Chlorotoxin derivatives and their use

Abstract

The present invention relates to a method for producing a compound having the general sequence X0X1CMPCX S1 X S2 X S3 DHX S4 X S5 A chlorotoxin derivative comprising the amino acid sequence of ARRCX2X3CCGGYGX4CFGYQCLCX5X6X7X8, wherein (i) the N-terminal XOX1 cluster is selected from the group consisting of AM, 0M, or 00; (ii) the soluble XS1XS2XS3XS4XS5 cluster is selected from the group consisting of FTTQT, FTTES, SSSQT, SSSES, FSSQT, FSSES, or FSSQS, (iii) the internal X2X3X4 cluster is selected from the group consisting of DKR, RDK, KDR, IKY, HKW, DRK, LKQ, KKK; and (iv) the C-terminal X5X6X7X8 cluster is selected from the group consisting of N000, R000, NR00, NRG0, NRGY, NRRR, or RRRR; where 0 indicates a position where no amino acid is present. The invention further relates to conjugates comprising said chlorotoxin derivatives and methods for their preparation, theranostic pairs consisting of said conjugates, kits comprising said conjugates or said theranostic pairs, pharmaceutical compositions comprising said conjugates, methods for treating cancer, nucleic acid molecules encoding chimeric antigen receptors comprising said chlorotoxin derivatives, vectors comprising said nucleic acids, and human populations transfected or transduced with said vectors.

Description

Technical Field

[0001] The present invention relates to chlorotoxin derivatives. In particular, the present invention relates to novel chlorotoxin derivatives useful for targeting cancer cells in mammals, particularly humans. The present invention also relates to methods of targeting cancer cells using the novel chlorotoxin derivatives.

Background Art

[0002] Chlorotoxin Characteristics Chlorotoxin (hereinafter referred to as CTX) is a small protein of 36 amino acids and is a cystine knot miniprotein [Moore 2012], having a compact tertiary structure stabilized by four disulfide bonds. CTX was first isolated from the venom of the scorpion Leiurus quinquestriatus and was named for its ability to block chloride channels. CTX can cause neurotoxicity in arthropods [DeBin 1993]. The term chlorotoxin or CTX refers to the wild-type protein shown in SEQ ID NO: 1. The disulfide pattern of wild-type CTX and several CTX variants / derivatives is shown in FIG. 1. The disulfide pattern of wild-type CTX is as follows: C 2 -C 19 、C 5 -C 28 、C 16 -C 33 、およびC 20 -C 35 is.

[0003] When studying glioma-specific chloride currents, it was found that CTX shows specific and selective binding to glioma cells in vitro and in vivo, in contrast to cells of normal brain tissue [Soroceanu 1998]. This discovery is 131The concept of using CTX as a targeting molecule to concentrate cytotoxic radiotherapy based on iodine radioisotope on target tumor cells was proposed [Mamelak 2006]. Also, immunohistochemical studies have shown that CTX-like molecules with fluorescent or biotin labels can be successfully used for immunohistochemical labeling of gliomas [Soroceanu 1998]. Subsequently, tumor-specific binding by CTX has been more widely observed in various types of tumors classified as neuroectodermal-derived tumors [Lyons 2002]. In addition to gliomas including glioblastoma multiforme (GBM), anaplastic astrocytoma, astrocytoma, and oligodendroglioma, Lyons et al. described numerous other primary brain tumors such as sarcoma, ganglioglioma, meningioma, epithelioma, etc., as well as peripheral neuroectodermal tumors such as medulloblastoma, neuroblastoma, ganglioneuroma, melanoma (primary and metastatic), pheochromocytoma, Ewing sarcoma, small cell lung carcinoma, and schwannoma as CTX positive (i.e., specific binders). Lyons et al. also showed high specific binding of biotinylated CTX to immortalized cell lines of different human tumor origins, including 7 glioblastoma cell lines and 6 peripheral neuroectodermal tumor cell lines. Also, in living cells at physiological temperature, CTX has been shown to be taken up by tumor cells and non-tumor cells by different internalization processes that result in different cellular localization patterns [Wiranowska 2011]. In this study, it was demonstrated that in glioma cells, CTX is taken up by receptor-mediated (also called clathrin-mediated) endocytosis because it is processed to lysosomes in the endosomal compartment, and as a result, is retained intracellularly for a long time. On the other hand, in normal cells (astrocytes and fibroblasts), CTX is taken up by non-receptor-mediated macropinocytosis and then rapidly released, so the net uptake is low and the retention is not prolonged. These characteristics may play an important role in the selective tumor targeting properties of CTX.

[0004] Native CTX derived from the original toxin has a carboxamide at the C-terminus (i.e., the native protein denoted as CTX), while a variant of recombinantly produced CTX (hereinafter denoted as rCTX) ends with the carboxyl group of arginine at the C-terminus.

[0005] The tumor selectivity of CTX was originally due to binding to the chloride ion channel of glioma cells. Subsequently, matrix metalloproteinase 2 (MMP-2) was identified as a receptor for CTX, either alone or as a protein complex with MMP-14 (matrix metalloproteinase 14, also known as membrane-type-1 matrix metalloproteinase (MT1-MMP)), TIMP-2 (tissue inhibitor of metalloproteinase 2), and avβ3 integrin [Deshane 2003]. This MMP-TIMP-integrin complex is thought to play an important role in the regulation (maturation, immobilization, activation, and inhibition) of cell surface-bound and released active MMP-2, and these factors may cooperate to promote tumor invasion, metastasis, and angiogenesis [Yosef 2018].

[0006] Nevertheless, in subsequent studies, a direct and specific interaction between CTX and recombinantly produced MMP-2 could not be established in pull-down assays [Veiseh 2007]. Prompted by this ambiguity, other research groups reexamined the identification of the glioma CTX receptor using affinity columns, cross-linking reagents, and mass spectrometry, and concluded that the direct binding partner protein containing the CTX receptor was probably annexin 2 (Anx2) [Kesavan 2010]; they acknowledged the possibility of other binding partner proteins and did not directly demonstrate the CTX-Anx2 protein-protein interaction using recombinantly produced and purified Anx2 protein.

[0007] Yet another research group identified neuropilin-1 (NRP1), an endocytosis receptor of tumor cells and endothelial cells, as a novel CTX target [McGonigle 2019]. They found that only rCTX binds to NRP1, and native (i.e., C-terminus is carboxamide) CTX does not bind to NRP1, but is metabolized to the carboxyl variant by deamidation in the cellular environment.

[0008] Thus, in view of the proposals that are debatable, the true target protein of CTX remains largely unknown. The domain or site of the target protein that specifically binds to CTX is often called the CTX receptor [e.g., Soroceanu 1998; Dardevet 2015], and this term is also used herein for simplicity.

[0009] CTX-containing constructs and CTX variants in preclinical and clinical trials Compared with specific antibody target molecules, the small protein CTX can cross the blood-brain barrier. Therefore, CTX is promising as a starting point for creating new targeted molecules.

[0010] In some initial preclinical studies, it has been reported in several patent applications that synthetically produced CTX (TM-601) is effective against glioblastoma xenograft tumor growth in vivo in mice, either alone or in combination with temozolomide [U.S. Patent Publication No. 20100215575 and U.S. Patent Publication No. 20100210546]. However, these findings were not later clearly confirmed in scientific papers or follow-up clinical trials, and rather, the interest has turned towards the use of CTX as a target molecule.

[0011] Despite the fact that the true target of CTX has not been clearly defined, this molecule has gathered much interest in the field of cancer research as a diagnostic or therapeutic tool, or as a theranostic that combines such uses. The multiple uses outlined in [Dardevet 2015] are made possible by the fact that the amino acid sequence of CTX provides several opportunities for useful chemical modifications. Such modifications include conjugation with a number of compounds, or the addition of further peptide sequences as linkers for conjugation or cyclization, which enable CTX to be used as a targeted carrier molecule. The use of targeting is made possible by the distinct feature that such modifications often do not significantly alter the target recognition (CTX receptor binding) properties of the modified CTX compound. For example, CTX contains one tyrosine residue at position 29 that can be used for iodination [Soroceanu 1998], and specific radioactive iodine isotopes ( 123 I, 125 I, and 131 I) are routinely used for binding to targeted molecules for any of various preclinical studies (e.g., 125 I in binding studies) or clinical imaging (e.g., gamma scan or single photon emission computed tomography - SPECT or SPECT / CT) or radiotherapy applications. 131 A clinical phase 1 / 2 trial using

[0012] I-labeled CTX (designated TM-601 in the literature) was initiated for both local (intratumoral) treatment administration and intravenous imaging (low dose) administration. The results of the first phase of the treatment trial were published as showing promising signals with regard to efficacy and safety [Mamelak 2006]; however, the approved phase 2 treatment trial and the development of the diagnostic agent were discontinued without any results being published. The first phase diagnostic (imaging) trial was clearly discontinued early [clinicaltrials.gov reference number NCT00683761].

[0012] WO2011094671 pamphlet describes a polypeptide conjugated to the N-terminus, which may be CTX or its variant. Here, it is described that CTX helps to target the conjugate to a population of cancerous cells and that CTX can specifically bind to MMP-2.

[0013] The lysine residues of the CTX molecule can also be utilized to facilitate the conjugation of active substances due to the availability of a wide range of cross-linking reagents. There are three lysine residues at positions 15, 23, and 27 of CTX, which have been utilized for conjugation with NHS-ester modified cyanine 5.5 (Cy5.5) and other fluorescent molecules [Akcan 2011; International Publication WO2011142858 pamphlet]. However, since wild-type CTX has three lysine residues, some modifications of CTX were considered appropriate to reproducibly produce homogeneous conjugate derivatives at specific position(s) in high yield, rather than a mixture of various single-site and multi-site conjugation products. Although it is possible to use mixtures approved by the US Food and Drug Administration (FDA) and other similar regulatory agencies, it can be costly and difficult to match the ratios of mono-, di-, and tri-labeled batches, which may hinder commercialization. Therefore, Olson and co-workers reported an example of CTX re-engineering. They showed that by substituting lysine 15 and 23 with either alanine or arginine, the mixed bioconjugate species in the resulting batch were eliminated, while the in situ tumor staining activity of the mutant CTX protein was retained compared to CTX [Akcan 2011; International Publication WO2011142858 pamphlet]. As a result, the monolisine (K27) mutant of CTX (hereinafter referred to as mCTX; SEQ ID NO: 2) was obtained by lysine-arginine substitution (K15R, K23R) at positions 15 and 23. This mCTX protein was conjugated with indocyanine green (ICG), designated as BLZ-100 (INN name tozuleristide; CAS registration number: 1673565-40-6). This is currently in Phase 2 clinical development [clinicaltrials.gov reference number NCT03579602] for intraoperative visualization of human tumors [Dintzis 2019; Patil 2019], i.e., as a "tumor paint".While studying the fluorescent conjugates of CTX and mCTX, it was observed that these conjugates are internalized by HeLa (cervical cancer) cells, and that the cellular uptake of the Cy5.5- and Alexa Fluor 488-conjugates of mCTX is 1.5- and 2-fold higher than that of the analogous conjugates of CTX [Ojeda 2017]. However, this difference is a feature of CTX conjugated to a fluorophore and not necessarily of non-conjugated targeted CTX variants. However, for intraoperative visualization (“fluorescence-guided surgery”), in addition to the above Cy5.5 and ICG, several other fluorophore-conjugated CTX variants may be used, such as IRDye 800 CW, DyLight 750, or VivoTag-S 750, and the use of near-infrared dyes is most preferred [Stroud 2011].

[0014] International Publication WO2015042202 discloses conjugates and kits comprising CTX variants. According to some embodiments, the CTX variants form conjugates with a fluorescent moiety. The conjugates described herein are useful for the treatment and imaging of tumors.

[0015] A review on CTX-specific inventions was published in 2014 [Cheng 2014]. Herein, it is described that CTX can specifically inhibit the growth and metastasis of glioma cells and accelerate tumor apoptosis. Several references regarding CTX bioconjugates are provided. It is also described herein that CTX specifically binds to the MMP-2 receptor.

[0016] Cyclization is a method often used in vitro or in vivo to extend the half-life of polypeptide drug candidates in blood serum. Akcan et al. showed that the cyclic variant of CTX retains the ability to bind to malignant tissues [Akcan 2011]. This cyclic variant was produced by linking the C-terminal arginine to the N-terminal methionine via a heptapeptide (GAGAAGG) linker. This modification not only extended the half-life in plasma but also promoted single-site conjugation. Thus, cyclization using several extended peptide segments or incorporating extra disulfide bridges is an obvious modification of the CTX variant parent protein to those skilled in the art, and such modifications would retain the targeting ability of the parent protein.

[0017] Another use of CTX as a targeting molecule is to apply CTX in chimeric antigen receptor T cell (CAR-T cell) therapy instead of a specific protein recognition antibody. A chimeric antigen receptor (CAR) is a receptor protein that has been genetically engineered to give T cells the new ability to target specific proteins present on the surface of tumor cells. The receptor is chimeric because it incorporates both an antigen-binding function and a T cell activation function into one receptor. Thus, a CAR is composed of an extracellular tumor recognition / targeting domain, an extracellular linker / spacer, a transmembrane domain, and an intracellular T cell activation and costimulatory signaling domain. The patent application International Publication No. WO2017 / 066481 describes CAR-T constructs in which the extracellular tumor recognition / targeting domain is CTX, a related toxin, or a CTX variant. Currently, the development of CAR-T cell immunotherapy using CTX as a tumor targeting domain is in Phase 1 clinical trials [NCT04214392 on clinicaltrials.gov] for the treatment of MMP-2 positive glioblastoma patients.

[0018] Another use of CTX as a targeting molecule is to conjugate CTX to an anti-proliferative or cytotoxic payload molecule. To achieve specificity by the targeting molecule, various anti-proliferative agents have been conjugated to antibodies or smaller targeting polypeptides. Initially, this attempt was directed at enhancing the specificity of existing chemotherapeutic agents such as vinca alkaloids and doxorubicin. However, antibody-drug conjugates (ADCs) have evolved significantly in recent years, with some having received FDA approval and many others being in clinical development [Lambert 2018]. The cytotoxic compounds used in most of the currently clinically developed ADCs are either the potent anti-mitotic microtubule disruptor dolastatin 10 (auristatin) or a derivative of maytansine, or some highly cytotoxic DNA-damaging agents: calicheamicin, duocarmycin, a pyrrolobenzodiazepine dimer, and a derivative of one of the indolinobenzodiazepine pseudodimers [Lambert 2018]. These carrier-linker-payload constructs are based on the premise that the cytotoxic compound acts on an intracellular target and the targeting molecule facilitates specific internalization into cells recognized by the surface presence of a cell-specific target protein. Internalization occurs via the endosome-lysosome pathway, where the linker is cleaved and / or the antibody is degraded to release the payload. Thus, such ADC constructs were assembled with either lysosome-enzyme-cleavable or non-cleavable linkers [Lambert 2018]. In the published international patent application WO2017 / 136769, a conjugate of CTX and a cryptophycin derivative is described as an effective anti-cancer therapeutic agent. In addition to auristatin (e.g., monomethyl auristatin E or F - MMAE or MMAF) and maytansine, cryptophycin is also another microtubule disruptor. A notable example of this patent application is a carrier-linker-payload construct of CTX conjugated to an analog of cryptophycin via a cleavable dimethyldisulfide linker at lysine 27.This compound has excellent potent antitumor activity compared to the efficacy of similar linker-payload constructs without CTX in subcutaneous xenograft mouse models of glioblastoma, pancreatic cancer, prostate cancer, and breast cancer.

[0019] Furthermore, as diagnostic and therapeutic applications utilizing CTX as a targeting molecule, platinum(IV)-CTX; conjugate for polymer dot bioconjugate; conjugate upconversion nanoprobe; Ag-In-S / ZnS quantum dot; dendrigraft polylysine having a gadolinium magnetic nanovector compatible with a combination of cancer cell targeting, imaging, and siRNA delivery; nanoparticle-based targeted gene delivery; combination of drug delivery and enhancement of MRI contrast agent by CTX-conjugated iron oxide nanoparticles; CTX-modified doxorubicin-loaded liposomes; other nanoparticles and nanoprobes have been proposed and developed as reviewed in [Dardevet 2015; Cohen 2018].

[0020] Disadvantages of existing CTX constructs and variants Numerous attempts have been made to utilize CTX as a diagnostic or therapeutic targeting agent, including in early-stage clinical development, but to date, no developed product has reached regulatory approval (marketing approval) for introduction into the medical field. Two approaches using CTX as a targeting agent (CAR-T and tozuleristide) appear to have been actively developed in recent years. Some of the other developed products are apparently discontinued according to information publicly available in essential clinical trial databases such as clinicaltrials.gov. However, the reasons for failure or discontinuation have not been disclosed and are only speculative. In addition to potential financial or managerial issues, one obvious reason for failure may be the insufficient affinity or strength of CTX for useful receptors and / or the insufficient selectivity against unwanted binding or effects on other molecular or cellular targets.

[0021] The 50% inhibitory effect (IC 50 ) of CTX on the invasion and migration of glioma cell lines of U251MG, D54MG and U87MG is brought about by a concentration of approximately 600 nM [Soroceanu 1999], which is in the range of approximately 500 - 700 nM of EC 50 and IC 50 values measured in the MMP-2 binding and displacement experiments of the present inventors (see below). Such moderate potency and high submicromolar effective concentration levels mean that it is difficult to achieve a systemic concentration in vivo that is sufficiently effective for treatment or labeling. Nevertheless, the selectivity of a targeting molecule that delivers more payload molecules to tumor cells than to healthy normal cells is even more important for the effective diagnostic and / or therapeutic application of the targeting molecule. Thus, it is an obvious assumption to those skilled in the art that the vast unmet needs in the field of CTX-related research for the diagnosis and treatment of malignant tumors may be better met by more potent and / or more selective compounds, in terms of either target protein-related or cell-binding and functional effects. Furthermore, even in the same type of tumor, the expression of marker proteins shown by tumor cell lines varies widely. Therefore, there is a need for a variety of new tumor cell marker recognition molecules with different recognition profiles, and also for the combination of cell-specific diagnostic tools and matching therapeutic tools linked by a common pattern recognition target molecule, i.e., "seranostics". Specific antibodies are widely used for such purposes, but smaller-sized specific targeting molecules may have advantages over antibodies, such as better penetration characteristics and production cost effectiveness.

Summary of the Invention

Problems to be Solved by the Invention

[0022] The idea underlying the present invention is that the CTX derivatives of the present invention (hereinafter referred to as CTXD or chlorotoxin derivatives) bind more strongly to matrix metalloproteinase 2 (MMP-2) protein than CTX. As a result, these compounds are taken up more strongly by a number of neuroectodermal tumor cells that overexpress MMP-2, including but not limited to breast cancer, glioblastoma, melanoma and pancreatic cancer. Surprisingly, representative compounds of the present invention conjugated with a fluorophore (i.e., CTXD5 and CTXD8, see Table II) showed not only stronger cellular uptake but also higher selectivity for the tested neuroectodermal tumor cells compared to non-tumor cells such as fibroblasts, astrocytes and endothelial cells. Furthermore, in purified form, when examined for binding to a panel of putative target proteins, surprisingly, CTX binds to other proteins other than MMP-2, i.e., NRP-1, with approximately the same strength, and also binds moderately to MMP-9, TIMP-2, and chloride channel CLC3. At the same time, CTXD5 and CTXD8 also showed an increase in target protein selectivity, showing distinct binding only to MMP-2 and very weak binding to TIMP-2 and CLC3. Furthermore, the affinity of CTXD5 and CTXD8 for MMP-2, their major target protein, did not change significantly even when conjugated with lysine residues to relatively large molecules such as chemotherapeutic cytostatic compounds or fluorescently labeled fluorophore compounds having a designed linker structure. Taken together, these observations suggest that the compounds of the present invention are useful when conjugated with other molecules, nanoparticles, CAR-T cell constructs that provide additional features enabling applications, also called "theranostic", that will be apparent from the prior art related to such putative uses of CTX, either alone or in combination with diagnosis, or treatment, or a combination of diagnosis and treatment, and are superior to CTX or mCTX as targeting molecules. The proteins of the present invention can serve as a starting point for further modification or extension of CTX derivatives, such as cyclization, for those skilled in the art to extend the half-life of protein drug candidates in blood serum.

Means for Solving the Problems

[0023] From the above viewpoints, the present invention relates to a general sequence X0X1CMPCX S1 X S2 X S3 DHX S4 X S5 ARRCX2X3CCGGYGX4CFGYQCLCX5X6X7X8 (SEQ ID NO: 43) and relates to a chlorotoxin derivative comprising the amino acid sequence of Here, (i) The N-terminal X0X1 cluster is selected from the group consisting of AM, 0M, or 00, (ii) the solubility X S1 X S2 X S3 X S4 X S5 [[ID=3③]]cluster is selected from the group consisting of FTTQT, FTTES, SSSQT, SSSES, FSSQT, FSSES, or FSSQS; (iii) The internal X2X3X4 cluster is selected from the group consisting of DKR, RDK, KDR, IKY, HKW, DRK, LKQ, KKK; and, (iv) The C-terminal X5X6X7X8 cluster is selected from the group consisting of N000, R000, NR00, NRG0, NRGY, NRRR, or RRRR; here, 0 indicates a position where no amino acid is present Shi; A chlorotoxin derivative having a relative human MMP-2 binding property that is at least 1.62 times higher than that of the wild-type chlorotoxin of SEQ ID NO: 1 Relates to .

[0024] According to a preferred embodiment, the present invention relates to a general sequence X0X1CMPCFTTDHQTARRCX2X3CCGGYGX4CFGYQCLCX5X6X7X8 (SEQ ID NO: 35) and relates to a chlorotoxin derivative comprising the amino acid sequence of Here, (i) The N-terminal X0X1 cluster is selected from the group consisting of AM, 0M, or 00, (ii) The internal X2X3X4 cluster is selected from the group consisting of DKR, RDK, KDR, IKY, HKW, DRK, LKQ, and KKK; and (iii) The C-terminal X5X6X7X8 cluster is selected from the group consisting of N000, R000, NR00, NRG0, or NRGY; wherein 0 indicates a position where no amino acid is present.

[0025] According to a preferred embodiment, the internal X2X3X4 cluster of the chlorotoxin derivative is DKR.

[0026] According to a more preferred embodiment, the chlorotoxin derivative is selected from chlorotoxin derivatives having any of the sequences set forth in SEQ ID NO: 3 to SEQ ID NO: 18 and SEQ ID NO: 36 to SEQ ID NO: 42. According to an even more preferred embodiment, the chlorotoxin derivative has the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 42.

[0027] According to another preferred embodiment, the chlorotoxin derivative is cyclic.

[0028] The present invention further relates to a conjugate, wherein the conjugate comprises a chlorotoxin derivative of the present invention and a family of deficiency molecules.

[0029] The family of deficiency molecules is preferably selected from the following agents: (i) A visualizing agent, preferably a visualizing agent selected from fluorescent labels, radioactive labels, magnetic resonance imaging labels, and agents that enable indirect labeling by high-affinity binding to a labeled molecule; (ii) A therapeutic agent, preferably a therapeutic agent selected from chemotherapeutic agents and biological therapeutic agents; (iii) A targeting agent, preferably a targeting agent selected from antibodies, polypeptides, polysaccharides, and nucleic acids; (iv) A part that increases the circulation half-life, preferably selected from a part that enables cyclization of the chlorotoxin derivative according to any one of claims 1 to 5 via a PEG part, a glycosyl part, a glycosyl PEG part, and a linker extension.

[0030] According to a more preferred embodiment of the present invention, the conjugate includes one or more linker(s) and optionally one or more spacer(s) between the chlorotoxin derivative and the family of compensatory molecules. The linker is preferably selected from the group consisting of a peptide, a dimethyldisulfide linker, a glutaric linker, and a cathepsin-cleavable linker.

[0031] The present invention further relates to a seranostic pair of conjugates including a first conjugate and a second conjugate, where both conjugates are conjugates of the present invention. According to a preferred embodiment of the present invention, the chlorotoxin derivative parts of the two conjugates have the same amino acid sequence, and the two conjugates have different families of compensatory molecules. According to another preferred embodiment, the chlorotoxin derivative parts of the first conjugate and the second conjugate have different amino acid sequences, and the first conjugate and the second conjugate have the same or different families of compensatory molecules.

[0032] The present invention further relates to a kit including the conjugate of the present invention and an instruction manual. According to other embodiments, the kit includes the seranostic pair of the present invention and an instruction manual.

[0033] The present invention further relates to a pharmaceutical composition including the conjugate of the present invention, where the family of compensatory molecules of the conjugate is a therapeutic agent or a visualization agent, and the pharmaceutical composition further includes a pharmaceutically acceptable excipient.

[0034] The present invention further relates to a conjugate of the present invention, which conjugate is for use in the treatment of cancer or for use in the diagnosis of cancer. According to a preferred embodiment, the cancer is selected from the group consisting of breast cancer, cervical cancer, colon cancer, epithelioma, Ewing's sarcoma, ganglioglioma, ganglioma, glioma, glioblastoma, gliosarcoma, medulloblastoma, meningioma, neuroblastoma, melanoma (primary and metastatic), pancreatic cancer, pheochromocytoma, prostate cancer, schwannoma and small cell lung carcinoma. According to the most preferred embodiment, the cancer is selected from the group consisting of breast cancer, glioblastoma, melanoma and pancreatic cancer.

[0035] The present invention further relates to a conjugate of the present invention, which conjugate is for use in the visualization of cancerous tissue. According to a preferred embodiment, the cancerous tissue is selected from the group consisting of breast cancer, cervical cancer, colon cancer, epithelioma, Ewing's sarcoma, ganglioglioma, ganglioma, glioma, glioblastoma, gliosarcoma, medulloblastoma, meningioma, neuroblastoma, melanoma (primary and metastatic), pancreatic cancer, pheochromocytoma, prostate cancer, schwannoma and small cell lung carcinoma. According to the most preferred embodiment, the cancerous tissue is selected from the group consisting of breast cancer, glioblastoma, melanoma and pancreatic cancer.

[0036] The present invention further relates to a method of treating a patient suffering from cancer by administering an effective amount of a conjugate according to the present invention, wherein the family of missing molecules is a therapeutic agent suitable for the treatment of cancer. According to a preferred embodiment, the cancer is selected from the group consisting of breast cancer, cervical cancer, colon cancer, epithelioma, Ewing's sarcoma, ganglioglioma, ganglioma, glioma, glioblastoma, gliosarcoma, medulloblastoma, meningioma, neuroblastoma, melanoma (primary and metastatic), pancreatic cancer, pheochromocytoma, prostate cancer, schwannoma and small cell lung carcinoma. According to the most preferred embodiment, the cancer is selected from the group consisting of breast cancer, glioblastoma, melanoma and pancreatic cancer.

[0037] The present invention further relates to a method for treating a patient suffering from cancer by administering the pharmaceutical composition according to the present invention, wherein the complementing molecular family of the conjugate is a therapeutic agent suitable for the treatment of cancer. According to a preferred embodiment, the cancer is selected from the group consisting of breast cancer, cervical cancer, colon cancer, epithelioma, Ewing's sarcoma, ganglioglioma, ganglioneuroma, glioma, glioblastoma, gliosarcoma, medulloblastoma, meningioma, neuroblastoma, melanoma (primary and metastatic), pancreatic cancer, pheochromocytoma, prostate cancer, schwannoma, small cell lung carcinoma. According to the most preferred embodiment, the cancer is selected from the group consisting of breast cancer, glioblastoma, melanoma and pancreatic cancer.

[0038] The present invention further relates to a method for preparing a conjugate, comprising: a) providing a chlorotoxin derivative according to the present invention; b) optionally binding a linker group to the chlorotoxin derivative of step a); c) binding a complementing molecular family to the linker portion of the compound formed in step b) or directly binding to the protein of step a) in the absence of a linker.

[0039] The present invention further relates to a method for visualizing cancerous tissue, comprising contacting the tissue to be examined with the conjugate of the present invention, wherein the complementing molecular family of the conjugate is a visualization agent suitable for the visualization of cancerous tissue.

[0040] The present invention further relates to a nucleic acid molecule encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: a) a chlorotoxin derivative according to the present invention; b) optionally, a spacer region between the chlorotoxin derivative and the transmembrane domain; c) a transmembrane domain; d) one or two co-stimulatory domains; e) a signaling domain and wherein the chlorotoxin derivative enables the chimeric antigen receptor, when expressed on the surface of a T cell, to direct the activity of the T cell towards cancerous cells.

[0041] According to a preferred embodiment, the nucleic acid molecule of the present invention encodes a chimeric antigen receptor, wherein the chlorotoxin derivative is selected from chlorotoxin derivatives having any of the sequences set forth in SEQ ID NO: 3 to SEQ ID NO: 18, and SEQ ID NO: 36 to SEQ ID NO: 42, preferably SEQ ID NO: 4 or SEQ ID NO: 42.

[0042] According to another preferred embodiment, the nucleic acid molecule of the present invention encodes a chimeric antigen receptor, wherein i) the transmembrane domain is selected from the group consisting of the CD4 transmembrane domain or a variant thereof, the CD8 transmembrane domain or a variant thereof, the CD28 transmembrane domain or a variant thereof, and the CD3 zeta transmembrane domain or a variant thereof; ii) one or two co-stimulatory domains are selected from the group consisting of the CD28 co-stimulatory domain or a variant thereof, the 4-1BB co-stimulatory domain or a variant thereof, and the OX40 co-stimulatory domain or a variant thereof; and iii) the signaling domain is the CD3 zeta signaling domain or a variant thereof.

[0043] The present invention further relates to a vector comprising the nucleic acid molecule of the present invention.

[0044] The present invention further relates to a population of human cells, i) transfected with an RNA or DNA vector comprising an expression cassette containing the nucleic acid of the present invention, wherein the transfection is performed in vivo or ex vivo; or ii) transduced with a viral vector comprising an expression cassette containing the nucleic acid of the present invention, wherein the transduction is performed in vivo or ex vivo, Here, the viral vector is preferably a retroviral or lentiviral vector; the human cells are selected from the list consisting of autologous human T cells, autologous human CD4+ helper T cells, autologous human CD8+ cytotoxic T cells, a mixture of autologous human CD4+ helper T cells and CD8+ cytotoxic T cells in any ratio, allogeneic human T cells, allogeneic human CD4+ helper T cells, allogeneic human CD8+ cytotoxic T cells, a mixture of allogeneic human CD4+ helper T cells and CD8+ cytotoxic T cells in any ratio, autologous primary human natural killer (NK) cells, allogeneic primary human natural killer (NK) cells, allogeneic cells of the NK-92 cell line, autologous human monocytes, autologous human macrophages, allogeneic human monocytes, and allogeneic human macrophages.

[0045] The present invention further relates to a method for treating cancer in a patient, comprising administering one or more populations of human cells according to the present invention.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0047] In the context of this specification, under the terms "variant", "chlorotoxin variant", or "CTX variant", these proteins are understood to be proteins different from wild-type CTX and are part of the art.

[0048] In the context of this specification, under the terms "derivative", "chlorotoxin derivative", "CTX derivative", or "CTXD", these proteins are understood to have been developed during the projects that led to the present invention.

[0049] The term CTXD with a running number at the end refers to a chlorotoxin derivative (protein) or a phage clone that produces said chlorotoxin derivative. Each specific CTXD referred to in this specification has a specific amino acid sequence. For example, CTXD5 in this specification refers to a protein having the sequence according to SEQ ID NO: 4, and CTXD1 in this specification refers to a protein having the sequence according to SEQ ID NO: 17.

[0050] The term "cancer" is used for diseases in which abnormal cells divide without control and can invade nearby tissues (this definition is based on the description at https: / / www.cancer.gov / publications / dictionaries / cancer-terms / def / cancer as of the filing date of this patent application). Cancer cells can also spread to other parts of the body via the blood and lymphatic systems. There are several main types of cancer. Carcinoma is cancer that starts in the tissues that line or cover the skin or internal organs. Sarcoma is cancer that starts in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissues. Leukemia is cancer that starts in hematopoietic tissues such as the bone marrow and produces too many abnormal blood cells. Lymphoma and multiple myeloma are cancers that start in cells of the immune system. Cancers of the central nervous system are cancers that start in the tissues of the brain and spinal cord. Cancer is also called a malignant tumor.

[0051] The term "cancerous" is understood to be a condition of a mammalian or human patient that includes cancer.

[0052] The term "theranostics" is derived from the combination of therapeutics and diagnostics. This term refers to a specific combination of diagnostic and therapeutic technologies, whereby a combination of a specific diagnostic technology and a method or product for treatment provides guidance for identifying patients, tissues, or cell populations sensitive to appropriate treatment [Jeelani 2014].

[0053] The chlorotoxin derivative of the present invention has the following general amino acid sequence: X0X1CMPCX S1 X S2 X S3 DHX S4 X S5 ARRCX2X3CCGGYGX4CFGYQCLCX5X6X7X8 (SEQ ID NO: 43) comprising, wherein (i) the N-terminal X0X1 cluster is selected from the group consisting of AM, 0M, or 00, (ii) the solubility X S1 X S2 X S3 X S4 X S5 cluster is selected from the group consisting of FTTQT, FTTES, SSSQT, SSSES, FSSQT, FSSES, or FSSQS; (iii) the internal X2X3X4 cluster is selected from the group consisting of DKR, RDK, KDR, IKY, HKW, DRK, LKQ, KKK; and (iv) the C-terminal X5X6X7X8 cluster is selected from the group consisting of N000, R000, NR00, NRG0, NRGY, NRRR, or RRRR; wherein 0 indicates a position where no amino acid is present Shi; A relative human MMP-2 having a binding property that is at least 1.62 times higher than that of the wild-type chlorotoxin of SEQ ID NO: 1 Having .

[0054] According to a preferred embodiment, the present invention relates to a chlorotoxin derivative comprising the amino acid sequence of a general sequence X0X1CMPCFTTDHQTARRCX2X3CCGGYGX4CFGYQCLCX5X6X7X8 (SEQ ID NO: 35) wherein (i) the N-terminal X0X1 cluster is selected from the group consisting of AM, 0M, or 00, (ii) the internal X2X3X4 cluster is selected from the group consisting of DKR, RDK, KDR, IKY, HKW, DRK, LKQ, KKK; and (iii) the C-terminal X5X6X7X8 cluster is selected from the group consisting of N000, R000, NR00, NRG0, or NRGY; Here, 0 indicates the position where no amino acid is present.

[0055] Note that not all possible variations of SEQ ID NO: 35 and SEQ ID NO: 43 shown in the sequence listing fall within the scope of the present invention, and only those that satisfy the above cluster definition fall within the scope of the present invention.

[0056] The general sequences SEQ ID NO: 35 and SEQ ID NO: 43 have three and four variable clusters, respectively. The N-terminal N-terminal cluster (X0X1 cluster), the solubility cluster (X S1 X S2 X S3 X S4 X S5 cluster, only for SEQ ID NO: 43), the internal cluster (X2X3X4 cluster), and the C-terminal C-terminal cluster (X5X6X7X8 cluster). All of these clusters have specific limited amino acid compositions. However, it has been revealed that the compositions of the clusters are independent of each other.

[0057] The N-terminal cluster consists of two positions, namely the positions of X0 and X1. The composition of the N-terminal cluster can be AM, 0M, or 00. Here, A represents alanine (Ala), M represents methionine (Met), and 0 indicates the position where no amino acid is present. Therefore, the N-terminal cluster may have only an Ala-Met dimer or a Met monomer, or this N-terminal cluster may remain empty, that is, have no amino acid at these positions.

[0058] The solubility cluster is the X S1 X S2 X S3 X S4 X S5 cluster (only for SEQ ID NO: 43). This is the X S1 、X S2 X S3 X S4 and X S5consists of five positions, where the subscript "S" refers to the characteristic of "solubility". Amino acids forming a solubility cluster are not necessarily adjacent amino acids in the sequence, and there are two sub - clusters, namely X S1 X S2 X S3 and X S4 X S5 It is clear that there exist. The solubility cluster can have the following compositions: FTTQT, FTTES, SSSQT, SSSES, FSSQT, FSSES, or FSSQS. For example, X S1 X S2 X S3 X S4 X S5 When the composition of the solubility cluster is FTTQT, the amino acid at the X S1 position is F, which represents phenylalanine (Phe), and X S2 , X S3 and X S5 positions are T, which represents threonine (Thr), and the amino acid at the X S4 position is Q, which represents glutamine (Gln). Further amino acids that can be found in this solubility cluster are E, which represents glutamic acid (Glu), and S, which represents serine (Ser).

[0059] The internal cluster is an X2X3X4 cluster approximately in the center of the amino acid sequence and consists of three positions, X2, X3, and X4. It is clear that the amino acids forming the internal cluster are not necessarily adjacent amino acids in the sequence. The amino acids at positions X2 and X3 are adjacent, but the amino acid at position X4 is seven positions away in the C-terminal direction. The internal X2X3X4 cluster can have the following compositions: DKR, RDK, KDR, IKY, HKW, DRK, LKQ, KKK. For example, when the composition of the internal X2X3X4 cluster is said to be DKR (which is one of the preferred embodiments of the present invention), the amino acid at position X2 is D representing aspartic acid (Asp), the amino acid at position X3 is K representing lysine (Lys), and the amino acid at position X4 is R representing arginine (Arg). Further amino acids that can be found in this internal cluster are I representing isoleucine (Ile), Y representing tyrosine (Tyr), H representing histidine (His), W representing tryptophan (Trp), L representing leucine (Leu), and Q representing glutamine (Gln).

[0060] The C-terminal cluster consists of four positions, namely X5, X6, X7, and X8. The composition of the C-terminal cluster can be N000, R00D, NR00, NRG0, or NRGY. Here, N represents asparagine (Asn), R represents arginine (Arg), G represents glycine (Gly), and Y represents tyrosine (Tyr). 0 represents a position where no amino acid is present. This C-terminal cluster can have only one amino acid N or R (i.e., when the meaning of X5X6X7X8 is N000 or R00D), and in these cases, it is clear that the chlorotoxin derivative of the present invention ends at the C-terminus having position X5. Further, the C-terminal cluster can have two amino acids (i.e., when the meaning of X5X6X7X8 is NR00), or three amino acids (i.e., when the meaning of X5X6X7X8 is NRG0), or four amino acids (i.e., when the meaning of X5X6X7X8 is NRGY in the case of SEQ ID NO: 35, and NRGY, NRRR, or RRRR in the case of SEQ ID NO: 43).

[0061] According to a preferred embodiment of the present invention, the chlorotoxin derivative is selected from chlorotoxin derivatives having any of the sequences set forth in SEQ ID NOs: 3 to 18 and SEQ ID NOs: 36 to 42. The chemical space defined by SEQ ID NO: 43 covers all chlorotoxin derivatives set forth in SEQ ID NOs: 3 to 18 and SEQ ID NOs: 36 to 42. Apart from the description in the sequence listing, the chlorotoxin derivatives corresponding to these sequences are shown in Table I and Table II.

[0062] According to a more preferred embodiment of the present invention, the chlorotoxin derivative is selected from chlorotoxin derivatives having any of the sequences set forth in SEQ ID NOs: 3 to 18. The chemical space defined by SEQ ID NO: 35 covers all chlorotoxin derivatives set forth in SEQ ID NOs: 3 to 18. Apart from the sequence listing, the chlorotoxin derivatives corresponding to these sequences are shown in Table I.

[0063] According to an even more preferred embodiment of the present invention, the chlorotoxin derivative has the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 42. The protein having the amino acid sequence of SEQ ID NO: 4 is also referred to herein as CTXD5. The protein having the amino acid sequence of SEQ ID NO: 42 is also referred to herein as CTXD8.

[0064] The chlorotoxin derivatives of the present invention can be cyclic proteins. It is well known to those skilled in the art that proteins can be more stable in a cyclized form than in a linear form. As described above, the cyclic variants of CTX maintained the ability to bind to malignant tissues [Akcan 2011]. Therefore, it is clearly foreseeable to those skilled in the art that any chlorotoxin derivative can be cyclized, thereby increasing its half-life. Methods for cyclizing proteins are well known to those skilled in the art [Di 2015]. Within the scope of the present invention, the term "cyclic" refers to a chlorotoxin derivative that is cyclized by itself or by inserting a linker, provided that a given cyclization does not significantly affect the desired effect of the chlorotoxin derivative, such as the affinity of the chlorotoxin derivative for the MMP-2 protein, in a negative manner.

[0065] The chlorotoxin derivatives of the present invention can effectively recognize and bind to cancerous cells. Therefore, they are suitable for delivering a chemical entity to the cancerous cells, and this chemical entity can exert a desired function once bound by the cancerous cells. Within the scope of the present invention, under the term "conjugate", a construct is understood to include at least a chlorotoxin derivative and a family of compensatory molecules. According to the present invention, the chemical entity is in the form of a family of compensatory molecules. Within the scope of the present invention, under the term "family of compensatory molecules", any chemical entity is understood to bind to the chlorotoxin derivative according to the present invention via a primary bond (i.e., a covalent bond or an ionic bond). The scope of the family of compensatory molecules can range from a single atom to a large protein.

[0066] The moiety to be supplemented is preferably a visualizing agent. That is, in a preferred embodiment of the present invention, the chlorotoxin derivative according to the present invention forms a conjugate with the moiety to be supplemented, where the moiety to be supplemented is a visualizing agent. Thereby, such a conjugate can be used to visualize the part of the body (e.g., the human body) that binds to the chlorotoxin derivative. Methods for chemical bonding to visualizing agents and specific proteins are known to those skilled in the art. Preferred visualizing agents in the sense of the present invention are i) Fluorescent labels such as cyanine dyes (e.g., indocyanine green); ii) 123 I, 125 I and 131 Radioactive labels such as iodine isotopes of I; iii) Magnetic resonance imaging labels such as boron nanoparticles, boron and carbon nanoparticles, boron carbide nanoparticles, boron-containing polymers, boron and carbon-containing polymers, boron carbide polymers, any of these nanoparticles or polymers further containing gadolinium; iv) Agents that enable indirect labeling by high-affinity binding to labeling molecules such as biotin, avidin, his tag.

[0067] When the visualizing agent is a fluorescent label, it can be used as preferably in fluorescence-guided surgery. During this surgical procedure, the conjugate of the present invention visualizes the cells or part of the tissue that binds to the conjugate via the chlorotoxin derivative moiety. Thereby, these cells or part of the tissue are visualized to the surgeon performing the surgery. Preferably, near-infrared dyes including, but not limited to, indocyanine green, cyanine 5.5, IRDye 800 CW, DyLight 750 or VivoTag-S 750 can be used for this purpose.

[0068] Another preferred example of using the conjugate of the present invention where the moiety to be supplemented is a fluorescent label is when analyzing target cells during a flow cytometry method or a histological staining process. In such a case, the conjugate of the present invention is used as a diagnostic agent.

[0069] According to another preferred embodiment, the moiety is a therapeutic agent. That is, in a preferred embodiment of the present invention, the chlorotoxin derivative according to the present invention forms a conjugate with the moiety, where the moiety is a therapeutic agent. Thereby, such a conjugate can be used for treatment, where the chlorotoxin moiety of the conjugate recognizes and binds to cancer tissue. The therapeutic moiety of the conjugate thereby approaches or internalizes into the cancerous tissue cells and exerts a therapeutic effect. Methods for chemical conjugation to therapeutic agents and specific proteins are known to those skilled in the art. Preferred therapeutic agents in the context of the present invention are i) chemotherapeutic agents such as auristatin, cryptophycin, calicheamicin, duocarmycin, pyrrolobenzodiazepine dimer, indolinobenzodiazepine pseudodimer, maytansine, methotrexate, docetaxel, cisplatin and etoposide; ii) biological therapeutic agents such as cDNA, siRNA, shRNA, and RNAi.

[0070] A conjugate formed with monomethyl auristatin F (MMAF) is an example of a conjugate of the present invention suitable for therapeutic purposes (for details, see, for example, item 3.3 of the following "Chemical Toolbox" or Example 4).

[0071] According to a further preferred embodiment, the moiety is a targeting agent. That is, in a preferred embodiment of the present invention, the chlorotoxin derivative according to the present invention forms a conjugate with the moiety, where the moiety is another targeting agent. Thereby, such a conjugate having a double binding site on the target cell provides a targeting molecule with a higher affinity for the targeted cell or tissue. Methods for chemical conjugation to targeting agents and specific proteins are known to those skilled in the art. Preferred targeting agents in the context of the present invention are antibodies, polypeptides, polysaccharides and nucleic acids.

[0072] According to another preferred embodiment, the moiety family is a moiety that increases the circulation half-life. That is, in a preferred embodiment of the present invention, the chlorotoxin derivative according to the present invention forms a conjugate with the moiety family, where the moiety family is a moiety that increases the circulation half-life. Thereby, such a conjugate can alleviate the metabolism of CTXD. Methods for increasing the circulation half-life moiety and chemical bonding to specific proteins are known to those skilled in the art. Preferred sites for increasing the circulation half-life in the context of the present invention are PEG moieties, glycosyl moieties, glycosyl PEG moieties, and moieties that enable cyclization of the chlorotoxin derivative of the present invention via linker extension.

[0073] The chlorotoxin derivative and the moiety family together form the conjugate of the present invention. In some cases, it is necessary to construct a linker portion between the chlorotoxin derivative and the moiety family. For example, if the moiety family cannot be directly chemically bonded to the chlorotoxin derivative, a linker must be used. If the moiety family needs to be released when it reaches a target site, such as a cancerous cell, a specially cleavable linker can be used. Linkers are known to those skilled in the art, and examples of linkers are peptides, dimethyldisulfide linkers, glutaric linkers, and cathepsin-cleavable linkers. As will be apparent to those skilled in the art, it is possible to use two or more linkers in a particular chlorotoxin derivative-moiety family construct.

[0074] Despite the presence of one or more linkers, some types of moiety families need to be extended away from the chlorotoxin derivative in order to exert their effect. For this purpose, one or more spacers can be applied between the chlorotoxin derivative and the moiety family. The spacer portion is bonded to the moiety family, linker, or chlorotoxin derivative via a primary bond (i.e., a covalent or ionic bond).

[0075] It will be apparent to those skilled in the art that the families of compensatory molecules, linkers and spacers may in some cases not be strictly distinguishable and that their boundaries may in some cases not be strict. For example, some families of compensatory molecules may have a tail region that can function as a linker.

[0076] The seranostic pair can be selected from the conjugates of the present invention. These two conjugates, i.e., the first conjugate and the second conjugate, may be the same or different. According to a preferred embodiment of the present invention, the chlorotoxin derivative moieties of the two conjugates have the same amino acid sequence, and the two conjugates have different families of compensatory molecules.

[0077] The term "seranostic" refers to a combination of diagnostic and therapeutic applications, as is known to those skilled in the art. Briefly, one member of the seranostic pair is suitable for diagnostic purposes, and the other member of the seranostic pair is suitable for therapeutic purposes.

[0078] The members of the seranostic pair can be used separately, spatially and / or temporally, or simultaneously. The seranostic pairs of the present invention typically have the same chlorotoxin derivative (i.e., the same amino acid sequence), whereby both conjugates bind to the same cancerous tissue. However, typical seranostic pairs of the present invention have different families of compensatory molecules, one for diagnostic purposes (such as a visualization agent) and the other for therapeutic purposes (such as a chemotherapeutic agent). As will be apparent to those skilled in the art, the same conjugate of the present invention can also form a seranostic pair, where the family of compensatory molecules of the conjugate has both diagnostic and therapeutic characteristics. On the other hand, the members of the seranostic pairs of the present invention may have different amino acid sequences in the chlorotoxin derivative moiety and may likewise have different families of compensatory molecules.

[0079] The present invention also relates to a kit comprising the conjugate of the present invention and an instruction manual. The conjugate of the present invention can be provided in the kit in the form of a solution or a lyophilized product. The instruction manual describes the use of the conjugate of the kit at a level understandable by those skilled in the art. The instruction manual may be in the form of paper, an electronic data carrier, or may be available online. The kit may further include solutions, buffers, reagents, and disposable parts of experimental instruments.

[0080] The present invention also relates to a kit comprising the seranostic pair of the present invention and an instruction manual. The seranostic pair of the present invention can be provided in the kit in the form of a solution or a lyophilized product. The instruction manual describes the use of the seranostic pair of the kit at a level understandable by those skilled in the art. The instruction manual may be in the form of paper, an electronic data carrier, or may be available online. The kit may further include solutions, buffers, reagents, and disposable parts of experimental instruments.

[0081] The present invention further relates to a pharmaceutical composition comprising the conjugate of the present invention and a pharmaceutically acceptable excipient, wherein the moiety of the conjugate lacking the molecule is a therapeutic agent or a visualizing agent. When the pharmaceutical composition of the present invention is used for treatment, the moiety lacking the molecule is a therapeutic agent. When the pharmaceutical composition of the present invention is used for visualization, the moiety lacking the molecule is a visualizing agent.

[0082] The pharmaceutical composition of the present invention can be administered by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal or buccal routes. Since this pharmaceutical composition is suitable for the treatment of tumors, the preferred administration method is microinjection which is applied locally to the tumor to be treated. The dosage of the pharmaceutical composition to be administered depends on the type of tumor, the age, health status, and weight of the patient, the type of any concurrent treatment, the frequency of treatment, and the nature of the effect to be achieved. Those skilled in the art can determine the dosage considering the above data. Typical dosages include from 1.0 pg / kg body weight to 100 mg / kg body weight. Preferred dosages for systemic administration include from 100.0 ng / kg body weight to 10.0 mg / kg body weight. Preferred dosages for direct administration to the site via microinjection include from 1 ng / kg body weight to 1 mg / kg body weight.

[0083] The pharmaceutically acceptable excipients in the pharmaceutical composition of the present invention facilitate the processing of the conjugate into a formulation that can be pharmaceutically used for delivery to the site of action. Formulations suitable for parenteral administration include aqueous solutions of the conjugate in water-soluble form (e.g., water-soluble salts). Further, a suspension of the conjugate (e.g., an oily injection suspension) may be administered. Suitable lipophilic solvents or vehicles include fatty oils (e.g., sesame oil) or synthetic fatty acid esters (e.g., ethyl oleate) or triglycerides. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol and dextran. Optionally, the suspension can also contain stabilizers. Liposomes may be used to encapsulate the conjugate for delivery to tumor cells.

[0084] The pharmaceutical preparation for systemic administration according to the present invention can be formulated for enteral, parenteral or topical administration. Any common topical preparation such as a solution, suspension, gel, ointment or salve can be employed. The preparation of such topical preparations is described in Remington’s Pharmaceutical Sciences (Gennaro ed, 1995, Mack Publishing). For topical application, the pharmaceutical composition can also be administered as a powder or a spray, particularly in the form of an aerosol. The pharmaceutical composition can be administered by inhalation. For inhalation therapy, the conjugate can be a solution useful for administration by a metered dose inhaler or in a form suitable for a dry powder inhaler. The pharmaceutical composition is also suitable for administration by bronchial lavage.

[0085] Formulations suitable for oral administration include hard or soft gelatin capsules, pills, tablets such as coated tablets, elixirs, suspensions, syrups or inhalants, and their controlled release forms.

[0086] The CTXD conjugate of the present invention can be used for the diagnosis of cancer, visualization of cancerous tissues, and treatment of cancer. All of these uses are based on the phenomenon that the conjugate selectively binds to cancerous tissues / cells. Such cells can be cells that overexpress the MMP-2 protein. Once the conjugate binds to cancerous cells, depending on the function of the family of complementing molecules of the conjugate, the specific binding can be used for diagnostic purposes, visualization, or killing of the cells (i.e., treatment of cancer). Diagnostic use can serve either to confirm or locate the presence of cancerous cells / tissues in the body or on epithelial surfaces (skin or mucosa), or to identify the types of cancerous tissues that may be sensitive to specific treatments using the CTXD receptor as a targeting motif. When the conjugate has a visualization agent as the family of complementing molecules, the cancerous tissue can be visualized. Visualization can help, for example, in fluorescence-guided surgery to distinguish cancerous cells or tissues from surrounding healthy (non-cancerous) cells / tissues. The diagnosis, visualization, and treatment of cancerous cells and tissues with conjugates of specific proteins and appropriate families of complementing molecules are well known to those skilled in the art.

[0087] The conjugate of the present invention is useful for the diagnosis and treatment of substantially all types of malignant cancers that express the CTX binding site. These types of cancers include glioma, astrocytoma, oligodendroglioma, medulloblastoma, choroid plexus carcinoma, epithelioma, meningioma, glioblastoma, ganglioma, pheochromocytoma, and metastatic brain tumors, other brain tumors, neuroblastoma, head and neck cancers, small cell lung carcinoma, breast cancer, intestinal cancer, pancreatic cancer, colon cancer, liver cancer, kidney cancer, skin cancer, sarcomas (more than 30 types), osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma, carcinoma, melanoma, ovarian cancer, cervical cancer, lymphoma, thyroid cancer, anal cancer, colorectal cancer, endometrial cancer, germ cell tumor, laryngeal cancer, multiple myeloma, prostate cancer, retinoblastoma, gastric cancer, testicular cancer, and Wilms' tumor. The conjugate of the present invention is particularly useful for the treatment and diagnosis of cancers selected from the following group: breast cancer, cervical cancer, colon cancer, epithelioma, Ewing's sarcoma, ganglioglioma, ganglioma, glioma, glioblastoma, gliosarcoma, medulloblastoma, meningioma, neuroblastoma, melanoma (primary and metastatic), pancreatic cancer, pheochromocytoma, prostate cancer, schwannoma, and small cell lung carcinoma. In the experiments of the present invention, therefore, a particularly high intensity uptake of the CTXD of the present invention into breast cancer, glioblastoma, melanoma, and pancreatic cancer cells, which are the most preferred cancerous tissues according to the present invention, was demonstrated.

[0088] The conjugate of the present invention is useful for the visualization of substantially all types of malignant cancers that express the CTX binding site. The conjugate of the present invention is particularly useful in the visualization of cancerous tissues, and the tissues are derived from the following cancers: breast cancer, cervical cancer, colon cancer, epithelioma, Ewing's sarcoma, ganglioma, ganglioglioma, glioma, glioblastoma, gliosarcoma, medulloblastoma, meningioma, neuroblastoma, melanoma (primary and metastatic), pancreatic cancer, pheochromocytoma, prostate cancer, schwannoma, small cell lung carcinoma; breast cancer, glioblastoma, melanoma, and pancreatic cancer are the most preferred cancerous tissues according to the present invention.

[0089] The present invention further relates to a method for treating a patient suffering from cancer. In this context, under the term "patient", any animal is understood, such as a human, sheep, horse, cow, pig, dog, cat, rat and mouse. The present invention is particularly useful for the treatment of human patients suffering from cancer. The method comprises administering to the patient an effective amount of the conjugate according to the present invention. The conjugate for the purpose of treating a cancer patient has a family of deficiency molecules of a therapeutic agent suitable for the treatment of said cancer. In a preferred embodiment, the conjugate used in the treatment method is in the form of a pharmaceutical composition of the present invention.

[0090] According to a preferred embodiment of the present invention, the patient treated with the conjugate or with the pharmaceutical composition of the present invention suffers from a cancer selected from the group consisting of glioma, pancreatic cancer, melanoma and breast cancer.

[0091] The present invention further relates to a method for preparing the conjugate of the present invention. This method comprises at least the following steps: a) providing a chlorotoxin derivative according to any one of claims 1 to 5; b) optionally attaching a linker group to the chlorotoxin derivative of step a); c) attaching a family of deficiency molecules to the linker moiety of the compound formed in step b) or directly to the protein of step a) in the absence of a linker.

[0092] Briefly, the starting material for the preparation method is the chlorotoxin derivative of the present invention (i.e., step "a"). Since these proteins constitute a simple amino acid sequence consisting of the usual amino acids that make up the protein, the synthesis of the chlorotoxin derivative can be carried out using methods and reagents well known in the art.

[0093] The following step of the preparation method is optional because the presence of the linker in the conjugate is optional. That is, if the conjugate to be prepared is a conjugate having a linker between the chlorotoxin derivative and the family of defective molecules, the step "b" must be carried out. The linker can be attached to the protein by methods well known in the art.

[0094] As the next step "c", a family of defective molecules is attached to a chlorotoxin derivative in the absence of a linker or to the construct that is the result of step "b". The attachment of the family of defective molecules to the protein strongly depends on the family of defective molecules itself. However, constructing such a construct is known to those skilled in the art.

[0095] The method for preparing the conjugate may further include well-known steps such as isolation, purification, analysis, etc., all of which are generally known to protein chemists skilled in the art.

[0096] The present invention further relates to a method for visualizing cancerous tissue, the method including the step of contacting the tissue to be examined with the conjugate of the present invention, wherein the family of defective molecules of the conjugate is a visualization agent suitable for visualizing cancerous tissue. Such a tissue can be a tissue overexpressing the MMP-2 protein.

[0097] As detailed above, when the family of defective molecules of the conjugate of the present invention is a visualization agent, the conjugate can be used to visualize the tissue to which the chlorotoxin derivative portion of the conjugate binds. By performing this method, when the chlorotoxin derivative specifically binds to cancerous tissue, the cancerous tissue can be visualized. By this method, tumors can be visualized, which is very useful for surgical operations aimed at removing the tumors.

[0098] By the visualization method described above, a specific tumor can be located in a patient's body. This method is useful when the location of a specific type of tumor is uncertain or needs to be confirmed. By using the visualization and localization tools provided by the present invention, a physician is supported during the surgical procedure to remove cancerous tissue.

[0099] A further useful application of the visualization method described above is to examine the patient to determine whether there is a specific tumor in the patient's body. That is, the present invention provides a useful tool for cancer diagnosis by detecting cancerous tissue with the aid of the selective binding of the conjugate of the present invention.

[0100] Another useful application of the visualization method described above is to locate tumor cells, whereby a physician can confirm whether the tumor has been completely removed from the patient.

[0101] The present invention further relates to a chimeric antigen receptor (CAR) incorporating an extracellular domain derived from a chlorotoxin derivative according to the present invention. CARs constructed using chlorotoxin or similar toxins are disclosed in the pamphlet of International Publication No. WO2017 / 066481, which is an international patent application. Regarding the synthesis method, characteristics and use of CARs containing the chlorotoxin domain, this international patent application is referred to as information known to those skilled in the art. CARs are expressed on the surface of genetically modified T cells. CAR T cells can be redirected to specifically recognize antigenically distinct tumor populations.

[0102] Briefly, chimeric antigen receptor (CAR)-modified T cells can specifically recognize and kill various tumor cell lines by binding to their specific tumor-associated antigen (TAA). The CAR is a transmembrane chimeric protein that comprises an extracellular TAA recognition / targeting domain, which is a chlorotoxin derivative according to the present invention that provides specific and efficient binding to a molecular target, and an intracellular effector domain (usually the TCR zeta (TCRζ) chain) capable of efficient activation of T lymphocytes [June 2018]. T cells can express the CAR when they have the CAR encoded by the corresponding nucleic acid, for example, after retroviral or lentiviral transduction.

[0103] According to one embodiment of the present invention, there is provided a nucleic acid encoding a chimeric antigen receptor, i.e., a CAR, which CAR comprises at least the following components: a) a chlorotoxin derivative according to the present invention; b) optionally, a spacer region between the chlorotoxin derivative and the transmembrane domain; c) a transmembrane domain; d) one or two co-stimulatory domains; e) a signaling domain; and wherein the chlorotoxin derivative, when expressed on the surface of a T cell, enables the CAR to direct the activity of the T cell towards cancerous cells.

[0104] According to this construct, the CAR encoded by the nucleic acid of the present invention has an extracellular chlorotoxin derivative domain corresponding to the chlorotoxin derivative of the present invention. This chlorotoxin derivative forms, or is part of, an extracellular domain that functions as an extracellular recognition domain. Its recognition element specifically binds to a molecule present on the cell surface of the target cell. The extracellular recognition domain is linked to the intracellular effector domain by a hinge (also known as a linker or spacer) and an associated transmembrane domain. That is, the transmembrane domain anchors the CAR to the membrane of the T cell. The intracellular domain contains at least one co-stimulatory domain and a signaling domain, the functions of which are well known to those skilled in the art.

[0105] The extracellular domain, or in other words the extracellular recognition domain, may comprise one or more, identical or different, chlorotoxin derivatives of the present invention. According to a preferred embodiment, the CAR construct comprises one chlorotoxin derivative.

[0106] The chlorotoxin derivative is preferably selected from chlorotoxin derivatives having any of the sequences set forth in SEQ ID NO: 3 to SEQ ID NO: 18 and SEQ ID NO: 36 to SEQ ID NO: 42, preferably SEQ ID NO: 4 or SEQ ID NO: 42.

[0107] The CAR construct encoded by the nucleic acid of the present invention optionally comprises a spacer region between the chlorotoxin derivative and the transmembrane domain. As will be apparent to those skilled in the art, a spacer region is required when the transmembrane domain and the chlorotoxin derivative need to have a certain spatial arrangement relative to each other for the CAR construct to function properly. The composition and length of the spacer region determine the mobility of the antigen recognition domain and the physical distance between the T cell and the target. The spacer region may comprise amino acids having a length of 5 to 300 residues. For example, the spacer region may comprise an IgG (IgG1 or IgG4) or a CD8 hinge region.

[0108] The transmembrane domain of the CAR construct encoded by the nucleic acid of the present invention is preferably selected from the group consisting of the CD4 transmembrane domain or a variant thereof, the CD8 transmembrane domain or a variant thereof, the CD28 transmembrane domain or a variant thereof, and the CD3 zeta transmembrane domain or a variant thereof [Fujiwara 2020]. Under the term "variant" in relation to the transmembrane domain, a protein is understood to have 1 to 5 amino acid modifications (e.g., substitutions, insertions, deletions) compared to the original protein, provided that the cysteine residues are not modified.

[0109] The co-stimulatory domain of the CAR construct encoded by the nucleic acid of the present invention is preferably selected from the group consisting of the CD28 co-stimulatory domain or a variant thereof, the 4-1BB co-stimulatory domain or a variant thereof, and the OX40 co-stimulatory domain or a variant thereof [Zhong 2010]. Under the term "variant" in relation to the co-stimulatory domain, a protein is understood to have 1 to 5 amino acid modifications (e.g., substitutions) compared to the original protein, provided that the cysteine residues are not modified.

[0110] The signaling domain of the CAR construct encoded by the nucleic acid of the present invention is preferably selected from the group consisting of the CD3 zeta signaling domain or a variant thereof. Under the term "variant" in relation to the signaling domain, a protein is understood to have 1 to 5 amino acid modifications (e.g., substitutions) compared to the original protein, provided that the cysteine residues are not modified.

[0111] The co-stimulatory domain and the signaling domain may have a short (i.e., less than 10 amino acids) linker amino acid sequence therebetween.

[0112] The chlorotoxin derivative of the CAR construct encoded by the nucleic acid of the present invention, when expressed on the surface of T cells, enables the CAR to redirect T cell activity towards cancerous cells, preferably glioblastoma cells.

[0113] The nucleic acids of the present invention can be prepared by standard techniques of molecular cloning known to those skilled in the art and assembled into the complete coding sequence.

[0114] The present invention further relates to a vector comprising a nucleic acid molecule encoding the CAR construct of the present invention. The vector may be a plasmid, a nucleic acid (RNA or DNA) molecule, which is used as a vehicle for carrying foreign genetic material in other cells. In this case, the foreign genetic material is the nucleic acid of the present invention encoding the CAR construct, and the other cells are specific immune cells suitable for the purpose (suitable immune cells are autologous human T cells, autologous human CD4+ helper T cells, autologous human CD8+ cytotoxic T cells, any mixture of autologous human CD4+ helper T cells and CD8+ cytotoxic T cells in any ratio, allogeneic human T cells, allogeneic human CD4+ helper T cells, allogeneic human CD8+ cytotoxic T cells, any mixture of allogeneic human CD4+ helper T cells and CD8+ cytotoxic T cells in any ratio, autologous primary human natural killer (NK) cells, allogeneic human primary natural killer (NK) cells, allogeneic cells of the NK-92 cell line, autologous human monocytes, autologous human macrophages, allogeneic human monocytes, allogeneic human macrophages). This vector is suitable for transfecting the immune cells and thereby introducing the nucleic acid molecule of the present invention into the immune cells. The transfected immune cells can then express the CAR construct on their surface. The introduction of the vector can be easily performed by transfection reagents such as electroporation or lipofectamine. The entry of foreign genetic material into the host cell and its integration into the host cell's genetic material can also be facilitated by viral vectors, in which case this process is called transduction instead of transfection.

[0115] The present invention further relates to a population of human cells, wherein the cells are transfected by an RNA or DNA vector comprising an expression cassette containing the nucleic acid of the present invention, wherein the transfection is carried out in vivo or ex vivo; or ii) transduced by a viral vector comprising an expression cassette containing the nucleic acid of the present invention, wherein the transduction is carried out in vivo or ex vivo. In the case of transduction, the viral vector is preferably a retroviral vector or a lentiviral vector, since this type of viral vector is widely used and has proven useful for the purpose of transduction. In the case of transduction, the population of human cells of the present invention is preferably transduced by a viral vector which is a retroviral vector or a lentiviral vector, said vector comprising an expression cassette containing the nucleic acid of the present invention. Here, the vector is a viral vector, and in this case, the term transduction is used instead of transfection. This population of virus-transduced human cells will be able to express the CAR construct on the surface of the transduced cells. The transduction is carried out in vivo or ex vivo.

[0116] As will be apparent to those skilled in the art, in this context, the term in vivo means that transfection or transduction is performed on cells present in a living human. As will be apparent to those skilled in the art, the term ex vivo means that in this context, transfection or transduction is performed on cells extracted from a living human, and these cells may later be returned to the living human or introduced into another living human. The human cells shall be selected from the following list of human cell types: autologous human T cells, autologous human CD4+ helper T cells, autologous human CD8+ cytotoxic T cells, a mixture of autologous human CD4+ helper T cells and CD8+ cytotoxic T cells in any proportion, allogeneic human T cells, allogeneic human CD4+ helper T cells, allogeneic human CD8+ cytotoxic T cells, a mixture of allogeneic human CD4+ helper T cells and allogeneic human CD8+ cytotoxic T cells in any proportion, autologous primary human natural killer (NK) cells, allogeneic primary human natural killer (NK) cells, allogeneic cells of the NK-92 cell line, autologous human monocytes, autologous human macrophages, allogeneic human monocytes, allogeneic human macrophages.

[0117] The above-listed cells are useful for the purposes of the present invention for the following reasons: i) The effectiveness of CAR T cell therapy most often results from CD8+ cytotoxic T cells, and CD4+ helper T cells are known for their helper function and have been shown to essentially induce cytolytic activity by enhancing the activity of CD8+ T cells through cytokine production [Zhang 2020]. ii) Chimeric antigen receptor-modified natural killer (CAR-NK) cell-based immunotherapy has been shown to be a promising and advanced option in the context of cancer immunotherapy for either solid tumors or hematological malignancies [Marofi 2021]. iii) The NK-92 cell line is derived from non-Hodgkin lymphoma patients and is characterized by being similar to activated human primary NK cells obtained from peripheral blood. However, isolation and ex vivo expansion of primary cells are difficult. Therefore, the NK-92 cell line provides a valuable alternative as it can be easily expanded in an IL-2-supplemented environment. iv) Furthermore, it has been demonstrated that chimeric antigen receptor-modified macrophages infiltrate solid tumor tissues and can interact with almost all cell components of the tumor microenvironment (tumor cells, immune cells such as T cells, NK cells, dendritic cells, and other resident non-immune cells, etc.) [Mukhopadhyay 2020]. Monocytes are considered precursors of macrophages present in the blood.

[0118] The said population of human cells is transfected or transduced by the said vector containing the expression cassette comprising the nucleic acid of the present invention. The population of human cells of the present invention carries the nucleic acid of the present invention in the form of an expression cassette. These human cells can be prepared by isolating appropriate cells from the human patient to be treated and transfecting or transducing these appropriate human cells with a vector containing an expression cassette comprising the said nucleic acid. Transfection or transduction is carried out in vivo or ex vivo.

[0119] The present invention further relates to a method for treating cancer in a patient. This method includes the step of administering a population of human cells of the present invention. According to the present invention, for the purpose of treating cancer in a patient, a combination of different populations of human cells of the present invention can also be administered.

[0120] The method for treating cancer includes, for example, the step of administering a population of autologous or allogeneic human T cells transduced by a vector containing an expression cassette comprising the nucleic acid of the present invention.

[0121] The method for treating cancer includes, for example, the step of administering a population of autologous or allogeneic human natural killer (NK) cells or the NK-92 cell line transduced by a vector containing an expression cassette comprising the nucleic acid of the present invention.

[0122] This method involves administering a population of autologous or allogeneic human monocytes or macrophages transduced with a vector containing an expression cassette comprising the nucleic acid of the invention.

[0123] The inventors' approach (process to the invention) The goal of the inventors was to find a new CTX derivative with improved affinity and selectivity for MMP-2, which is used to distinguish tumor cells from healthy tissue cells. Although it was strongly suggested from the underlying science that MMP-2 is the target of the true binding protein of CTX, this finding has not been confirmed until now in pull-down assays using recombinantly produced MMP-2 [Veiseh 2007]. Therefore, the inventors first established a "Magnabead assay", which is a quantitative flow cytometry method for evaluating the binding of CTX to recombinantly produced purified MMP-2 protein. Using this new assay, the inventors confirmed that the fluorophore-labeled CTX specifically binds to the beads covered with MMP-2 immobilized on the beads by an anti-MMP-2 antibody. This binding is detected by measuring the fluorescence intensity accumulated on the beads, saturates by increasing the incubation concentration of the labeled CTX, and completely disappears ("is displaced") by incubating with an excess amount of unlabeled CTX, and thus, this shows that it specifically binds to the CTX receptor on the MMP-2 protein.

[0124] Since it was confirmed that purified MMP-2 binds specifically to CTX alone, i.e., not necessarily in a protein complex, the inventors initiated a screening program for more potent MMP-2-binding CTXDs. For this purpose, the inventors applied designer toxin platform technology [Takacs et al., 2009]. This technology uses a method called phage display, invented by G.P. Smith in 1985 [Smith 1985], to create a library of toxin proteins displayed on the surface of the filamentous bacteriophage M13 using existing toxin protein sequences and to identify high-affinity conjugates by iterative selection. The inventors designed and constructed a library of 1,376,254 permutation derivatives from 32 scorpions while retaining the disulfide bridge pattern of CTX. The displayed proteins were fused to the P3 coat protein at the C-terminus via a GSASSA (peptide sequence) linker. The inventors screened this library against human recombinant MMP-2 and identified a novel compound (hereinafter referred to as CTXD1 or SEQ ID NO: 17) that binds more strongly to MMP-2 than CTX.

[0125] To quantitatively evaluate the relative binding strength of the proteins presented on the phage, the inventors developed a new flow cytometry-based assay called the "cobalt bead test" or simply the "Co-bead test" (unpublished). This assay is based on the use of pre-fabricated cobalt-coated beads (Dynabeads-His-Tag Isolation & Pulldown from ThermoFisher Scientific) that are manufactured in a factory and are designed to isolate his-tagged proteins according to the principle of immobilized metal affinity chromatography (IMAC). These beads immobilize different his-tagged target proteins on their surface, thereby enabling the evaluation of the amount of phage particles presenting ligand peptides bound to the target proteins after fluorescent staining of the phage against the M13 antigen. This method also made it possible to examine the binding of synthetic or recombinantly produced purified and fluorophore-labeled ligand peptides to various his-tagged target proteins. The details of this novel flow cytometry-based method are described in Example 5; the preparation of phages tested in the Co-bead test is described in Example 7.

[0126] Using the support of the Co-bead test, a directed mutagenesis test starting from the sequence of CTXD1 was performed to clarify the structure-activity relationship (SAR), and the superiority or inferiority of the binding of different CTXD-presenting phages to MMP-2 ( "relative binding") was quantified compared to the CTX-presenting phage as a reference. Relative binding was evaluated by incubating with equal concentrations of CTXD- and CTX-expressing phages, performing staining for M13 antigen detection, and then measuring the relative amount of M13-related fluorescence increase on beads coated with MMP-2 relative to the fluorescence signal from beads not coated with MMP-2. The amino acid sequences and the results examined in these SAR tests are shown in Table I.

Table 1

[0127] CTXD proteins that are not substantially more potent conjugates than CTX itself are listed in SEQ ID NOs: 19 to 34. The "-" in the middle of the sequences are merely apparent gaps for aligning the sequences with each other. "N=" refers to the number of measurements of relative MMP-2 binding, and "relative MMP-2 binding" is the average based on N experiments. The protein encoded by SEQ ID NO: 4 is referred to herein as CTXD5, and the protein encoded by SEQ ID NO: 17 is referred to as CTXD1.

[0128] Since monorisin CTXD proteins may be advantageous for the purpose of conjugate products for labeling or targeted delivery, first, the inventors examined various monorisin derivatives of CTXD1 by substituting two of the three lysine residues. Surprisingly, among the monorisin derivatives of CTXD1, seven derivatives (SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16) were found that are substantially more potent MMP-2 binders than CTXD1. Next, the inventors aimed to determine the minimum protein length essential for the observed excellent MMP-2 binding strength while maintaining the disulfide bridge pattern. Therefore, CTXD5 was selected for further modification, and the effects of N-terminal and C-terminal truncations (deletion mutations) up to the first cysteine residue were examined. It is considered that even if the C-terminus is truncated stepwise, the excellent binding ability of the CTX derivative is not lost until the glutamine following the last cysteine is removed, as shown in Figure 3. In this figure, the bar graph shows the different binding strengths and selectivities of CTX and different CTXD5 derivatives for MMP-2 and NRP1 target proteins, as shown by phage display assays using the Co-bead test. Since this experiment aimed to examine the effect of C-terminal modification of CTXD5, different C-terminal sequences starting from the last cysteine residue of the proteins derived from CTX and CTXD5 tested are also shown. Control beads (CONT) were not exposed to phage but were coated with the target protein and subjected to the whole staining procedure. The other beads had a uniform phage concentration of 2.6×10 14The cells were exposed to the indicated peptide-displaying phages at phage particles / mL.

[0129] Our quantitative phage binding assay using C-terminal truncation mutants was also extended to measure binding to NRP1 protein, as McGonigle et al. [McGonigle 2019] suggested that the C-terminal portion of rCTX plays an important role in binding to NRP1. Phage-displayed CTX showed approximately equal binding to both MMP-2 and NRP1, whereas CTXD5 and its truncated variants were selective for MMP-2 and did not bind to NRP1, except when the C-terminal portion was truncated to the cysteine residue. However, when all amino acids after the cysteine were deleted, binding to NRP1 reappeared, resulting in relatively reduced MMP-2 binding (Figure 3) or reaching a level nearly equivalent to that of CTX. Therefore, these phage display results indicated that CTXD5 and its truncated derivatives, excluding those terminating at the cysteine, are not only stronger binders of MMP-2 than CTX, but are also selective for MMP-2 relative to NRP1. This prediction from the phage display studies was also confirmed by examining recombinantly produced pure CTXD5 protein material (see below).

[0130] Deletion of one or two N-terminal amino acids from CTXD5 (i.e., SEQ ID NO: 4) resulted in the chlorotoxin derivatives SEQ ID NO: 6 and SEQ ID NO: 8, respectively (see Table I). However, these mutations did not significantly alter the binding strength of the resulting chlorotoxin derivatives.

[0131] Deletion of the N-terminal alanine of CTXD1 (i.e., SEQ ID NO: 17) resulted in the chlorotoxin derivative of SEQ ID NO: 18. This mutation also did not significantly alter the binding strength of the resulting protein.

[0132] Based on the SAR results obtained from the quantitative evaluation of the target protein binding of phage-displayed compounds, a series of chlorotoxin derivatives were identified, which are substantially more potent than CTX or mCTX in terms of binding to MMP-2 and more selective in terms of target protein selectivity, which may lead to improved selectivity for tumor cells / tissues compared to normal cells / tissues.

[0133] To confirm the characteristics of the protein predicted from the phage display assay, CTXD5 was recombinantly produced and obtained as a purified chlorotoxin derivative. Using CTXD5 as a lead compound, the inventors characterized its target protein and cell binding properties compared to CTX and / or mCTX in either the single or conjugate form. However, the water solubility of this lead compound was moderate and could be further reduced by various conjugations, which could potentially be an inhibitory factor for the formulation of diagnostic or therapeutic agents for parenteral use.

[0134] Therefore, the inventors investigated the effects of the planned solubility-enhancing mutations of the lead compounds SEQ ID NO: 4 (CTXD5) and SEQ ID NO: 10. Based on the published scientific literature data [Trevino 2007], the sequences of FTT and QT starting from the amino acid positions 7 and 12 were replaced with the sequences of SSS and ES, respectively, and a polyarginine sequence was applied to the C-terminal region, assuming that the solubility of the resulting CTXD protein could be significantly improved while enhancing the MMP-2 affinity of the lead compound. To evaluate the MMP-2 affinity of the new solubility-enhanced mutants, the inventors quantified the superiority of the MMP-2 binding of different solubility-enhanced CTXD-displayed phages ( "relative MMP-2 binding") to the CTX-displayed phages as described above. The results of this solubility-enhanced mutant phage-display assay for this relative MMP-2 binding are shown in Table II.

Table 2

[0135] As a result, all of the designed solubility-enhancing mutants showed binding strengths approximately three times or slightly higher than that of CTX, maintaining high MMP-2 binding. Therefore, the inventors selected a protein (SEQ ID NO: 42) containing all of the tested solubility-enhancing mutants as a secondary lead compound for test substance production and named it CTXD8. Using CTXD5 as the primary lead compound and CTXD8 as the secondary lead compound, the inventors characterized the target protein and cell binding properties in comparison with CTX and / or mCTX alone or in conjugate form.

[0136] The chemical toolbox for these studies included the following compounds:

[0137] Chemical Toolbox 1. Unconjugated CTX derivatives: Some unconjugated CTX variants / derivatives are shown in Figure 1.

[0138] Synthetic CTX was purchased from Iris Biotech GMBH (Germany). rCTX and mCTX were recombinantly produced as described in Example 1.

[0139] 2. Unconjugated CTXD derivatives: CTXD5 and CTXD8 were recombinantly produced as described in Example 1.

[0140] 3. Conjugated CTX and CTXD Derivatives The following conjugation products were purchased or produced by conjugating chemical reactions with the unconjugated CTX and chlorotoxin derivatives described above.

[0141] 3.1. Cyanine-5 (-Cy5) conjugates We used CTX-Cy5, rCTX-Cy5, mCTX-Cy5, CTXD5-Cy5, and CTXD8-Cy5 in this study. All of these were monoconjugated CTX derivatives selected by HPLC separation. CTX-Cy5 and rCTX-Cy5 were mixtures of molecules randomly monoconjugated at one of the three lysine residues of CTX. In the case of CTXD5, CTXD8, and mCTX, conjugates linked at only one lysine residue were isolated and used. The conjugation reaction was performed using cyanine 5 NHS ester as described in Example 2.

[0142] 3.2.Alexa 488 (-A488) conjugates The CTX-A488 conjugate of CTX was applied as a fluorescently labeled ligand, operating at sufficiently different excitation / emission wavelengths to allow comparative evaluation of the displacement strength (affinity) of different Cy5-labeled and unlabeled CTX and CTXD ligands without interference from other (Cy5) fluorophores. The excitation / emission maxima of A488 and Cy5 are 495 / 519 and 647 / 665, respectively. [ka]

[0143] 3.3. Cleavable Cytostatic Conjugates with Monomethyl Auristatin F (MMAF) as the Payload The widely used valine-citrulline-p-aminobenzyloxycarbonyl (VC-PABC) dipeptide linker has gained popularity as a means of maintaining a stable covalent bond of a payload drug to a specifically targeted antibody that can be preferentially cleaved by cathepsin B, an intracellular protease of the lysosomal degradation pathway [Doronina 2008]. To demonstrate this type of targeting utility of CTXD5 compared to mCTX, the inventors synthesized a cleavable cell division inhibitory conjugate bound to a target protein via a glutaryl linker. Both mCTX-glutaryl-valine-citrulline-p-aminobenzyloxycarbonyl-MMAF (mCTX-G-VC-PAB-MMAF) and CTXD5-glutaryl-valine-citrulline-p-aminobenzyloxycarbonyl-MMAF (CTXD5-G-VC-PAB-MMAF) were synthesized in both the form of the lx conjugate and the 2x conjugate, i.e., mCTX(-G-VC-PAB-MMAF)2 and CTXD5(-G-VC-PAB-MMAF)2, such that they were recovered in the final purification step. The second conjugation site is the N-terminal amine. The synthesis of the G-VC-PAB-MMAF conjugate is described in Example 4.

[0144] The structure of the CTXD5-G-VC-PAB-MMAF conjugate is shown in Figure 2. This construct is a preferred example of the conjugate of the present invention. That is, the chlorotoxin derivative moiety is CTXD5 (SEQ ID NO: 4). This conjugate has two linkers, namely a glutaryl linker and a cathepsin-cleavable linker. It further has a spacer. The payload family of this conjugate is monomethyl auristatin F (MMAF).

[0145] 3.4. Biotinylated CTX and CTX Derivatives Biotinylated CTX and CTXD5 were used for cytochemical and histochemical applications. Randomly monobiotinylated CTX was purchased from Iris Biotech GMBH (Germany). Monobiotinylated CTXD5 was prepared by conventional biotinylation procedures, the monoconjugate at the only lysine residue was purified by HPLC, and lyophilized as described in Example 3.

[0146] Profiling of CTXD5 Compared to CTX as a Targeting Molecule The inventors examined the key features of conjugates of CTXD5, CTXD8 and related examples using the compounds of the detailed chemical toolbox described above, and compared them to CTX and in some aspects to mCTX and their conjugates.

[0147] To examine the binding affinity of test substances for the selected target proteins, in addition to the Co-bead test, another flow cytometry-based assay, the "Magnabead test," was established. This test uses specific antibodies to immobilize the target protein on the bead surface and has advantages and disadvantages compared to the Co-bead test. The advantages are higher sensitivity, i.e., acting at low effective concentrations, and a non-significant level of non-specific binding without a blocking incubation step. The disadvantage was that it was not applicable to binding assays of phage-displayed proteins or uniform investigations of the binding of labeled ligands to a wide panel of immobilized target proteins. The Magnabead test was used to examine the affinity of ligands in two different test modes, i.e., binding and displacement. To evaluate the affinity of a fluorophore-labeled ligand for the CTX receptor, the "binding mode" was applied, and the amount of bound ligand was directly estimated from the fluorescence intensity after incubation at various labeled ligand concentrations. To evaluate the affinity of an unlabeled ligand for the CTX receptor, the "displacement mode" was applied, in which the beads were exposed to a fixed concentration of labeled ligand (1 μM CTX-Cy5) after pre-incubation and co-incubation with the tested unlabeled ligand, and the bound fluorescence was measured. The relative affinity of the unlabeled ligand was estimated by a concentration displacement test that characterized the 50% displacement concentration (IC 50 value) of the test ligand. The detailed protocol for the Magnabead test for comparative measurement of the binding affinity of labeled and unlabeled CTX and CTXD for MMP-2 and NRP1 proteins is described in Example 6.

[0148] MMP-2 Binding Affinities of CTXD5 and CTXD8 Compared with CTX, rCTX, and mCTX The binding affinities of CTXD5, CTXD8, and CTX were compared by concentration-response testing of unlabeled ligands obtained by displacing 1 μM of the labeled ligand CTX-Cy5 (Figure 4). Figure 4 shows the concentration-displacement relationship of CTX, CTXD5, and CTXD8 for 1 μM CTX-Cy5 as the displacing ligand in the Magnabead assay, demonstrating the differences in affinity for MMP-2 protein. Results are shown as the mean ± SD of two independent experiments comparing two compounds, and as sigmoidal curve fitting to the mean values.

[0149] The binding affinities of CTXD5, CTXD8, and CTX were also compared by displacement of 1 μM CTX-A488 from MMP-2 in a Magnabead assay. The results are shown in Table III. [Table 3]

[0150] In the latter experiment, comparisons were also made to rCTX and mCTX. The strength of binding of labeled CTX was concentration-dependently displaced by all five ligands. Inhibition followed a sigmoidal concentration-response relationship, reaching complete displacement, suggesting a common binding site among the five protein ligands. However, CTXD5 and CTXD8 displaced the labeled ligand CTX-Cy5 4.4-fold more potently than CTX, with IC values of 1.0 and 1.2, respectively. 50 The IC values were 0.16 μM, 0.16 μM, and 0.71 μM (see Figure 4). The CTX-A488 displacement assay showed that the IC values of rCTX and mCTX were 50 The values were not substantially different from those of CTX, and the IC 50 The values were 0.68 μM, 0.77 μM, and 0.68 μM, respectively. Therefore, these results confirmed that the MMP-2 binding affinities of CTXD5 and CTXD8 were almost as high as those of CTX, rCTX, and mCTX, as predicted by the phage display assay.

[0151] MMP-2 binding affinity of conjugate derivatives CTXD5, CTX, and mCTX The binding affinities of CTXD5-Cy5, CTXD8-Cy5, and CTX-Cy5 were compared by the concentration-response test of the binding of these labeled ligands to MMP-2 in the Magnabead assay (Figure 5). This figure shows the relationship between the MMP-2 binding concentration and fluorescence of CTX-Cy5, CTXD5-Cy5, and CTXD8 fluorophore-labeled ligands in the Magnabead assay, indicating the differences in the affinities of these fluorophore-labeled ligands for the MMP-2 protein. The results are shown as the mean ± SD of three independent experiments comparing the three compounds and sigmoid curve fitting to the mean values. The concentration-response relationship fit well to a sigmoid curve and reached saturation at a similar level, suggesting that they bind to approximately the same number of binding sites on MMP-2. CTXD5-Cy5 and CTXD8-Cy5 were 3.7-fold and 3.1-fold more potent than CTX-Cy5, and their EC 50 values were 0.15 μM, 0.18 μM, and 0.56 μM, respectively. These results indicate that all of the conjugate ligands of CTXD5, CTXD8, and CTX maintain the affinity of the unlabeled protein. This conclusion was also confirmed in a substitution test where CTX-A488 (1 μM) was the labeled ligand, and the IC 50 values of CTXD5, CTXD8, CTXD5-Cy5, and CTXD8-Cy5 were 22 μM, 18 μM, 18 μM, and 18 μM, respectively. For comparison, the unlabeled and Cy5-labeled derivatives of CTX, rCTX, and mCTX were also examined (see Table III). As a result, there was no substantial difference in the MMP-2 affinity of CTX, rCTX, and mCTX, and the Cy5 conjugates showed similar or slightly higher affinities, but the largest difference was observed between the labeled and unlabeled proteins, and the IC 50 value of mCTX-Cy5 was 0.40 μM compared to 0.77 μM of mCTX.

[0152] The maintenance of the MMP-2 binding affinity of the cell division inhibitory conjugate of CTXD5, namely CTXD5-G-VC-PAB-MMAF and CTXD5(-G-VC-PAB-MMAF)2, was also confirmed in a displacement test using CTX-Cy5 as a displacement ligand. The IC 50 values were equivalent, namely 0.20 μM, 0.23 μM, and 0.22 μM, respectively.

[0153] Changes in target protein selectivity of CTXD5 and CTXD8 compared to CTX Using the Co-bead test, the inventors examined the binding of CTX-Cy5, CTXD5-Cy5, and CTXD8-Cy5 to a panel of target proteins suggested as direct or indirect binding partners of CTX. The panel included the his-tagged proteins MMP-2, MMP-9, MMP-14, TIMP-2, α v β3 integrin, Anx2, NRP1, CLC3 (chloride channel), and human serum albumin (HSA), the latter being a kind of negative control. The results are shown in FIG. 6. Here, the bar graph shows the different binding strengths of CTX-Cy5, CTXD5-Cy5, and CTXD8-Cy5 labeled test substances (all 1 μM) to various putative target proteins, measured by the bead-binding fluorescence intensity in the Co-bead test. The triplets of columns represent control beads (CONT) blocked with casein, which were not coated with any of the target proteins but were the same as all other target protein-coated beads coated with MMP-2, NRP1, MMP-9, TIMP-2, MMP-14, Anx2, αvβ3 integrin (INT), human serum albumin (HSA), and CLC3 chloride channel. The results are shown as the mean ± SD of three experiments. From the results shown in FIG. 6, CTX-Cy5 bound to MMP-2 and NRP1 with approximately equal strength at a test concentration of 1 μM and also showed a weak but clear binding to MMP-9, TIMP-2, and CLC3. Nevertheless, for CTX-Cy5, MMP-14, α vThe binding to β3 integrin, Anx2, and HSA was negligible. In contrast, CTXD5-Cy5 and CTXD8-Cy5 bound to MMP-2 at least 3-fold more strongly than CTX-Cy5, but the binding to all other proteins in the test panel, except for CLC3 and TIMP-2, was negligible. Even the minimal binding to CLC3 and TIMP-2 was clearly weaker than that of CTX-Cy5. The target protein panel binding and selectivity profiles of the two lead compounds appeared qualitatively and quantitatively similar. Since CTX-Cy5 showed significant affinity for NRP-1, the inventors compared the binding concentration-response relationships of CTX-Cy5 and CTXD5-Cy5 in the Magnabead assay. The results are shown in Figure 7. This figure shows the NRP1 binding concentration-fluorescence relationship for the CTX-Cy5 fluorophore-labeled ligand, indicating that no obvious binding of CTXD5-Cy5 to NRP1 was seen in the Magnabead assay. The results are shown as the mean ± SD of two independent experiments comparing the two compounds, and the EC 50 calculated value, together with the mean value of the relative fluorescence intensity of CTX-Cy5, to characterize the affinity of CTX-Cy5 for NRP1. From the results shown in Figure 7, CTX-Cy5 bound to NRP1 in a concentration-dependent manner, with an EC 50 value of 0.41 μM, while CTXD5 showed no binding to NRP1 up to the test concentration of 10 μM. The lack of binding of unlabeled CTXD5 and CTXD8 to NRP1 was also confirmed in the CTX-Cy5 displacement assay (not shown).

[0154] Usefulness of CTXD5-Cy5 and CTXD8-Cy5 for selective staining of tumor cells compared to CTX-Cy5 and mCTX-Cy5 The inventors examined how higher MMP-2 binding affinity and selectivity are reflected in the cell uptake intensity and selectivity for tumor cells versus non-tumor cells. For this purpose, several representative tumor cell lines were examined for qualitative and semi-quantitative characterization by fluorescence microscopy and image analysis, and for quantitative characterization by flow cytometry.

[0155] Cell culture image results Fluorescence microscopy images of adherent cultures of human U251MG glioblastoma cells and HDFa fibroblasts incubated with mCTX-Cy5 or CTXD5-Cy5 (2 μM) for 1 hour at 37 °C were examined using an ImageXpress Nano Automated Imaging System and analyzed with MetaXpress software (Molecular Devices). To visualize cell bodies and nuclei, the cultures were stained with Calcein AM (Invitrogen #C1430) and Hoechst 33342 (Life Technologies #H3570), respectively. As a result, both mCTX-Cy5 and CTXD5-Cy5 were taken up by the cells, showing a perinuclear large granule localization consistent with clathrin-mediated endocytosis with trans-Golgi accumulation in glioblastoma cells, i.e., characteristic intracellular distribution described by Wiranowska et al. [Wiranowska 2011], while showing a diffuse cytoplasmic distribution consistent with micropinocytosis in fibroblasts. By semi-quantitative analysis, it was shown that the uptake of both labeled CTXDs was higher in glioblastoma cells than in fibroblasts, the uptake of CTXD5-Cy5 was higher than that of mCTX-Cy5 in both cell types, and also that the CTXD5 / mCTX uptake ratio was higher in glioblastoma cells than in fibroblasts.

[0156] Analysis by flow cytometry A detailed protocol for flow cytometric investigation of cellular uptake is shown in Example 8. Briefly, test cells were incubated (double stained) with a viability marker (PO-PRO-1 dye) and Cy5-labeled CTX, mCTX, CTXD5, or CTXD8. Uptake of the Cy5-conjugated protein was measured as the increase in the median fluorescence intensity (ΔMFI) relative to Cy5-protein unstained cells after gating out non-viable cells based on the height of PO-PRO-1 uptake. As a representative set of tumor cell lines, in this study, human glioblastoma (U251 MG and U87 MG), pancreatic cancer (Panc-1), melanoma (A375) and breast cancer (SK-Br) cells were included, which have been classified as "CTX positive" in previous studies [Lyons 2002]. Additionally, the malignant tumor cell line TE 671 human rhabdomyosarcoma, reported as "CTX negative" [Soroceanu 1998], and normal non-tumor cells, namely rat astrocytes, human dermal fibroblasts (HDFa) and human umbilical vein cells (HUVEC) were also investigated to clarify changes in tumor selectivity. First, the inventors performed comparative concentration uptake tests on U251 MG and Panc-1 cells using fluorophore-labeled CTXD5 and CTX. As a result, CTXD5-Cy5 was taken up 4-fold more potently, i.e., at 4-fold lower equipotent concentrations, than CTX-Cy5 in both cell lines (Figure 8). This figure shows the relationship between intracellular uptake concentration - fluorescence of CTX-Cy5 and CTXD5-Cy5 fluorophore-labeled ligands in a cell staining intensity test measured by flow cytometry. Data are shown as mean ± SD of N = 2 and N = 3 experiments comparing the staining intensities of the two compounds in two malignant tumor cell lines: U251 and Panc-1. Based on the concentration-uptake relationship, 300 nM was selected as the fixed test concentration, and uptake of the conjugated proteins of CTXD5-Cy5, CTX-Cy5 and mCTX-Cy5 was investigated in a simultaneous comparison experiment in a selected set of cell lines.As a result, relatively large amounts of labeled CTX, mCTX, and CTXD5 were taken up by "CTX-positive" tumor cells: glioblastoma, melanoma, pancreatic cancer, and breast cancer cells, and uptake into "normal cells" and "CTX-negative" rhabdomyosarcoma cell lines was much less (see Table IV). [Table 4]

[0157] Uptake of CTXD5-Cy5 was higher in all cell types than that of CTX-Cy5. However, from the CTXD5 / CTX uptake ratio, it was shown that CTXD5 was taken up 2.4 - 3.5 times more by tumor cells than CTX, but only 1.1 - 1.9 times by normal cells, indicating that the fluorophore-labeled CTXD5 not only stains tumor cells more strongly than labeled CTX, but also stains the examined tumor cells more selectively than normal cells. The intensity of mCTX-Cy5 cell uptake was not substantially different from that of CTX-Cy5 in all cell lines tested.

[0158] A second series of experiments was performed to compare the intracellular uptake of the two labeled lead compounds CTXD8-Cy5 and CTXD5-Cy5. The results of this comparative study are shown in Table V. [Table 5]

[0159] As a result, it was shown that the uptake intensity of CTXD5-Cy5 in this new test was very similar to that in the previous test (see Table IV). Furthermore, the uptake intensity of CTXD8-Cy5 was almost the same as that of CTXD5-Cy5, indicating very similar cell uptake and tumor-to-non-tumor selectivity profiles.

[0160] Use of CTXD5 in Targeted Intracellular Delivery of Chemotherapeutic Agents To investigate whether the targeting efficacy of CTXD5 is improved compared to mCTX conjugated with chemotherapeutic agents, the inventors compared the growth inhibitory efficacy of intracellularly cleavable MMAF conjugates of two target proteins, CTXD5 and mCTX. Both mono-conjugate and bi-conjugate forms of CTXD5 and mCTX with -GVC-PAB-MMAF were tested. The investigation protocol is described in Example 9. As a result, the mono-conjugate CTXD5-G-VC-PAB-MMAF with an IC 50 of 585 nM was shown to be a 2.4-fold more potent inhibitor of U251 MG cell proliferation than the mono-conjugate mCTX-GVC-PAB-MMAF. The IC 50 of the bi-conjugate CTXD5-(GVC-PAB-MMAF)2 of 235 nM was 9.7-fold more potent than the bi-conjugate mCTX-(GVC-PAB-MMAF)2. The difference between the two targeted proteins was more prominent in the comparison of the minimum effective concentrations, which means that the minimum effective concentration of the CTXD5 bi-conjugate compound was 10 nM, while the superior mCTX conjugate, i.e., the mCTX mono-conjugate, had a minimum effective concentration of 1000 nM, indicating that the chemotherapeutic conjugate of CTXD5 begins to have the desired therapeutic effect at a much lower concentration level.

[0161] In vitro test on the usefulness of CTXD8 in CAR T cell therapy To investigate whether CTXD8- or classical CTX-recognition domain-redirected effector cells are likely to induce cytolysis of target cells, we generated retroviral vectors encoding CTXD8-CAR or CTX-CAR and transduced primary human T lymphocytes derived from peripheral blood mononuclear cells (PBMCs) isolated from the blood of three human donors. Both CTXD8- and CTX-CAR were found to be stably expressed on T cells. The average expression efficiency was 54.4% and 51.2% for CTXD8- and CTX-CAR T cells. To investigate the effector function and potential cytotoxicity of CTXD8- and CTX-redirected CAR T cells, we used a HER2+ trastuzumab-resistant breast cancer cell line (MDA-HER2) with high MMP-2 expression as a target. We also examined cytotoxicity against the HEK293T cell line as a known CTX-CAR-insensitive control (International Publication No. WO 2017 / 066481). To assess their effector function and cytotoxic activity, CTXD8- and CTX-expressing CAR T ("effector") cells were cocultured with MDA-HER2 cells at different effector-to-target cell ratios. We examined effector function by determining the production of interferon gamma (IFNγ) cytokines. CTXD8- and CTX-derived CAR T cells produced significant amounts of IFNγ. No cytokine production was observed with non-modified T cells (NT) or in the absence of target antigen (HEK293T cell line). The viability of tumor cells treated with CTXD8- or CTX-CAR effectors was expressed as a percentage of the mean viability of tumor cells normalized to the mean viability measured in coculture with non-transduced ("NT") lymphocytes. The study protocols are described in Examples 10 and 11. The results shown in Figures 9 and 10 indicated that both CTX- and CTXD8-CAR induced significant cytotoxic effects (reduced viability) that depended on the effector-to-target ratio. No cytotoxicity was observed in coculture with NT control T cells. However, neither CTXD8- nor CTX-reprogrammed CAR T lymphocytes exhibited cytotoxic effects against HEK293T cells.The inventors concluded that CTXD8-CAR T cells induce greater cytotoxicity than CTX-CAR T cells. The effect of CTXD8-CAR T cells was statistically significant (two-way ANOVA, Bonferroni test, Figure 10).

[0162] Conclusion on the use of CTXD of the present invention In summary, the inventors developed a novel CTXD lead compound (i.e., CTXD1) with substantially higher affinity for MMP-2 protein than CTX and mCTX using designer toxins and phage display technology. Using a novel flow cytometry method suitable for quantitative evaluation of the binding strength of different phage-displayed CTXD proteins to MMP-2 and other potential target proteins, the inventors established the structure-activity relationship around this lead compound. These structure-activity correlation studies revealed a set of CTXD compounds that are more potent and also more selective for binding to MMP-2 compared to CTX and mCTX and do not bind to NRP1. The observations obtained by investigating phage-displayed proteins were further confirmed by investigations using recombinantly produced and purified test substances of two selected exemplary chlorotoxin derivatives, CTXD5 and CTXD8. These studies were consistent with the phage display results and showed that the MMP-2 activity and selectivity of CTXD5 and CTXD8 were 3- to 4-fold higher. Surprisingly, CTXD5 and CTXD8 were demonstrated to be more selective not only for NRP1 but also for other target proteins to which CTX shows some binding. Fluorophore and chemotherapeutic agent conjugate derivatives of the lead compound maintained the MMP-2 binding affinity of the parent protein. Furthermore, fluorophore-labeled CTXD5 and CTXD8 were taken up by tumor cells at much higher intensities than CTX, and the selectivity for tumor cells over normal cells was increased. Additionally, the test chemotherapeutic conjugate of CTXD5 inhibited the growth of glioblastoma cells with higher potency than the similar conjugate of mCTX. CAR T cells assembled with CTXD8 or CTX as the antigen recognition domain showed a selective cytotoxic effect against high-MMP-2-expressing breast cancer-derived cell lines but were non-cytotoxic to control cell lines. The selective cytotoxic effect of CTXD8-CAR cells was greater than that of CTX-CAR cells.Combining these observations, the compounds of the present invention, alone or conjugated with other molecules, nanoparticles, or as a targeting recognition domain in CAR cell constructs that provide additional features also referred to as theranostic applications, which may be apparent from the prior art related to such putative uses of CTX, for diagnostic, therapeutic, or combined diagnostic and therapeutic applications, are more useful and superior as targeting molecules than CTX or mCTX. The chlorotoxin derivatives of the present invention can serve as a starting point for further modification.

[0163] Such uses include, but are not limited to, the use of the chlorotoxin derivatives of the present invention as diagnostic targeting molecules in various conjugate forms for intraoperative visualization of intraocular tumor staining, or tumor staining of the skin or mucosal surface, or for radiological and imaging applications, or for histochemical staining.

Examples

[0164] Hereinafter, the present invention will be described in more detail and specifically with reference to examples, which are not intended to limit the present invention.

Examples

[0165] Recombinant production and purification of rCTX, mCTX, CTXD5, and CTXD8 Cloning The DNA sequences encoding rCTX, mCTX, and CTX derivatives were cloned into a pET-based expression vector that also encodes the N-terminal His-tagged fusion partner DsbC (disulfide bond isomerase C). The CTX derivative was amplified by PCR using a forward primer containing the recognition site for BamHI restriction endonuclease and the amino acid sequence of the cleavage site (WELQ) of SplB (serine protease-like protein B). The reverse primer contained two stop codons and the recognition site for XhoI. The PCR product was digested with BamHI and XhoI restriction endonucleases and ligated into the linearized vector downstream of the DsbC fusion partner protein coding segment. The ligation reaction was transformed into chemically competent DH5α cells (Thermo Fisher Scientific). The DNA sequence of the construct was determined by Sanger sequencing.

[0166] Expression Chemically competent SHuffle T7 Express cells (New England Biolabs) were transformed with the plasmid and streaked onto LB agar medium supplemented with ampicillin (100 μg / mL). The next day, a single colony was inoculated into 20 mL of LB medium supplemented with ampicillin and incubated overnight at 37°C with shaking at 160 rpm. The next day, 1 mL of the overnight culture was inoculated into 1 L of auto-inducing terrific broth supplemented with ampicillin and grown at 30°C with shaking at 160 rpm in an Ultra Yield flask (Thomson Instrument Company). After 30 hours of incubation, the bacterial cells were harvested by centrifugation at 7460×g for 10 minutes. The cell pellet of recombinant CTX was resuspended in 140 mL of 50 mM Tris-HCl pH 8.0, 300 mM NaCl, and 0.5% Triton X-100. The cell pellet of recombinant CTX derivative was resuspended in 140 mL of lysis buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 30 mM imidazole, and 0.25% Triton X-100). The samples were stored at -20°C.

[0167] Purification of rCTX After thawing, the cells were disrupted by sonication. Cell debris was pelleted by centrifugation at 48400×g for 20 minutes, and the supernatant was loaded onto a column packed with 17 mL of BioRad Nuvia IMAC chromatography resin using a peristaltic pump to capture the 6xHis-tagged DsbC-CTX fusion protein. After washing with 5 column volumes of IMAC wash buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, and 30 mM imidazole), the sample was eluted from the column with IMAC elution buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 250 mM imidazole). The eluate was dialyzed overnight at 25 °C against 2 L of 20 mM Tris-HCl pH 8.0 buffer. The dialyzed sample was centrifuged at 20000×g for 10 minutes, filtered through a 0.2 μm membrane, and applied to a 5 mL HiTrap Q HP column (GE Healthcare) pre-equilibrated with 20 mM Tris-HCl pH 8.0 buffer (buffer A). After washing with buffer A, the DsbC-CTX fusion protein was eluted at a flow rate of 2 mL / min over 25 minutes with a 0-50% linear gradient of 20 mM Tris-HCl pH 8.0, 1 M NaCl (buffer B). The eluted fractions were analyzed on a 10% tricine-SDS gel. Fractions containing DsbC-CTX were pooled, SplB protease (non-cleavable His-tagged) was added at a fusion protein:protease ratio of 2:1, and tris(2-carboxyethyl)phosphine (TCEP) was added at a final concentration of 100 μM. The sample was incubated overnight at 25 °C. The next day, the cleavage rate was determined on a 10% tricine-SDS PAGE gel. NaCl and imidazole were added to the sample to final concentrations of 0.5 M and 15 mM, respectively, and the solution was filtered through a syringe filter with a pore size of 0.2 μm. Next, using an Akta purification chromatography system, the sample was applied to 8 ml of BioRad Nuvia IMAC resin at 2 ml / min. The column was washed with 20 mM Tris-HCl pH 8.0, 0.5 M NaCl, and 15 mM imidazole buffer, and the flow-through was collected.The 6xHis-DsbC and 6xHis-SplB proteins were eluted from the column using 20 mM Tris-HCl pH 8.0, 0.5 M NaCl, and 250 mM imidazole buffer. The flow-through containing the target (rCTX) protein was concentrated using an Amicon Ultra-15 centrifugal filter unit (MWCO = 3000 Da). This filter unit was also used to exchange the wash buffer into 10 mM Tris-HCl pH 8.0, 0.5 M NaCl buffer for subsequent size-exclusion chromatography. A HiLoad 16 / 600 Superdex 30 pg column (GE Healthcare) connected to an Akta Pure chromatography system was equilibrated with 10 mM Tris-HCl pH 8.0, 0.5 M NaCl buffer. The sample was applied to the column in 1 mL aliquots at 1 mL / min. The sample was then eluted with one column volume of buffer at a flow rate of 1 mL / min, and the absorbance was monitored at 280 nm. Chlorotoxin-containing fractions were determined by PAGE on 10% Tricine-SDS gels. The Tris-HCl and NaCl contents of the chlorotoxin-containing fractions were reduced to less than 0.01 mM and 0.5 mM, respectively, by repeatedly concentrating the samples with Amicon Ultra-15 centrifugal filter units (MWCO = 3000 Da) and diluting them with USP test WFI water. The concentration of the samples was determined by measuring the absorbance at 214 nm before lyophilization. The purity and molecular weight of the protein were determined by subsequent HPLC-UV / MS analysis.

[0168] Purification of mCTX and CTXD After thawing, phenylmethylsulfonyl fluoride (PMSF, a protease inhibitor) at a final concentration of 1 mM was added to the sample, and the cells were disrupted by sonication. Cell debris was pelleted by centrifugation at 48400×g for 20 minutes, and the supernatant was loaded onto a column packed with 25 mL of BioRad Nuvia IMAC chromatography resin using a peristaltic pump to capture the 6xHis-tagged DsbC-CTX derivative fusion protein. After washing with 5 column volumes of IMAC wash buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, and 30 mM imidazole), the sample was eluted from the column with IMAC elution buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, and 250 mM imidazole). The eluate was dialyzed overnight at 25 °C against 2 L of 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) pH 7.5 buffer using a Spectra / Por dialysis membrane (MWCO: 12000 Da, Fisher Scientific). After dialysis, SplB protease and TCEP (tris(2-carboxyethyl)phosphine) were added to the sample at a molar ratio of fusion protein:protease:TCEP of 3:1:1 and incubated overnight at 25 °C. The next day, the extent of cleavage was examined on a 10% tricine-SDS PAGE gel. The digested sample was centrifuged at 20000×g for 10 minutes, filtered through a 0.2 μm membrane, and subjected to cation exchange chromatography performed on an Akta pure system. The sample was loaded onto a 5 mL HiPrep SP HP column (GE Healthcare) pre-equilibrated with wash buffer (20 mM HEPES pH 7.5), and the column was washed with 5 column volumes of wash buffer. Next, the sample was eluted using a linear gradient of 0–50% elution buffer (20 mM HEPES pH 7.5, 1 M NaCl) over 25 minutes at a flow rate of 2 mL / min. The eluted fractions were analyzed on an SDS-PAGE gel. Fractions containing the CTX derivative were pooled and loaded onto a Jupiter 10μ C5 300Å column (Phenomenex) connected to an Agilent 1100 series HPLC system (Agilent Technologies).The mobile phase contained HPLC-grade water with 0.1% formic acid (buffer A) and acetonitrile with 0.1% formic acid (buffer B). Proteins were eluted from the column with a linear gradient of 15 - 45% buffer B over 30 minutes at a flow rate of 2 ml / min. The eluted fractions were analyzed on a 10% tricine-SDS gel, and the fractions containing CTXD were pooled. The concentration of the sample was determined by measuring the absorbance at 280 nm before subjecting it to lyophilization. The purity and molecular weight of the protein were determined by subsequent HPLC-UV / MS measurements.

[0169] Examples 2 - 4 show the synthesis of conjugate CTX, rCTX, mCTX, CTXD5, and CTXD8.

Example

[0170] Synthesis of Fluorophore-Labeled CTX, rCTX, mCTX, CTXD5, and CTXD8 Synthesis of Cy5-Labeled CTX, and CTX Variants and Derivatives CTX-Cy5 (monoconjugate) was synthesized using a mixture of CTX (10 mg / mL) dissolved in 0.1 M sodium bicarbonate adjusted to pH 8 with 0.1 N HCl and cyanine 5 N-hydroxysuccinimide ester (Cy5-NHS ester, Lumiprobe) dissolved in anhydrous dimethylformamide (DMF) supplemented with 0.1% (v / v) diisopropylethylamine (DIPEA). The dye solution concentrations were adjusted, and the two solutions were mixed at a molar ratio of 1:0.9 (CTX / dye) and a solvent ratio of 1:10 (bicarbonate / DMF). Conjugation was performed at room temperature for 1 h. The reaction sample was purified to homogeneity using reverse-phase chromatography. The sample was loaded onto an XBridge BEH C18 column (3.5 μm, 4.6 x 50 mm) connected to an Alliance 2795 HPLC system equipped with a PDA 996 detector. The mobile phase contained HPLC-grade water with 0.1% formic acid (buffer A) and acetonitrile (buffer B). CTX-Cy5 was eluted from the column using a linear gradient of 5–70% buffer B from 0.5 min to 4.5 min at a flow rate of 2 mL / min by collecting 20-s fractions. Absorbance chromatograms were monitored at 650 nm. Relevant fraction samples were analyzed by mass spectrometer (Waters Acquity SQD; ionization: ES+ / ES-, source block temperature: 150 °C, desolvation temperature: 250 °C, desolvation gas: 650 L / h, cone gas: 80 L / h, capillary: 3000 V, cone: 30 V, extractor: 6 V, RF lens: 0.1 V, scan: 80-1000 m / z per second, inter-scan delay: 0.1 s). Fractions containing pure monoconjugate CTX-Cy5 were pooled, and the molecular weight and purity of the sample were confirmed by HPLC mass spectrometry. Finally, the resulting sample was lyophilized. Cy5-labeled rCTX, mCTX, CTXD5, and CTXD8 were synthesized using essentially the same process.

[0171] Synthesis of Alexa 488-labeled CTX, and CTX variants and derivatives Alexa 488-labeled CTX (CTX-A488) was also synthesized by a similar process except that the starting reactive dye compound was Alexa 488 tetrafluorophenyl (TFP) ester.

Example

[0172] Synthesis of biotinylated CTXD Biotinylated CTXD (monoconjugate) was synthesized using a mixture of CTXD (10 mg / mL) dissolved in 0.1 M sodium bicarbonate adjusted to pH 8 with 0.1 N HCl and biotin 3-sulfo-N-hydroxysuccinimide ester (Sigma) dissolved in anhydrous dimethylformamide (DMF) with 0.1% (v / v) DIPEA added. The concentration of the activated biotin solution was set, and the two solutions were mixed to a molar ratio of 1:1 (CTX / biotin) and a solvent ratio of 1:10 (bicarbonate / DMF). The conjugation was carried out at room temperature for 1 hour. The post-reaction sample was purified uniformly using reverse-phase chromatography. The sample was loaded onto a Phenomenex Jupiter C5 column (300 Å 4.6 × 250 mm) connected to an Alliance 2795 HPLC system equipped with a PDA 996 detector. The mobile phase consisted of HPLC-grade water containing 0.1% formic acid (eluent A) and acetonitrile (eluent B). CTXD-biotin was eluted from the column at a flow rate of 1.8 mL / min for 7 minutes from 5 to 12 minutes using a linear gradient of 5 to 70% of eluent B by collecting the fraction at 27 seconds. The absorbance chromatogram was monitored at 280 nm. The relevant fraction samples were analyzed by mass spectrometry, and the fractions containing pure monoconjugate CTXD-biotin were pooled, and the molecular weight and purity of the sample were confirmed by HPLC mass spectrometry. Finally, the obtained sample was lyophilized.

Example

[0173] Synthesis of CTXD5-glutaryl-Val-Cit-PAB-MMAF and mCTX-glutaryl-Val-Cit-PAB-MMAF A CTX receptor-based targeted chemotherapeutic agent was designed, which contains a CTX derivative as the targeting protein, an intracellular cleavage cathepsin B enzyme-sensitive linker (glutaryl-valine-citrulline-P-aminobenzoyloxycarbonyl, abbreviated as G-VC-PAB), and a cell division inhibitory monomethyl auristatin F (MMAF) as the payload. For the test, the G-VC-PAB-MMAF conjugate was synthesized according to the following procedure using either CTXD5 or mCTX as the target molecule:

[0174] Step 1: Synthesis of VC-PAB-MMAF The starting materials were Fmoc-Val-Cit-PAB-PNP (Fmoc = fluorenylmethoxycarbonyl protecting group; PNP = paranitrophenyl) purchased from MedChemExpress and MMAF (MedChemExpress). The starting materials (28 mg of Fmoc-Val-Cit-PAB-PNP and 27 mg of MMAF, molar ratio 1.25:1) were dissolved in 3.5 mL of DMF containing 5.6 mg of 1-hydroxybenzotriazole (HOBt) and 3 μL of DIPEA. After reacting at room temperature for 1 hour, 20% (v / v) piperidine was added and reacted at room temperature for 20 minutes to remove the Fmoc protecting group, and the sample was purified on a Sun Fire C18 column (5 μm, 4.6 × 250 mm) connected to a Waters 600 semi-preparative reverse-phase HPLC system equipped with a 2487 dual-wavelength absorbance detector.

[0175] The mobile phase contained HPLC-grade water containing 0.1% formic acid (eluent A) and acetonitrile (eluent B). The product was eluted from the column at a flow rate of 13 mL / min using a linear gradient of 5 - 70% eluent B from 5 to 16 minutes. The absorbance chromatogram was monitored at 280 nm. The relevant fraction samples were analyzed by a mass spectrometer, and the fractions containing purely the target compound (VC-PAB-MMAF) were pooled and lyophilized.

[0176] Step 2: Synthesis of NHS-G-VC-PAB-MMAF The starting materials for this reaction are VC-PAB-MMAF synthesized previously and diglutaryl acid di(N-succinimidyl) purchased from Sigma. Both starting materials were dissolved in 4 - 4 mL of dimethyl sulfoxide (DMSO), and 3 μL of DIPEA was added to the former. Next, the VC-PAB-MMAF solution was added dropwise to the diglutaryl acid disuccinimidyl solution over about 30 minutes and allowed to react overnight. The next day, the target compound was purified by HPLC as described above, lyophilized, and stored at -20 °C until coupling with CTXD.

[0177] Step 3: Coupling to G-VC-PAB-MMAF and CTX derivatives The coupling reaction was carried out in the same manner using CTXD5 or mCTX. The coupling reaction was performed using a mixture of CTXD5 (10 mg / mL) dissolved in 0.1 M sodium bicarbonate adjusted to pH 8 with 0.1 N HCl and NHS-G-VC-PAB-MMAF dissolved in anhydrous dimethylformamide (DMF) with 0.1% (v / v) DIPEA added. The concentration of the linker-payload solution was set, and the two solutions were mixed to a molar ratio of 1:2 (CTXD / linker-payload) and a solvent ratio of 1:10 (bicarbonate / DMF). The conjugation was carried out at room temperature for 1 hour. The reacted samples were purified homogeneously using reverse-phase chromatography as described above for the Cy5 conjugation reaction. The NHS ester linker-payload molecule can conjugate mainly with the primary amine of the only lysine residue in the monomethylated CTX derivative, but also secondarily with the N-terminal amine, so both mono-conjugate (e.g., CTXD5-G-VC-PAB-MMAF) and bi-conjugate (CTXD5(-G-VC-PAB-MMAF)2) derivatives were formed. Therefore, both the mono-conjugate compound and the bi-conjugate compound were separately recovered in the purification process and subjected to biological investigation after lyophilization.

[0178] Examples 5 - 9 show several test protocols.

Example

[0179] Co-bead assay The Co-bead assay was used to measure the binding strength of phage-displayed or purified CTXD ligand proteins to various target proteins. This assay is based on the principle of immobilizing various His-tagged target proteins (receptors) on immobilized cobalt-coated His-Tag Isolation & Pulldown Dynabeads (ThermoFisher Scientific, catalog number: 10104D), binding a fluorophore-labeled ligand to the target protein, and measuring the bead-bound fluorescence intensity by flow cytometry.

[0180] Preparation of Co-beads Beads with a volume of 0.5 μL (specified as 1 μm in diameter) were pipetted into each reaction tube and washed three times. Each wash involved resuspending the beads in 2 mL of Tris-buffered saline aqueous solution (TBS, 20xTBS from ThermoFisher Scientific, catalog number: 28360) containing 0.05% (v / v) Tween-20 (hereinafter, the washing solution is abbreviated as TT), placing the tube in a magnetic concentrator (DynaMag-5, ThermoFisher Scientific, catalog number: 12303D), and discarding the supernatant after 10 minutes. Subsequently, the beads were incubated overnight at 4 °C in 50 μL of the target protein solution. In the study reported in this application, all target proteins were applied at the same molar concentration of 0.16 μM: this corresponds to 10 μg / mL in the case of active MMP-2 (MW 62 kDa). The target protein solution was dissolved or diluted in TBS. Negative control beads were incubated simultaneously with TBS without the target protein. The target proteins used were as follows (NCBI accession number [segment amino acid sequence]; position of his-tag: C-terminal or N-terminal; supplier; and catalog number # are described in parentheses): MMP2 (NP_004521.1 [110-660]; N-; ProSpec, #ENZ-769); NRP1 (NP_001019799.1 [1-644]; C-; Sino Biological; #10011-H08H); MMP-9 (NP_004985.2 [20-701]; C-; ProSpec; #ENZ-1091), TIMP-2 (NP_003246.1 [27-220]; N-; ProSpec; #ENZ-646), MMP-14 (NP_004986 [24-524]; C-; ThermoFisher; #RP77533), Anx2 (NP_001002857.1 [1-339]; N-; ProsPec; #PRO-777), α vβ3 integrin (AAA52589.1 & NP_002196.4 heterodimer; both with C-terminal his-tag; Native Antigen Company; #REC31719-100), CLC3 (NP_001820.2 [1-818]; C-; Creative Biomart; custom made by recombinant expression in E. coli), human serum albumin (NP_000468.1 [25-609]; C-; Abcam; #ab217817). After incubation of the target proteins, the beads were washed three times and then blocked by incubating with 100 μL of Casein Blocking Solution (Sigma, #B6429) for 1 hour at room temperature, followed by three washes. The subsequent process differed depending on the phage display protein, as well as the purified and labeled protein test substances.

[0181] Exposure and staining of phage display When testing the ligands of phage display proteins (CTX and CTXD), the prepared beads were resuspended in the test monoclonal phage solution and incubated for 1 hour at room temperature. The phage was dissolved in phage and antibody incubation buffer TBS containing 0.5% bovine serum albumin and 0.05% (v / v) Tween-20 (hereinafter abbreviated as TBT). After phage exposure, the beads were washed three times and then incubated for 30 minutes at 4 °C in 50 μL of TBT containing a 1:200 dilution of mouse monoclonal anti-M13 antibody (Sino Biological, #11973-MM05). Then, the beads were washed three times and incubated for 30 minutes at 4 °C in 50 μL of TBT containing a Cy5-labeled goat anti-mouse secondary antibody diluted 1:1000. After three final washes, the beads were resuspended in 2 mL of TT in preparation for flow cytometry measurement.

[0182] Exposure of fluorophore-labeled test substances To test the fluorophore-labeled test substance (e.g., CTX-Cy5 or CTXD-Cy5), beads coated with the target protein and blocked with casein were resuspended in 50 μL of a test solution containing the labeled test substance at a specific concentration and incubated at room temperature for 1 hour. The test solution was prepared, for example, by diluting a 100 μM stock solution of the test substance dissolved in water containing 10 - 14% (v / v) dimethyl sulfoxide (DMSO) with TT. After incubation with the test solution, the beads were washed three times, resuspended in 2 mL of TT, and prepared for flow cytometry measurement.

[0183] Measurement of bead-bound fluorescence intensity by flow cytometry Flow cytometry analysis was performed using a flow cytometer (MACSQuant Analyzer 10 from Miltenyi Biotec), with a 635 nm red laser for excitation and the R1 channel for fluorescence intensity measurement in pulse area mode. The aspiration volume was 200 μL per sample, set to mix gently, and 10,000 events were collected per sample. The flow rate was set slow, and the maximum number of events was 500 per second. This comprehensive elliptical gate was applied around the major bead population, which was set based on the forward scatter versus side scatter dot plot obtained with negative control beads, such that events related to doublets and multiplets were excluded from the analysis. Gating was kept constant throughout all experiments using the same type of beads. For the quantitative characterization of binding, the median fluorescence intensity (MFI) of the gated events was used. Results were expressed as MFI or relative fluorescence intensity (RFI), where the latter was defined as the ratio of the MFI of the sample to the MFI of a related negative control sample processed simultaneously. The negative control samples were subjected to all preparation, blocking, and staining procedures except that the beads were not coated with the target protein. To evaluate the binding strength (i.e., the number / amount of phage / protein bound to the beads), delta MFI values or delta RFI values were used, which represent the increase (difference) relative to the values of the negative control samples.

[0184] While evaluating the binding strength of phages presenting CTXD (e.g., the results shown in Tables I and II), the CTX-expressing phage solution was also tested at the same phage concentration (in the range of 1.08 - 4.08×10 14 phage particles / mL) in the same experiment, and the relative MMP-2 binding strength (delta RFI) of CTXD was normalized (divided) by the delta RFI of CTX. The values of the relative MMP-2 binding strength shown in Table I are the average of N experiments. N was at least 3 for mCTX and the claimed compounds (Compounds 3 - 18).

Example

[0185] Magnabead Assay The Magnabead Assay is based on the principle of immobilizing a target protein via a target protein-specific rabbit IgG antibody on microbeads factory-coated with MagnaBind Goat Anti-Rabbit IgG Beads (ThermoFisher, Scientific, catalog number: 21356), i.e., goat anti-rabbit antibody, binding a fluorophore-labeled ligand to the target protein, and measuring the bead-bound fluorescence intensity by flow cytometry. The Magnabead Assay was used to characterize the concentration-binding relationship properties of fluorophore-labeled CTX, mCTX, and CTXD ligand proteins against MMP-2 and NRP-1 target proteins, thereby characterizing the relative binding affinity by measurement of the half-maximal effective concentration (EC 50 value). This method is also useful for characterizing the relative affinity of unlabeled CTXD and conjugated CTX derivatives by measuring the displacement (inhibition) of the binding of fluorophore-labeled CTX from the receptor on the MMP-2 or NRP1 target protein. In this way, the half-maximal inhibitory concentration (IC 50 value) was measured to compare the affinities of various CTX, mCTX, and CTXD derivatives.

[0186] Magnabead Preparation In each reaction tube, 1 μL of beads (specified with bead diameter 1 - 4 pm) were pipetted and washed three times. Each wash involved resuspending the beads in 2 mL of Dulbecco's phosphate - buffered saline (DPBS, Sigma #D8662), placing the tube in a magnetic concentrator (DynaMag - 5; ThermoFisher Scientific, catalog number: 12303D), and discarding the supernatant after 10 minutes. Subsequently, the beads were incubated for 1 hour at 4°C in 50 μL of DPBS containing a rabbit antibody against the test target protein diluted 1:100. The anti - MMP - 2 antibody used was a polyclonal antibody (ThermoFisher Scientific, catalog number: PAI - 16667), which was produced against the synthetic sequence of amino acid positions 504 - 518 of the 660 - amino - acid - long human MMP - 2 protein (NP_004521.1). The MMP - 2 protein can be either the mature active enzyme (e.g., ProspecBio #Enz - 100 or Sigma #SRP3118) or the pro - enzyme (e.g., ProspecBio #Enz - 769 or Sigma #SRP6270), and these have very similar CTX - binding results. The anti - NRP1 antibody used was a polyclonal antibody (ThermoFisher Scientific, catalog number: PA5 - 26079), which was produced against the synthetic sequence of amino acid positions 722 - 750 of the 923 - amino - acid - long human NRP1 protein (NP_003864.5). The NRP1 protein immobilized on the beads by the antibody was recombinantly produced (ProsPec, catalog number: Cyt - 1059) as a single glycosylated polypeptide chain containing 843 amino acids (22 - 856) out of the 923 - amino - acid - long sequence. After the second antibody coating, the beads were washed three times and incubated for 1 hour at room temperature in 50 μL of DPBS containing the target protein (e.g., MMP - 2 or NRP1) selected at a concentration of 0.16 μM. For the Magnabead assay, no blocking incubation was required to remove non - specific binding.

[0187] Exposure to the test substance In the test of a fluorophore-labeled test substance (e.g., CTX-Cy5, or CTXD-Cy5, or CTX-A488), beads coated with the target protein were resuspended in a test solution containing the labeled test substance at a specific concentration and incubated at room temperature for 1 hour. The test solution was prepared by diluting a stock solution in which 100 μM of the test substance was dissolved in water containing, for example, 10 - 14% (v / v) dimethyl sulfoxide (DMSO) with DPBS. After incubation with the test solution, the beads were washed three times, resuspended in 2 mL of DPBS, and prepared for flow cytometry measurement.

[0188] When testing an unlabeled test substance, beads coated with the target protein were resuspended in 50 μL of a test solution containing a specific concentration of the unlabeled test substance dissolved in DPBS and incubated at room temperature for 1 hour. Next, 50 μL of a DPBS solution containing 2 μM of labeled CTX (CTX-Cy5 or CTX-A488) and a specific concentration of the unlabeled test substance was added, and the beads were incubated in 1 μM of labeled CTX (substitution-indicating ligand) and a specific concentration of the unlabeled test substance (substitution ligand). After incubating the beads at room temperature for an additional 1 hour, they were washed three times, resuspended in 2 mL of DPBS, and prepared for flow cytometry measurement.

[0189] Measurement of bead-bound fluorescence intensity by flow cytometry Flow cytometry was performed using a flow cytometer (MACSQuant Analyzer 10 from Miltenyi Biotec). For excitation, a 635 nm red laser was used, and the R1 channel was used to measure the fluorescence intensity of the Cy5-labeled ligand binding. Or for excitation, a 488 nm blue laser was used, and the Bl channel was used to measure the fluorescence intensity of the A488-labeled ligand binding. Flow cytometry analysis was performed in pulse area mode. The aspiration volume was set at 200 μL per sample and was set to mix gently, and 10,000 events per sample were collected. The flow rate was set slow, and the maximum number of events was 500 per second. This comprehensive elliptical gate was applied around the major bead population, which was set based on the forward scatter versus side scatter dot plot obtained with the negative control beads, and events related to doublets and multiplets were excluded from the analysis. Gating was kept constant throughout all experiments using the same type of beads.

[0190] For the quantitative characterization of the binding of the labeled ligand, the MFI of the gated events was used. The results were expressed as delta RFI (RFI minus 1) values. The relative affinity of the labeled test substance was plotted with the delta RFI values against the logarithmic concentration, and the half-maximal effective concentration (EC 50 ) was evaluated by determining it with sigmoid curve fitting (Boltzmann function).

[0191] In the displacement test, the results were converted to the percentage inhibition of the labeled ligand binding (displacement %), the displacement % was plotted against the logarithmic concentration, and the relative affinity of the unlabeled test substance was evaluated by determining the half-maximal inhibitory concentration (IC 50 ) with sigmoid curve fitting.

Example

[0192] Preparation of phage for phage display ligand peptide binding measurement The phage display test solution was prepared according to conventional phage display protocols [e.g., Tonikian 2007]. Oligonucleotides encoding the expressed peptides were phosphorylated and annealed and ligated to the pAS62 phagemid vector [U.S. Patent Publication No. 8,716,437, column 32, lines 48-49] at a vector / insert ratio of 1:3. The phagemid containing the insert encoded a display protein with a C-terminus extended with a linker peptide sequence (GSASSA) fused to the P3 coat protein. Electrocompetent bacterial cells (E. coli SS320) were transformed with the ligation product and then streaked onto lysogeny broth agar plates (LB / Amp) containing ampicillin (100 μg / mL) and incubated at 37 °C for 16 hours. Under these conditions, only phagemid-bearing cells survived and formed colonies. The nucleotide sequences of several clones were determined, and a single colony with the target sequence confirmed was inoculated into 3 mL of 2YT / Amp broth and incubated at 37 °C until mid-log phase of culture was reached. The culture was infected with M13KO7 helper phage (phage / cell ratio 10:1) and incubated at 37 °C for 30 minutes. 3 mL of the culture was transferred to 200 mL of 2YT / Amp / Kan broth and incubated overnight. The overnight cell culture (200 mL) was pelleted by centrifugation at 8000 × g for 10 minutes at 4 °C in a 400 mL centrifuge tube. The supernatant was transferred to a new 400 mL centrifuge tube containing 40 mL of PEG / NaCl (200 g / L PEG-8000, 146.1 g / L NaCl), mixed well, and then incubated at room temperature for 20 minutes. The precipitated phage was pelleted by centrifugation at 18000 × g for 15 minutes at room temperature. After discarding the supernatant, the pellet was recentrifuged at 1000 rpm for 1 minute with the pellet oriented tangentially to recover and remove residual fluid. The phage was solubilized in 4 mL of phage resuspension buffer (TBT) and centrifuged at 18000 × g for 10 minutes at 4 °C to remove all insoluble material. The supernatant was transferred to four 1.5 mL centrifuge tubes (4 × 1 mL), reprecipitated with 200 - 200 μL of PEG / NaCl, incubated for an additional 5 minutes, and then recentrifuged at 12000 × g.The pellets were resuspended in a small volume (150 - 300 μL) of phage resuspension buffer to form a single phage solution. The particle concentration of the phage solution was determined spectrophotometrically based on the linear relationship between the absorbance (OD 268 ) in the absorbance range of 0.5 - 10 using a NanoDrop One C (Thermo Scientific) spectrophotometer and the phage titer. The phage particle concentration was evaluated according to the formula: (OD 268 - OD 320 ) × 5 × 10 12 particles / mL [Tonikian 2007]; it was set to the target concentration by dilution with TBT.

Example

[0193] Analysis of the cell staining intensity of fluorophore-labeled test substances by flow cytometry Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2 in the recommended medium supplemented with 10% fetal bovine serum (FBS) and an antibiotic-antifungal solution (Ab / Am, Sigma #A5955), unless otherwise specified. The cells used (source #catalog number and culture medium in parentheses) were as follows: U251 MG human glioblastoma (Sigma #09063001, RPMI-1640), U87 MG human glioblastoma (Sigma #89081402, DMEM high glucose), PANC-1 human pancreatic cancer (Sigma #87092802, DMEM high glucose + Glutamax), A375 human melanoma (Sigma #88113005, DMEM high glucose + Glutamax), SK-Br-3 human breast cancer (AddexBio #C0006007, DMEM low glucose), TE-671 human rhabdomyosarcoma (Sigma #85111502, DMEM high glucose), HDFa human dermal fibroblasts (Sigma #106-05A, fibroblast growth medium without other additives), HUVEC human endothelial cells (ThermoFisher #C0035C, Medium 200 supplemented with only Large Vessel Endothelial Supplement), neonatal rat astrocytes (ThermoFisher Scientific #N7745100, DMEM high glucose + Glutamax, supplemented with 15% FBS).

[0194] After removing the culture medium, TrypLE Express cell dissociation enzyme solution was allowed to act for 5 - 10 minutes to detach the cells, and the enzyme was inactivated with a culture medium supplemented with FBS. The cells were washed 3 times by centrifugation, the supernatant was discarded, and the cells were resuspended in DPBS. After the third wash, the cell density of the suspension was determined from a 20 μL sample using a Countess II Automated Cell Counter (ThermoFisher Scientific), and it was also evaluated whether the criterion of a viability of over 95% was satisfied by counterstaining with 0.4% Trypan Blue. The cell density was set to 1 million - 1.5 million cells per mL by dilution with DPBS. The cell suspension was dispensed as 100 μL samples into 5 mL flow cytometry tubes, and a labeled test substance (for example, a diluted 100 μM stock solution as needed) or an equivalent vehicle (for example, a 10% DMSO solution) was added in a volume of 1 - 3 μL. Then the test tubes were incubated at 37 °C for 45 minutes in a humidified atmosphere containing 5% CO2. Thereafter, the test tubes were placed in a chill rack, 2.5 μM of Po-Pro-1 was added to each tube, and the tubes were incubated at 4 °C for 15 minutes to stain the non-viable cells. Then the cells were washed 3 times and resuspended in cold DPBS (2 mL / tube), and stored in the chill rack until the flow cytometry test, which was performed as soon as possible, usually within 1 hour.

[0195] Flow cytometry analysis was performed using a flow cytometer (MACSQuant Analyzer 10 from Miltenyi Biotec), with a 635 nm red laser for excitation, an R1 channel for measuring the fluorescence intensity of Cy5-labeled ligand uptake, a 405 nm violet laser for excitation, and a V1 channel for Po-Pro-1 fluorescence detection. Events in both channels were recorded in pulse area mode. The aspiration volume was set to 200 μL per sample, set to mix gently, and 10,000 events per sample were collected. The flow rate was set slow, and the maximum number of events per second was 500.

[0196] To analyze each set of experiments, first, gates were defined on the linear mode FSC vs SSC scatter plot obtained from non-stained control cells, and only single cells with sizes and granularities typical of the cell line were included in the analysis. Non-viable cells showing high Po-Pro-1 signal intensity were also excluded from the analysis. The set gate settings were kept constant throughout all experiments using the same type of cells. For the quantitative characterization of staining intensity, the median fluorescence intensity (MFI) of the gated events was used. The results were expressed as MFI or relative fluorescence intensity (RFI), where the latter was defined as the ratio of the MFI of the sample to the MFI of the simultaneously processed unstained vehicle control sample. To evaluate the uptake into cells, delta MFI values or delta RFI values were used, which mean the increase (difference) relative to the values of the simultaneously processed unstained control samples. Most experiments were conducted in a two-set format.

Example

[0197] Measurement of the cell division inhibitory efficacy of the test substance Cells were grown in monolayers and cultured at 37 °C in a humidified atmosphere containing 5% CO2 until they reached a confluent level of 75 - 90%. After detachment with TrypLE Express, the cells were seeded into 96-well plates at a density of 1,500 cells / well in 100 μL of cell culture medium supplemented with 10% FBS per well. After an incubation period of approximately 24 hours, the entire volume of the medium in each well was replaced with 90 μL of fresh medium, and 30 μL of the test substance-containing medium was added thereto, obtaining various test substance concentrations from 3.16 nM to 3.16 μM in 3.16-fold increments. The cells were then incubated for an additional 72 hours. The viability of the cells after treatment with the test substance was determined using a Cell counting Kit-8 (CCK-8) viability assay conducted according to the manufacturer's instructions. Briefly, the CCK-8 solution was added to the cells to a final volume of 10% of the culture volume. The cells were incubated at 37 °C for an additional 4 hours, and the formazan dye production was measured from the absorbance at 460 nm using a plate reader. All experiments were performed in parallel with six measurements.

[0198] For each treatment, the vehicle control group was also performed on the same plate simultaneously. The results for each treatment were calculated as % relative to the corresponding vehicle group and plotted against concentration on a logarithmic scale with sigmoid curve fitting according to the Boltzmann function. The 50% inhibitory concentration (IC 50 ) was determined at the inflection point, i.e., the midpoint between the vehicle control and maximum inhibition. The minimum effective concentration was defined as the lowest test concentration that resulted in a statistically significant inhibition of more than 20%. Statistically significant differences from the vehicle control were tested using Dunnett's test following one-way ANOVA.

Example

[0199] Generation of CTX-CAR / CTXD-CAR T cells Construction of transduction vector The inventors designed CTXD8-CAR and CTX-CAR gene cassettes containing cDNAs encoding the following protein domain sequences: IgG heavy chain signal peptide (GMCSFRa), CTXD8, or CTX, CD8 alpha hinge, transmembrane region of human CD28 having the CD28 intracellular co-stimulatory endodomain (CD28 transmembrane and CD28 cytoplasmic), (Gly)3 linker, cytoplasmic region of human CD3 zeta, T2A self-cleaving peptide, and truncated CD19. The amino acid sequence of the designed CTXD8-CAR construct is shown in FIG. 11, which is described in SEQ ID NO: 44. FIG. 11 depicts the above elements of the amino acid sequence, with the GMCSFRa signal peptide, CD8 alpha hinge, CD28 cytoplasmic, CD3 zeta, and truncated CD19 underlined. The non-functional truncated CD19 was added to enable detection of transduction efficiency. This cassette was obtained by insertion into the pUC57 cloning vector, then excised and ligated into the modified pMSGV retroviral vector. Retroviral particles encoding CTXD8-CAR or CTX-CAR were prepared by transient transfection of HEK 293T cells with the modified MSGV retroviral vector encoding CTXD8- and CTX-CAR, the Peg-Pam-e plasmid containing the sequence of MoMLV gag-pol, and the pMax.RD114 plasmid containing the sequence of RD114 using the jetPrime transfection reagent (Polyplus, Illkirch, France). The viral supernatant was collected 72 hours after transfection and used for T cell transduction on the same day or rapidly frozen and stored at -80°C until use.

[0200] Proliferation and transduction of T lymphocytes Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by density gradient separation using Histopaque®-1077 (Sigma-Aldrich, St. Louis, MO, USA). Subsequently, the cells were seeded into 24-well non-tissue culture treated plates pre-coated with 1 μg / mL of anti-CD3e (clone: OKT3; Thermo Fischer, Waltham, MA, USA) and anti-CD28 (R&D Systems, Minneapolis, MN, USA). The culture medium for initial stimulation was RPMI 1640 supplemented with 10% fetal bovine serum and 2 mmol / L GlutaMAX (Thermo Fisher, Waltham, MA). After 24 hours, human interleukin-7 (IL-7; 10 ng / mL) and human interleukin-15 (IL-15; 5 ng / mL) (Miltenyi Biotec, Bergisch Gladbach, Germany) were added to the cultures. The next day, the cells were transduced with retroviral particles on 20 μg / mL RetroNectin (Takara Bio, Mountain View, CA) coated plates in the presence of IL-7 (10 ng / mL) and IL-15 (5 ng / mL). After 48 hours of incubation, the cells were used for further experiments.

[0201] Evaluation of transduction efficiency The transduction efficiency of retrovirus was determined by detecting the cleaved CD19 molecule by flow cytometry using allophycocyanin-conjugated anti-human CD19 antibody (Beckman Coulter, Brea, CA). The purity of T cells was determined by Alexa Fluor 488-conjugated anti-human CD3 antibody (BD Biosciences, San Jose, CA, USA) staining. All antibodies used for flow cytometry analysis were applied at a final concentration of 10 μg / mL for 10 minutes on ice. The analysis was performed on at least 10,000 cells per sample using a NovoCyte flow cytometer and NovoExpress software (ACEA Biosciences, San Diego, CA, USA).

Example

[0202] Examination of the cytotoxicity of CTX-CAR / CTXD-CAR T cells The HER2+ trastuzumab-resistant breast cancer-derived cell line (MDA HER2) was prepared by transducing the HER2-negative MDA-MB-468 cell line (purchased from American Type Culture Collection (Manassas, VA, USA)) with human HER2 gene-encoding lentiviral particles followed by single-cell cloning. The HEK293T negative control cell line was purchased from American Type Culture Collection (Manassas, VA, USA). The firefly luciferase-expressing MDA-HER2 cell line and HEK293T cell line were prepared by transducing the MDA-HER2 cell line and HEK293T cell line with a retrovirus encoding eGFP.ffLuc, expressing the fusion gene of highly sensitive green fluorescent protein (eGFP) and firefly luciferase (ffLuc), and then by single-cell cloning.

[0203] The cytotoxic activity of CAR T cells against the target was determined by a luciferase-based cytotoxicity assay. MDA-HER2 and HEK293T cells expressing eGFP / ffLuc were seeded in pairs at a density of 10 5 cells / well in a 96-well flat-bottom plate. After 4 hours, various amounts of CTXD8- and CTX-derived CAR T cells were added to the tumor cells, and the cytotoxicity at a wide range of effector-to-target cell ratios was evaluated. Wells containing unmodified effector cells (NT) were used as a reference for the untreated control. After 24 hours, the medium and T cells were removed, and the remaining tumor cells were quantified by luciferase activity. The luciferase assay kit was used according to the manufacturer's instructions (Promega, Madison, WI, USA), and the detection was performed with a Synergy HT luminometer (BioTek, Winooski, VE, USA).

[0204] References Patent Documents US Patent Publication No. 20100215575 (TRANSMOLECULAR INC), August 26, 2010 US Patent Publication No. 20100210546 (TRANSMOLECULAR INC), August 19, 2010 International Publication No. WO2011094671 (THE UAB RESEARCH FOUNDATION), August 4, 2011 International Publication No. WO2011142858 (FRED HUTCHINSON CANCER RESEARCH CENTER), November 17, 2011 International Publication No. WO2015042202 (BLAZE BIOSCIENCE INC), March 26, 2015 International Publication No. WO2017066481 (CITY OF HOPE), April 20, 2017 International Publication No. WO2017136769 (EISAI R&D MANAGEMENT CO LTD), August 10, 2017 US Patent No. 8716437 (STEVEN A. GOLDSTEIN, ZOLTAN TAKACS), January 13, 2011 Non-Patent Literature AKCAN, M. et al. Chemical Reengineering of Chlorotoxin Improves Bioconjugation Properties for Tumor Imaging and Targeted Therapy, J. Med. Chem. 2011. Vol. 54, No. 3, pp. 782 - 787. CHENG, Y. et al. Recent Advances in the Diagnosis and Treatment of Gliomas Using Chlorotoxin - Based Bioconjugates, Am. J. Nucl. Med. Mol. Imaging 2014. Vol. 4, No. 5, pp. 385 - 405. COHEN, G. et al. Chlorotoxin - A Multimodal Imaging Platform for Glioma Tumors. Toxins (Basel) 2018. Vol. 10, No. 496.1 - 12 pp. Dardevet, L. et al. Chlorotoxin: A Useful Natural Scorpion Peptide for Glioma Diagnosis and Fighting Tumor Invasion. Toxins (Basel). 2015. Vol. 7, No. 4, pp. 1079 - 1101. Debin, J. A. et al. Purification and Characterization of Chlorotoxin, a Chloride Channel Ligand Derived from Scorpion Venom. Am. J. Physiol 1993. Vol. 264, No. 2, pp. C361 - C369. Deshane, J. et al. Chlorotoxin Inhibits Glioma Cell Invasion via Matrix Metalloproteinase - 2. J. Biol. Chem. 2003. Vol. 278, No. 6, pp. 4135 - 4144. Di, L. Strategic Approaches for Optimizing Peptide ADME Properties. AAPS J. 2015. Vol. 17, No. 1, 134 - 143. Dintzis, S. M. et al. Real - Time Visualization of Breast Cancer Tumors in Pathologic Specimens from Patients Administered the Fluorescent Tumor - Labeling Agent Tocilizumab. Arch. Pathol. Lab. Med. 2019. Vol. 143, No. 9, pp. 1076 - 1083. Doronina, S. O. et al. Enhanced Activity of Monomethylauristatin F via Monoclonal Antibody Delivery: Influence of Linker Technology on Efficacy and Toxicity. Bioconjug. Chem. 2006. Vol. 17, No. 1, pp. 114 - 124. Fujiwara, K. et al. The Hinge and Transmembrane Domains of Chimeric Antigen Receptors Control Receptor Expression and Signaling Thresholds. Cells, 2020. Vol. 9, No. 5, 1182. Jeelani, S. et al. Celanostics: A Crucial Tailor for Tomorrow. J. Pharm. Bioallied. Sci. 2014. Vol. 6, Suppl. 1, pp. S6 - S8. June, C. H. et al. Chimeric Antigen Receptor Therapy. N. Engl. J. Med. 2018. Vol. 379, No. 1, pp. 64 - 73. KESAVAN, K. et al. Annexin A2 is the molecular target of peptide TM601 with tumor targeting and anti-angiogenic effects. J. Biol. Chem. 2010. Vol. 285, No. 7, pp. 4366 - 4374. LAMBERT, J. M. et al. Antibody-drug conjugates for cancer therapy. Annu. Rev. Med. 2018. Vol. 69, pp. 191 - 207. LYONS, S. A. et al. The scorpion-derived peptide chlorotoxin specifically binds to gliomas and neuroectodermal-derived tumors. Glia 2002. Vol. 39, No. 2, pp. 162 - 173. MAMELAK, A.N. et al. Phase I single-dose trial of intracavitary iodine-131-TM-601 in adult patients with recurrent high-grade gliomas. J. Clin. Oncol. 2006. Vol. 24, No. 22, pp. 3644 - 3650. MAROFI, F. et al. CAR-NK cells: A new paradigm in tumor immunotherapy. Front. Oncol. 2021. Vol. 11, 673276. MCGONIGLE, S. et al. Neuropilin-1 drives tumor-specific uptake of chlorotoxin. Commun. Signal 2019. Vol. 17, No. 67, pp. 1 - 14. MOORE, S. J. et al. Engineered notchtins as novel binders. Methods Enzymol. 2012. Vol. 503, pp. 223 - 251. MUKHOPADHYAY, M. Macrophages infiltrate CAR immunotherapy. Nat.Methods. 2020. Vol. 17, No. 6, 561. OJEDA, P.G. et al. Mutating lysine to arginine in chlorotoxin enhances cellular uptake. Biopolymers 2017. Vol. 108, No. 5, e2305. Patil, C. G. et al. Phase 1 safety, pharmacokinetics, and fluorescence imaging trial of tozuleristide (BLZ-100) in adults with newly diagnosed or recurrent glioma. Neurosurgery 2019. Vol. 85, No. 4, pp. E641 - E649. Smith, G. P. Filamentous fusion phage: a novel expression vector that displays cloned antigens on the virion surface. Science 1985. Vol. 228, No. 4705, pp. 1315 - 1317. Sorocanu, L. et al. Use of chlorotoxin to target primary brain tumors. Cancer Res. 1998. Vol. 58, No. 21, pp. 4871 - 4879. Sorocanu, L. et al. Regulation of glioma cell migration and invasion using Cl(-) and K(+) ion channel blockers. J. Neurosci. 1999. Vol. 19, No. 14, pp. 5942 - 5954. Stroud, M. R. et al. In vivo bioimaging using chlorotoxin - based conjugates. Curr. Pharm. Des. 2011. Vol. 17, No. 38, pp. 4362 - 4371. Trevino, S. R. et al. Amino acid contributions to protein solubility: Asp, Glu, and Ser contribute more favorably than other hydrophilic amino acids in RNase Sa. J. Mol. Biol. 2007. Vol. 366, No. 2, pp. 449 - 460. Takacs, Z. et al. Designer ligands specific for the Kvl.3 channel from a scorpion neurotoxin - based library. Proc. Natl. Acad. Sci. U.S.A. 2009. Vol. 106, No. 52, pp. 22211 - 22216. Tonikian, R. et al. Identification of the specificity profile of peptide recognition modules from phage - displayed peptide libraries. Nat. Protoc. 2007. Vol. 2, No. 6, pp. 1368 - 1386. VEISEH, M. et al. Tumor paint: chlorotoxin: a Cy5.5 bioconjugate for intraoperative visualization of cancer foci. Cancer Res 2007. Vol. 67, No. 14, pp. 6882 - 6888. WIRANOWSKA, M. et al. Clathrin - mediated invasion and cellular localization of chlorotoxin in human gliomas. Cancer Cell Int. 2011. Vol. 11, No. 27, pp. 1 - 13. YOSEF, G. et al. 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Claims

1. General amino acid sequence X ０ X １ CMPCX Ｓ１ X Ｓ２ X Ｓ３ DHX Ｓ４ X Ｓ５ ARRCX ２ X ３ CCGGYGX ４ CFG YQCLCX ５ X ６ X ７ X ８ (SEQ ID NO: 43) A chlorotoxin derivative comprising: Where: (i) N-terminal X ０ X １ the cluster is selected from the group consisting of AM, 0M, or 00; (ii) Solubility X Ｓ１ X Ｓ２ X Ｓ３ X Ｓ４ X Ｓ５ The cluster is selected from the group consisting of FTTQT, FTES, SSSQT, SSSES, FSSQT, FSSES, or FSSQS; (iii) Internal X ２ X ３ X ４ The cluster is selected from the group consisting of DKR, RDK, KDR, IKY, HKW, DRK, LKQ, KKK; and (iv) C-terminal X ５ X ６ X ７ X ８ the cluster is selected from the group consisting of N000, R000, NR00, NRG0, NRGY, NRRR, or RRRR; where 0 indicates a position where no amino acid is present; A chlorotoxin derivative having a relative human MMP-2 binding affinity that is at least 1.62 times higher than the wild-type chlorotoxin of SEQ ID NO:

1.

2. General amino acid sequence X ０ X １ CMPCFTTDHQTARRCX ２ X ３ CCGGYGX ４ CFG YQCLCX ５ X ６ X ７ X ８ (SEQ ID NO: 35) Including, (i) the N-terminal X ０ X １ the cluster is selected from the group consisting of AM, 0M, or 00; (ii) the X ２ X ３ X ４ The cluster is selected from the group consisting of DKR, RDK, KDR, IKY, HKW, DRK, LKQ, KKK; and (iii) the C-terminal X ５ X ６ X ７ X ８ the cluster is selected from the group consisting of N000, R000, NR00, NRG0, or NRGY; The chlorotoxin derivative according to claim 1, wherein 0 indicates a position where no amino acid is present.

3. the internal X ２ X ３ X ４ The chlorotoxin derivative according to claim 1 or 2, wherein the cluster is DKR.

4. The chlorotoxin derivative according to claim 1, which is selected from chlorotoxin derivatives having any of the sequences set forth in SEQ ID NOs: 3 to 18, and SEQ ID NOs: 36 to 42.

5. The chlorotoxin derivative of claim 1, having the sequence set forth in SEQ ID NO: 4 or SEQ ID NO:

42.

6. The chlorotoxin derivative according to any one of claims 1 to 5, which is cyclic.

7. A conjugate comprising the chlorotoxin derivative of any one of claims 1 to 6 and a prosthetic group.

8. The prosthetic group may be selected from the following agents and moieties: (i) a visualization agent selected from a fluorescent label, a radioactive label, a magnetic resonance imaging label, and an agent that allows indirect labeling by high affinity binding to the target molecule; (ii) a therapeutic agent selected from a chemotherapeutic agent and a biological therapeutic agent; (iii) a targeting agent selected from an antibody, a polypeptide, a polysaccharide, and a nucleic acid; (iv) a moiety that increases the circulating half-life selected from a PEG moiety, a glycosyl moiety, a glycosyl-PEG moiety, and a linker peptide moiety that cyclizes the chlorotoxin derivative of any one of claims 1 to 6; The conjugate of claim 7, selected from:

9. 9. The conjugate of claim 7, comprising one or more linkers between the chlorotoxin derivative and the prosthetic group.

10. 10. The conjugate of claim 9, wherein the linker is selected from the group consisting of a peptide, a dimethyl disulfide linker, a glutaryl linker, and a cathepsin-cleavable linker.

11. A seragnostic pair of conjugates comprising a first conjugate and a second conjugate, wherein the first conjugate is the conjugate according to any one of claims 7 to 10, and the second conjugate is the conjugate according to any one of claims 7 to 10.

12. a) The chlorotoxin derivative moieties of the first conjugate and the second conjugate have the same amino acid sequence, and the two conjugates have different families of complementing molecules; or b) The chlorotoxin derivative moieties of the first conjugate and the second conjugate have different amino acid sequences, and the first conjugate and the second conjugate have the same or different families of complementing molecules. The seragnostic pair according to claim 11.

13. A kit comprising a conjugate according to any one of claims 7 to 10 and an instruction manual.

14. A kit comprising the seragnostic pair according to claim 11 or 12 and an instruction manual.

15. A pharmaceutical composition comprising a conjugate according to any one of claims 8 to 10, wherein the family of complementing molecules of the conjugate is a therapeutic agent or a visualization agent, and the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

16. A conjugate according to any one of claims 7 to 10 for use in the treatment of cancer.

17. A conjugate according to any one of claims 7 to 10 for use in the diagnosis of cancer.

18. A conjugate according to any one of claims 7 to 10, wherein the family of complementing molecules is a visualization agent and is for use in visualizing cancerous tissue.

19. The conjugate according to claim 16 or 17, wherein the cancer is selected from the group consisting of breast cancer, cervical cancer, colon cancer, epithelioma, Ewing's sarcoma, ganglioglioma, ganglioneuroma, glioma, glioblastoma, gliosarcoma, medulloblastoma, meningioma, neuroblastoma, melanoma (primary and metastatic), pancreatic cancer, pheochromocytoma, prostate cancer, schwannoma, and small cell lung carcinoma.

20. The conjugate according to claim 18, wherein the cancerous tissue is selected from the group consisting of breast cancer, cervical cancer, colon cancer, epithelioma, Ewing's sarcoma, glioma, ganglioma, glioblastoma, gliosarcoma, medulloblastoma, meningioma, neuroblastoma, melanoma (primary and metastatic), pancreatic cancer, pheochromocytoma, prostate cancer, schwannoma, and small cell lung carcinoma.

21. A method for preparing a conjugate according to any one of claims 7 to 10, comprising: a) providing a chlorotoxin derivative according to any one of claims 1 to 6; and c) directly binding a family of compensatory molecules to the chlorotoxin derivative of step a) or a) providing a chlorotoxin derivative according to any one of claims 1 to 6; b) binding a linker group to the chlorotoxin derivative of step a); and c) binding a family of compensatory molecules to the linker moiety of the compound formed in step b) A method.

22. A nucleic acid molecule encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: a) a chlorotoxin derivative according to any one of claims 1 to 6; c) a transmembrane domain; d) one or two co-stimulatory domains; e) a signaling domain and wherein the chlorotoxin derivative enables directing T cell activity towards cancer cells when the chimeric antigen receptor is expressed on the surface of T cells.

23. The nucleic acid molecule according to claim 22, wherein the chimeric antigen receptor further comprises b) a spacer region between the chlorotoxin derivative and the transmembrane domain.

24. The nucleic acid molecule according to claim 22 or 23, wherein the chlorotoxin derivative is selected from chlorotoxin derivatives having any of the sequences set forth in SEQ ID NO: 3 to SEQ ID NO: 18 and SEQ ID NO: 36 to SEQ ID NO:

42.

25. i) the transmembrane domain is selected from the group consisting of the CD4 transmembrane domain or a variant thereof, the CD8 transmembrane domain or a variant thereof, the CD28 transmembrane domain or a variant thereof, and the CD3 zeta transmembrane domain or a variant thereof; ii) the one or two co-stimulatory domains are selected from the group consisting of the CD28 co-stimulatory domain or a variant thereof, the 4-1BB co-stimulatory domain or a variant thereof, and the OX40 co-stimulatory domain or a variant thereof; and iii) the signal transduction domain is the CD3 zeta signal transduction domain or a variant thereof (wherein "variant" means that the protein has 1 to 5 amino acid modifications compared to the original protein, provided that the cysteine residues are not modified), the nucleic acid molecule according to any one of claims 22 to 24.

26. A vector comprising the nucleic acid molecule according to any one of claims 22 to 25.

27. A population of human cells, i) transfected with an RNA or DNA vector comprising an expression cassette comprising the nucleic acid according to any one of claims 22 to 25, said transfection being carried out in vivo or ex vivo, or ii) transduced with a viral vector comprising an expression cassette comprising the nucleic acid according to any one of claims 22 to 25, said transduction being carried out in vivo or ex vivo, said viral vector being a retroviral vector or a lentiviral vector; wherein the human cells are selected from the list consisting of autologous human T cells, autologous human CD4+ helper T cells, autologous human CD8+ cytotoxic T cells, any proportion mixture of autologous human CD4+ helper T cells and CD8+ cytotoxic T cells, allogeneic human T cells, allogeneic human CD4+ helper T cells, allogeneic human CD8+ cytotoxic T cells, any proportion mixture of allogeneic human CD4+ helper T cells and CD8+ cytotoxic T cells, autologous primary human natural killer (NK) cells, allogeneic primary human natural killer (NK) cells, allogeneic cells of the NK-92 cell line, autologous human monocytes, autologous human macrophages, allogeneic human monocytes, allogeneic human macrophages; provided that it is a population of human cells excluding the human itself obtained by genetic manipulation.

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