneic cells, which are cells derived from a donor other than the patient. Using IKZF dimerization inhibitors together with allogeneic cells in cancer cell therapy may offer certain advantages, including 1) enhanced cell proliferation and persistence, 2) improved immune cell functionality, 3) overcoming immunosuppression, and 4) achieving synergistic effects.

[0123] In another embodiment, adoptive transfer includes autologous cells. These are cells taken from the patient, possibly modified and proliferated ex vivo, and then reintroduced into the patient. The use of IKZF dimerization inhibitors in conjunction with autologous cells can yield individual benefits such as 1) enhanced cell proliferation and persistence, 2) improved immune cell functionality, 3) overcoming immunosuppression, and 4) achieving synergistic effects.

[0124] The choice between allogeneic and autologous cells depends on various factors, including the specific type of cancer, the stage of the disease, the patient's overall health, and, in the case of allogeneic cells, the availability of a suitable donor. Therefore, based on these considerations, the most appropriate type of cell for adoptive transfer can be selected.

[0125] According to one embodiment of this method, the cancer is selected from the group consisting of lymphoid tumors and immunogenic solid tumors, preferably selected from myeloma, renal cancer, melanoma, lung cancer, breast cancer, head and neck cancer, and pancreatic cancer, and more preferably myeloma.

[0126] In the method of the second embodiment, the IKZF dimerization inhibitor may be a therapeutic peptide as defined according to the first embodiment. All embodiments described above in relation to the first embodiment of the present invention are applicable to the method of the second embodiment of the present invention.

[0127] Polynucleotides According to a third aspect, the present invention provides an isolated polynucleotide comprising a nucleic acid sequence encoding a therapeutic peptide according to a first aspect of the present invention.

[0128] The isolated polynucleotide may be a DNA molecule or an RNA molecule. Preferably, the isolated polynucleotide is a DNA molecule, particularly a cDNA molecule. Techniques used to isolate or clone peptide-encoding polynucleotides are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. Cloning polynucleotides from such genomic DNA can be carried out, for example, by detecting cloned DNA fragments having shared structural features using well-known polymerase chain reaction (PCR) or antibody screening of expression libraries (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 also be used.

[0129] Expression vector In a fourth aspect, the present invention also relates to an expression vector comprising a polynucleotide according to a third aspect of the present invention.

[0130] The expression vector preferably further includes regulatory elements such as a promoter, as well as transcription and translation stop signals. A recombinant expression vector may be prepared by linking a polynucleotide and a regulatory element together according to a second embodiment, and containing one or more restriction sites so that a polynucleotide encoding a polypeptide can be inserted or substituted at the restriction site. The polynucleotide can be inserted into an expression vector suitable for expression. When preparing the expression vector, the coding sequence is placed within the expression vector so that the coding sequence is operably linked to a regulatory sequence suitable for expression.

[0131] The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can conveniently perform recombinant DNA procedures and result in the expression of a polynucleotide according to the fourth aspect of the present invention. The selection of the expression vector typically depends on the compatibility of the expression vector with the host cell into which it is introduced. The expression vector may be a linear plasmid or a closed circular plasmid.

[0132] The expression vector is preferably adapted for expression in mammalian cells. The expression vector may be a self-replicating vector, i.e., a vector whose replication exists as an extrachromosomal element independent of chromosomal replication, such as a plasmid, extrachromosomal element, microchromosome, or artificial chromosome. For self-replication, the vector may further include an origin of replication on which the vector can self-replicate in the host cell of interest. The origin of replication may be any plasmid replicator that functions within the cell and mediates self-replication. The terms “origin of replication” or “plasmid replicator” mean a polynucleotide capable of replicating a plasmid or vector in vivo.

[0133] The vector is preferably such that, upon introduction into a host cell, it is integrated into the genome and replicates along with the integrated chromosome. When integrated into the host cell genome, the expression vector may depend on any other elements of the expression vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides to guide homologous recombination integration into the host cell genome to a precise location within the chromosome.

[0134] The vector of the present invention preferably contains one or more (e.g., several) selection markers that enable easy selection of cells such as transformed cells, transfected cells, or transduced cells. The selection markers are genes whose products provide biocide or virus resistance, heavy metal resistance, prototrophicity to trophic requirement strains, and the like.

[0135] The procedure used to ligate the elements described above and construct the recombinant expression vector of the present invention is well known to those skilled in the art (see Green and Sambrook 2012; Chapter 3).

[0136] According to one embodiment, the vector backbone of the vector according to the third embodiment is selected from pCDNA3, pCDNA3.1, pCDNA4, pCDNA5, pCDNA6, pCEP4, pCEP-puro, pCET1019, pCMV, pEF1, pEF4, pEF5, pEF6, pExchange, pEXPR, pIRES, and pSCAS.

[0137] host cell According to a fifth aspect, the present invention relates to a host cell comprising a nucleic acid according to a third aspect or a vector according to a fourth aspect. The expression vector according to the third aspect is introduced into the host cell such that the expression vector is maintained as a chromosomal integration or as a self-replicating extrachromosomal vector as described above. The term “host cell” encompasses all progeny of a parent cell that are not identical to the parent cell due to mutations that occur during replication. The selection of the host cell depends largely on the genes encoding the polypeptide and their sources.

[0138] According to one embodiment, the fusion protein is produced by expression in a mammalian host cell line. Preferably, the fusion protein is produced in a human host cell line. In general, any human host cell line is suitable for expressing the fusion protein. Preferably, the host cell is of human origin to ensure that the fusion protein is properly processed during folding and undergoes appropriate post-translational modifications (e.g., glycosylation, hydroxylation, phosphorylation, and sulfation). A preferred glycosylation profile of the fusion protein is obtained particularly by human kidney cell lines. Preferred human kidney cell lines are HEK cell lines, particularly the HEK293 cell line.

[0139] Examples of HEK cell lines for producing glycosylated polypeptides include HEK293 F, Flp-In(trademark)-293 (Invitrogen, R75007), 293 (ATCC(registered trademark) CRL-1573), 293 EBNA, 293 H (Thermo Scientific 11631017), 293S, 293T (ATCC(registered trademark) CRL-3216(trademark)), 293T / 17 (ATCC(registered trademark) CRL11268(trademark)), 293T / 17 SF (ATCC(registered trademark) ACS4500(trademark)), HEK293 STF (ATCC(registered trademark) CRL 3249(trademark)), and HEK-293.2sus (ATCC(registered trademark) CRL-1573(trademark)). The preferred cell line for polypeptide production is the HEK293 F cell line.

[0140] Other suitable human cell lines for expression include, but are not limited to, cell lines derived from myeloid leukemia cells. Specific examples of host cells include K562, NM-F9, NM-D4, NM-H9D8, NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X, GT-5s, and cells derived from any of the aforementioned 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 are selected for their specific glycosylation characteristics.

[0141] Further mammalian host cell lines suitable for producing the fusion protein according to the present invention include cell lines derived from hamsters, mice, and monkeys. Suitable host cells include Chinese hamster ovary cells (CHO cells, e.g., DG44, DXB11, and K1 [ATCC CCL-61, including its glutamine-dependent derivatives CHOZn and SAFC CHOGS]) and baby hamster kidney (BHK) cells. [Examples]

[0142] Example 1 - Identification of binding partners for IKZF3 Exhaustion was induced by potent activation of CD3 / CD28 dynabeads using immunoprecipitation (co-IP) combined with mass spectrometry of CD8+ T cells from four selected healthy donors. Cells were used only in a pull-down co-IP assay to capture full-length IKZF3 protein. Mass spectrometry to identify binding proteins was performed and analyzed at the Proteomics Core Facility at EMBL Heidelberg. To determine potential interaction partners of IKZF3, the p-value threshold was set to <0.05 and the log2 multiplier (FC) threshold to 1, adjusted using the Benjamini Hochberg (BH) method. We found that IKZF1 was highly enriched as a heterodimer of IKZF3 (Figure 2). Furthermore, mass spectrometry showed that not only IKZF1, but also ribosomal proteins, proteins related to splice variant expression, and post-translational modification proteins were similarly increased in the samples.

[0143] Example 2 - Identification of the binding site of the heterodimerization domain in IKZF3 In the next step, the binding domain of IKZF1 to IKZF3 was searched for using a protein-protein binding assay. Since human fetal kidney 293T (HEK293T) cells do not express members of the IKZF protein family, the assay was constructed using HEK293T cells.

[0144] Cells were co-transfected with either a recombinant glutathione S-transferase (GST) (GST-IKZF3_V1) fused to the C-terminus of a full-length IKZF1 protein and a different variant of GST-tagged IKZF3 (GST-IKZF3_V1 to GST-IKZF3_V5). To express the IKZF3 variants and determine the binding site, we designed GST-fused variants lacking only one functional domain in each variant. GST-IKZF3_V1 contains aa1~aa519, GST-IKZF3_V2 contains aa1~aa240, GST-IKZF3_V3 contains aa1~aa119, GST-IKZF3_V4 contains aa118~aa240, and GST-IKZF3_V5 contains aa240~aa519. The complexes were isolated by a pull-down co-IP assay using anti-GST beads, and IKZF1 expression was captured by immunoblotting analysis.

[0145] The results showed that IKZF1 binds to the full-length IKZF3 protein, but also to GST-IKZF3 variant 5, which contains the two C-terminal zinc finger domains of IKZF3, due to protein-protein interactions (Figure 3).

[0146] In summary, the data revealed that IKZF1 and IKZF3 heterodimerize in exhausted CD8+ T cells and may bind to one or both of the two zinc finger domains at the C-terminus of the IKZF3 transcription factor. Furthermore, ribosomes and proteins for post-translational modification also associated with the heterodimer complex.

[0147] Example 3 - Design of a blocking peptide As a result, amino acids 240 to 519 were shown to be involved in binding, and the binding site was predicted to be one of the highly conserved ZF domains at the C-terminus. • C1: Represents the fifth ZF domain of the IKZF3 protein, consisting of amino acids 452 to 474: (YRCDHCRVLFLDYVMFTIHMGCH);

[0148] To narrow down the binding site, two peptide constructs were designed to mimic the native zinc finger domain at the C-terminus to inhibit the formation of the IKZF1-IKZF3 heterodimer. The native peptide derived from IKZF3 showed a very low net charge at pH 7. This net charge was expected to hinder intracellular translocation. Therefore, an arginine-rich derivative peptide was designed that showed a net charge of approximately 6 at pH 7. To monitor the internal translocation of the peptide by flow cytometry, the peptide was labeled with fluoroceine isothiocyanate (FITC). After confirming the internal translocation of the peptide, the peptide was tested in a cell model.

[0149] [Table 1]

[0150] Example 4 - Testing of a blocking peptide mimicking a natural domain C1 IKZF3 domain-modified peptides (C1M) (Table 1) were tested in cell culture models to investigate their ability to neutralize IKZF1-IKZF3-induced IL-2 suppression. T cells were isolated using magnetic cell sorting from 18 different healthy donors. The T cells were then cultured for 24 hours in serum-free T cell medium and in either 10 μM C1M peptide or scrambled C1M as a negative control, or 10 μM lenalidomide or 1 μM pomalidomide (as previously described IMiD controls).

[0151] After 24 hours, the supernatant was collected, centrifuged to remove all cells, and stored at -20°C. Then, an enzyme-linked immunosorbent assay (ELISA) was performed to accurately assess IL-2 secretion under each condition.

[0152] Interestingly, treatment of cells with C1M peptide increased IL-2 secretion in all cases, and the inventors confirmed this finding by repeating the experiment three times with cells from 18 different healthy donors (Figures 4A and 4B). To confirm that this increase in IL-2 was specific and not due to a random increase in intracellular secretion, the inventors performed a cytokine screening assay using Sciomics GMBH. Screening 119 different cytokines in the supernatant from the same previous experiments revealed a strong specific increase in the secretion of IL-2, IL-6, and INFγ from T cells (Figure 5).

[0153] Example 5 - Ala scanning peptide method Ala scanning, also known as alanine scanning mutagenesis, is a commonly used technique in the genetic engineering of peptides or proteins to identify which amino acid residues are essential for the function of a molecule. This technique involves systematically substituting each amino acid residue within a molecule with alanine, a nonpolar amino acid with minimal steric hindrance. The fundamental principle of Ala scanning is to identify which amino acid residues contribute to the overall function of a molecule by testing the activity of each variant against that of the original molecule. By comparing the activity of each alanine-substituted variant with that of the original molecule, it becomes possible to identify which amino acid residues are essential for the overall function of the molecule. Ala scanning typically involves generating a series of mutant peptides or proteins and replacing each amino acid residue of the original molecule with alanine one at a time. The resulting variants are then tested using functional assays, such as measuring the peptide's ability to bind to a target molecule or induce a specific cellular response.

[0154] Therefore, the inventors evaluated the peptides using two different methods:

[0155] 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 μM test peptide or 10 μM scrambled control peptide. After 24 hours, the supernatant was collected from the cells, and IL-2 levels were measured using a highly sensitive ELISA kit according to the manufacturer's instructions. This method has been used in our analyses to date and has been shown to yield reliable and reproducible results.

[0156] By comparing the IL-2 secretion levels of cells treated with the test peptide to those treated with the control peptide, we were able to determine the ability of the peptide to increase IL-2 secretion in T cells. This assay provided a quantitative reading of the peptide's activity, allowing for accurate evaluation of its effectiveness in promoting IL-2 secretion.

[0157] B) IKZF1 / IKZF3 peptide binding assay: To overcome the challenge of testing peptides that are insoluble in suitable solvents, the inventors utilized a method of coating polystyrene plates with peptides. This made it possible to test the functionality of peptides even if they are insoluble in solvents typically used in cell culture techniques. The inventors used a sandwich ELISA method, but simply put, polystyrene plates were coated with the test peptides and incubated overnight to bind the peptides to the plates. These coated plates were then used to test the ability of peptides to capture IKZF1 or IKZF3 from lysates of HEK293 cells transiently transfected with their respective IKZF proteins. HEK293T cells were chosen for this purpose because they do not normally express IKZF1 and IKZF3. To detect the captured IKZF proteins, the coated plates were incubated with antibodies specific to IKZF1 or IKZF3. Next, the primary antibody was detected using a secondary antibody conjugated with horseradish peroxidase (HRP), and the protein was quantified using a chemiluminescent detection substrate (Figure 6). This sandwich ELISA method made it possible to determine the ability of the test peptide to bind to IKZF1 or IKZF3. By comparing the signal obtained from the peptide with the signal obtained from the scrambled control, it was possible to determine whether the peptide had specific binding activity to the target protein. This method provided a quantitative reading of the peptide's binding activity, making it possible to accurately evaluate their effectiveness in capturing IKZF1 or IKZF3, which are hypothesized to be the mechanism of action.

[0158] Prior to the ALA scanning method, three peptides, Pep M1 (SEQ ID NO: 35), Pep M2 (SEQ ID NO: 36), and Pep M3 (SEQ ID NO: 37), each having a duplicate sequence spanning eight amino acids, were designed and synthesized, as detailed in the table in Figure 9. To enhance their intracellular translocation ability, all three peptides were modified with an arginine-rich domain. Solubility tests were performed to evaluate the solubility of the peptides. Pep M1 was found to be insoluble in the appropriate solvent, while Pep M2 and Pep M3 showed excellent solubility. Therefore, all peptides were analyzed for IKZF1 / IKZF3 binding, and similarly, only Pep M2 and Pep M3 were tested for IL-2 in T cells.

[0159] The results are shown in Figures 7A and 7B. Compared to the scrambled control peptide, only Pep M1 and Pep M2 showed activity similar to that observed in the C1M peptide binding assay, while Pep M3 did not show such activity (Figures 7A and 7B). This result was confirmed for Pep M2 by IL-2 ELISA (Pep M1 could not be tested due to its insolubility). These results suggest that amino acids L9-M20 of C1M may be important.

[0160] To identify functional peptides, the inventors considered a threshold corresponding to 50% of the activity of the original peptide, C1M. Therefore, peptide isoforms exhibiting approximately 50% of the activity of the original C1M were considered functional peptides. Thresholds are indicated as dotted lines in all figures. The cutoff values ​​were set to the following values ​​based on the inventors' previous experiments:

[0161] Ala-scanning groups were designed by substituting each amino acid from L9 to H23 (15 peptides; see Table in Figure 9: Ala Scanning Group). To improve internal migration, all peptides were modified with an arginine-rich domain. Based on solubility tests, PepAla 3, PepAla 4, and PepAla 11 were shown to be insoluble in solvents suitable for IL-2 analysis. All peptides were analyzed for their IKZF1 / IKZF3 binding and the increase in IL-2 in T cells (where soluble).

[0162] The analysis revealed the following: - Removing V14 slightly reduces the peptide's function (represented by Ala 6). - Removing F16~M20 strongly reduces / inactivates the peptide's function (represented by Ala 8~Ala 12). - Furthermore, removal of C22 resulted in a decrease in IL-2 function, even though there was no evidence observed from the perspective of IKZF1 / IKZF3 binding (represented by A14). (See Figures 8A, 8B, and 8C).

[0163] There is a contradiction between the reported binding data and the efficacy of the C1M peptide in promoting IL-2 secretion between Figure 7B and Figure 8B. It must be recognized that variability in in vitro assays is standard and an expected part of experimental work. This variability is largely due to differences between human donor cells and their responses, and therefore, rigorous statistical testing is applied to determine significance.

[0164] 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.

[0165] For example, when evaluating IL-2 secretion in IKZF inhibition and T cell activation, the behavior can vary significantly depending on the donor cells. It is not uncommon to observe a 20-fold variability in results between different donors. This variability reflects the complex nature of the human immune system, where individual responses to the same stimulus can vary considerably.

[0166] To address this variability and draw reliable conclusions, the inventors apply rigorous statistical tests to the data. These tests help identify significant differences and ensure that the inventors' findings are not due to random variability. By using appropriate statistical methods, the effectiveness of the C1M peptide in inhibiting IKZF dimerization and promoting IL-2 secretion can be confidently determined, despite the natural variability in donor cell responses.

[0167] Example 6 - FLIM assay to test inhibition of IKZF dimerization To confirm that the C1M peptide inhibits homodimerization or heterodimerization of the IKZF protein, the inventors used a fluorescence lifetime imaging (FLIM) assay. This is a powerful technique for studying protein-protein interactions in living cells by detecting energy transfer between two closely located fluorophores.

[0168] FLIM occurs when two fluorescent proteins are in close proximity (typically 1–10 nm), and an energy transfer occurs from one fluorophore to the other, resulting in a measurable shift in the fluorescence signal lifetime of one fluorophore compared to the lifetime of the fluorophore alone. In this application, FLIM is used to determine the dimerization state of IKZF proteins.

[0169] Experimental Design 1. Fluorescent tagging IKZF1 and IKZF3 were fused with fluorescent proteins such as green fluorescent protein (GFP) or mCherry (red fluorescent protein). Methods for tagging proteins with fluorescent tags are known to those skilled in the art.

[0170] 2. Verification of homodimerization and heterodimerization: Homodimerization To verify the homodimerization of IKZF1 and IKZF3, IKZF1-GFP was co-expressed with IKZF1-mCherry, and IKZF3-mCherry was co-expressed with IKZF3-GFP in cells. When these proteins dimerize, the proximity of the fluorophores leads to a detectable change in the lifetime of the fluorescence signal compared to the lifetime of the fluorophores of the unbound proteins.

[0171] Heterodimization In the case of heterodimerization, IKZF1-GFP was co-expressed with IKZF3-mCherry. Interaction between IKZF1 and IKZF3 brings GFP and mCherry into close proximity, resulting in a detectable change in the lifetime of the fluorescence signal compared to the lifetime of the fluorophore of the unbound protein.

[0172] 3. Peptide intervention: Upon introduction of the C1M peptide, if the peptide inhibits homodimerization or dimerization, a reduction in the FLIM signal shift should be observed. This indicates that the C1M peptide interferes with the interaction between IKZF1 and IKZF3, and its function of inhibiting heterodimerization will be verified.

[0173] result: The results are shown using the phase vector method in a phase vector plot. FLIM allows visualization of spectra and decay curves using phase vectors. This method calculates the Fourier transform of the spectrum or decay curve and plots the resulting complex number on a 2D plot where the X-axis represents the real component and the Y-axis represents the imaginary component. This makes analysis easier because each spectrum and decay is transformed into a unique position on the phase vector plot corresponding to the spectral width, emission maximum, or its average lifetime. The most important features of this analysis are its speed and the provision of a graphical representation of the measured curves.

[0174] 1. Homodimerization: As shown in Figure 11, FLIM experiments confirmed homodimerization of both IKZF1 and IKZF3.

[0175] Specifically, the signals for GFP (upper phase vector plot in Figure 11A) and IKZF1-GFP (center phase vector plot in Figure 11A) are in similar positions (close to the left vertical dotted line). In experiments where IKZF1-GFP was co-expressed with IKZF1-Cherry (lower phase vector plot in Figure 11A), the GFP signal shifted significantly to the right, i.e., to the center of the two vertical dotted lines. This shift demonstrates that IKZF1-GFP and IKZF1-Cherry interacted, i.e., homodimerized.

[0176] The results of the same experiment using IKZF3 are shown in the lower panel of Figure 11. The signals for GFP (upper phase vector plot in Figure 11B) and IKZF3-GFP (center phase vector plot in Figure 11B) are in the same position (close to the left vertical dotted line). In the experiment in which IKZF3-GFP was co-expressed with IKZF3-Cherry (lower phase vector plot in Figure 11B), the GFP signal shifted significantly to the right, i.e., to the center of the two vertical dotted lines. This shift demonstrates that IKZF3-GFP and IKZF3-Cherry interacted, and therefore homodimerized IKZF3.

[0177] 2. Heterodimization : Furthermore, as shown in Figure 12, heterodimerization of IKZF1 and IKZF3 was also confirmed by FLIM. Specifically, the results for GFP and IKZF1-GFP were examined, and these results indicate that the signals for GFP (upper phase vector plot in Figure 12) and IKZF1-GFP (center phase vector plot in Figure 12) are in the same position (close to the left vertical dotted line). In the experiment in which IKZF1-GFP was co-expressed with IKZF3-Cherry (lower phase vector plot in Figure 12), the GFP signal shifted significantly to the right, i.e., blurred towards the center of the two vertical dotted lines. This shift proves that IKZF1-GFP and IKZF3-Cherry interacted, i.e., that IKZF1 and IKZF3 homodimerized.

[0178] Co-expression of IKZF1-GFP and IKZF3-mCherry also resulted in a clear shift in the FLIM signal, indicating successful heterodimerization (the shorter the lifetime signal, the clearer the interaction, Figure 12).

[0179] 3. Peptide intervention: Next, we confirmed that C1M inhibits heterodimerization of IKZF1 and IKZF3. As shown in Figure 13, we examined the results for GFP, IKZF1-GFP, and IKZF3-GFP. This result means that the signals for GFP (first phase vector plot from the top in Figure 13), IKZF1-GFP (second phase vector plot from the top in Figure 13), and IKZF3-GFP (third phase vector plot from the top in Figure 13) are in the same position (close to the vertical dotted line on the left). In the experiment where IKZF1-GFP was co-expressed with IKZF3-Cherry and scrambled peptide (fourth phase vector plot from the top in Figure 13), the GFP signal shifted significantly to the right, i.e., blurred towards the center of the two vertical dotted lines. This shift confirmed that IKZF1-GFP and IKZF3-Cherry interacted, i.e., that IKZF1 and IKZF3 homodimerized. Since the signal is equivalent to that in the lower panel of Figure 12, the peptides do not affect the signal, which means they do not interfere with dimerization.

[0180] In contrast, the addition of the C1M peptide to the co-expression of IKZF1-GFP and IKZF3-Cherry resulted in a decrease in the shift caused by the IKZF1 / IKZF3 heterodimer. This clearly indicates that dimerization is inhibited.

[0181] Next, to demonstrate the inhibition of heterodimerization in the functional core, the effect in the nucleus was directly measured. In contrast to previous methods that measured the overall signal from the cell (signals from the cytosol and nucleus) (see Figure 13), this experimental setup measured only the signal from the nucleus. As shown in Figure 14, the nuclear measurement confirmed the previous measurements in the whole cell. The phase vector plot in Figure 14 shows similar results to those seen in the two lower plots in Figure 13. Thus, even when measured in the nucleus, the sample containing IKZF1-GFP / IKZF3-mCherry and scrambled peptide has a higher concentration and shorter lifetime than the sample containing IKZF1-GFP / IKZF3-mCherry and C1M (not shown, as with the control).

[0182] The results showed that C1M interfered with (i.e., further prevented) the interaction between IKZF1 and IKZF3, and did not result in a significantly shorter lifetime compared to the scramble case (Figure 14).

[0183] Therefore, C1M approaches the nucleus, which enables the IKZF1 / IKZF3 heterodimer to function, by binding to DNA, and inhibits dimerization in the nucleus as well.

[0184] Example 7 - Cytotoxicity of therapeutic peptides To explain the therapeutic relationship between the previous results, namely the inhibition of homodimerization and heterodimerization of IKZF1 / 3 and the resulting activation of IL-2, we provide additional experimental evidence.

[0185] method T cells derived from healthy donors were incubated for 24 hours with either scrambled peptide, C1M peptide, or no peptide. These T cells were then co-cultured with NCI-H929 tumor cells for either 24 or 48 hours.

[0186] Flow cytometry was used to analyze the proportion of tumor cells among viable cells (n=6).

[0187] result The results are shown in Figure 15. These results demonstrate that T cells pre-cultured with C1M peptide killed significantly more tumor cells than T cells treated with scrambled peptide or control, thus demonstrating the potent activating effect of C1M peptide on T cells.

[0188] This indicates that treating T cells with the C1M peptide strongly enhances the killing of tumor cells. Specifically, T cells treated with the C1M peptide showed improved cytotoxic activity compared to the control, demonstrating that this peptide not only activates T cells but also enhances their ability to eliminate tumor cells (Figure 15).

[0189] This enhanced tumor cell killing is likely due to improved activation and proliferation of cytotoxic T cells induced by the upregulation of IL-2.

[0190] C1M peptide prevents T cell exhaustion, maintains their potential cytotoxicity, and improves their ability to target tumor cells by inhibiting homodimerization and heterodimerization of IKZF proteins.

[0191] conclusion Increased IL-2 production is a well-established marker of T cell activation and has significant implications for enhancing the immune response against tumors. The cytotoxicity resulting from the therapeutic peptide according to the present invention has been experimentally confirmed.

[0192] Many modifications and other embodiments of the invention described herein will be conceivable to those skilled in the art to which the invention pertains, who benefit from the teachings presented in the foregoing description and the accompanying drawings. Therefore, it should be understood that the invention is not limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims. Certain terms are used herein, but these are used in a general and descriptive sense only and are not intended to be limiting.

[0193] References Carlson B.Next Generation Sequencing;The Next Iteration of Personalized Medicine;Next generation sequencing,along with expanding databases like The Cancer Genome Atlas,has the potential to aid rational drug discovery and streamline clinical trials.Biotechnology healthcare.2012;9(2):21-25. Gullapalli RR, Desai KV, Santana-Santos L, Kant JA, Becich MJ.Next generation sequencing in clinical medicine;Challenges and lessons for pathology and biomedical informatics.Journal of pathology informatics.2012;3:40. Jemal A, Ward EM, Johnson CJ, et al.Annual Report to the Nation on the Status of Cancer,1975-2014,Featuring Survival.Journal of the National Cancer Institute.2017;109(9). Jiang T,Zhou C.The past,present and future of immunotherapy against tumor.Translational Lung Cancer Research.2015;4(3):253-264. O’Donnell JS,Teng MWL,Smyth MJ.Cancer immunoediting and resistance to T cell-based immunotherapy.Nature reviews.Clinical oncology.2019;16(3). https: / / pubmed.ncbi.nlm.nih.gov / 30523282 / . Kroenke,Jan;Udeshi,Namrata D.;Narla,Anupama;Grauman,Peter;Hurst,Slater N.;McConkey,Marie et al.(2014);Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells.In Science(New York,N.Y.)343(6168),pp.301-305.DOI;10.1126 / science.1244851. Roser M,Ritchie H Cancer.Our World in Data.2015.https: / / ourworldindata.org / cancer#cancer-deaths-by-age. Siegel RL,Miller KD,Jemal A.Cancer statistics,2020.CA;A Cancer Journal for Clinicians.2020;70(1):7-30. Shahid K,Khalife M,Dabney R,Phan AT.Immunotherapy and targeted therapy-the new roadmap in cancer treatment.Annals of Translational Medicine.2019;7(20). Schreiber RD,Old LJ,Smyth MJ.Cancer immunoediting;integrating immunity’s roles in cancer suppression and promotion.Science(New York,N.Y.).2011;331(6024). St Laurent J,Luckett R,Feldman S.HPV vaccination and the effects on rates of HPV-related cancers.Current problems in cancer.2018;42(5):493-506. Vesely MD,Schreiber RD.Cancer Immunoediting;antigens,mechanisms and implications to cancer immunotherapy.Annals of the New York Academy of Sciences.2013;1284(1):1-5.

Claims

1. A therapeutic peptide comprising a site that inhibits homodimerization or heterodimerization of a member of the Ikaros family of zinc finger proteins (IKZF inhibitory site), wherein the length of the peptide is in the range of 9 to 100 amino acids, and the amino acid sequence of the IKZF inhibitory site comprises SEQ ID NO: 1 (FTIHM) or SEQ ID NO: 2 (YTIHM).

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

3. The amino acid sequence of the IKZF inhibition site is, Sequence ID 3 (XXXTIHM) (wherein the formula, the first Xaa is Val or Ile, preferably Val; the third Xaa is Phe or Tyr, preferably Phe; and the second Xaa is preferably Met or Leu, more preferably Met); or A therapeutic peptide according to claim 1 or 2, comprising Sequence ID No. 4 (XXXTIHMXX) (wherein the formula, the Xaa at position 1 is V or I, preferably V; the Xaa at position 3 is Phe or Tyr, preferably Phe; the Xaa at position 9 is Cys; preferably the Xaa at position 2 is Met or Leu, more preferably Met; and preferably the Xaa at position 8 is Gly).

4. The therapeutic peptide according to any one of claims 1 to 3, wherein the IKZF inhibition site 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 (YRCDHCRRVLFLDYVMFTIHMGCH).

5. The IKZF inhibition site is a) Amino acid sequences that are at least 90% identical to AA 452-474 of SEQ ID NO: 13, and optionally, amino acid sequences that are at least 90% identical to AA 480-504 of SEQ ID NO: 13; b) An amino acid sequence that is at least 90% identical to AA 452-504 of SEQ ID NO: 13; c) Amino acid sequences that are at least 90% identical to AA 462-484 of SEQ ID NO: 11, and optionally, amino acid sequences that are at least 90% identical to AA 490-514 of SEQ ID NO: 11; d) An amino acid sequence that is at least 90% identical to AA 462-514 of SEQ ID NO: 11; e) Amino acid sequences that are at least 90% identical to AA 471-493 of SEQ ID NO: 12, and optionally, amino acid sequences that are at least 90% identical to AA 499-523 of SEQ ID NO: 12; f) Amino acid sequences that are at least 90% identical to AA 471-523 of SEQ ID NO: 12; g) Amino acid sequences that are at least 90% identical to AA 530-552 of SEQ ID NO: 14, and optionally, amino acid sequences that are at least 90% identical to AA 558-582 of SEQ ID NO: 14; h) Amino acid sequences that are at least 90% identical to AA 530-582 of SEQ ID NO: 14; i) an amino acid sequence that is at least 90% identical to AA 364-386 of SEQ ID NO: 15, and an amino acid sequence that is at least 90% identical to AA 392-416 of SEQ ID NO: 15; and j) A therapeutic peptide according to any one of claims 1 to 4, comprising 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%, and most preferably 100%.

7. The therapeutic peptide according to any one of claims 1 to 6, wherein the length of the IKZF inhibition site 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 the length of the IKZF inhibition site is greater than 12 amino acids, preferably greater than 15 amino acids, more preferably greater than 20 amino acids, most preferably greater than 25 amino acids.

8. The therapeutic peptide according to any one of claims 1 to 7, 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, and most preferably less than 30 amino acids.

9. A therapeutic peptide according to any one of claims 1 to 8, further comprising one or more elements selected from a charge modulating element, a charge modulating element, a half-life extension portion, a stability enhancing element, and a chemical staple, wherein the charge modulating element is preferably selected from the group consisting of arginine residues, histidine residues, or lysine residues, and / or the half-life extension element is preferably selected from albumin, PEG, and PAS, and / or the stability enhancing element is preferably selected from methylation or acetylation, and the chemical staple is a hydrocarbon chain that forms a crosslink between two amino acids on a hydrocarbon chain that forms a crosslink between two amino acids on the therapeutic peptide.

10. The therapeutic peptide according to claim 9, wherein at least one arginine residue, preferably at least two arginine residues, is located at 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 a therapeutic peptide according to any one of claims 1 to 10.

12. IKZF dimerization inhibitors for use in the treatment of a target cancer, wherein the use comprises administering the IKZF dimerization inhibitor to the target, and the inhibitor inhibits homodimerization or heterodimerization of a member of the IKZF family.

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

14. The IKZF dimerizing inhibitor for use according to claim 12 or 13, further comprising administering a therapeutic agent selected from the group consisting of an immune checkpoint inhibitor such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-TIGIT antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, or any cancer immunomodulator.

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

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