TRUNCATED CHLOROTOXIN FUSION PROTEINS AND METHODS OF USE THEREOF

Abstract

The present invention relates to a truncated form of chlorotoxin and chlorotoxin-like protein that are useful for glioma immunotherapy, in particular the treatment of glioblastoma. Thus, the invention also encompasses a therapeutic fusion protein comprising the truncated form of chlorotoxin and chlorotoxin-like protein, its methods of use, and its method of production.

Claims

1. A polypeptide of 13-18 amino acids residues comprising a sequence with at least 75% identity to SEQ ID NO:1.

2. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:1.

3. The polypeptide of claim 1, further comprising a therapeutic agent against glioma, wherein the therapeutic agent against glioma is linked to the polypeptide and the polypeptide binds to glioma cells.

4. The polypeptide of claim 3, wherein the therapeutic agent against glioma is linked to the C-terminus of the polypeptide.

5. A therapeutic fusion protein comprising: a truncated form of chlorotoxin being 13-18 amino acid residues comprising a sequence with at least 75% homology to SEQ ID NO:1; a V.sub.H domain of an antibody against CD3; and a V.sub.L domain of an antibody against CD3, wherein a first amino acid linker sequence links the C-terminus of the V.sub.H domain the antibody against CD3 to the N-terminus of V.sub.L domains of the antibody against CD3 and a second amino acid linker sequence links the C-terminus of the truncated form of chlorotoxin to the N-terminus of the V.sub.H domain of an antibody against CD3.

6. The therapeutic fusion protein of claim 5, wherein the truncated form of chlorotoxin comprises the sequence of SEQ ID NO:1.

7. The therapeutic fusion protein of claim 5, wherein the V.sub.H domain of the antibody against CD3 comprises the sequence set forth in SEQ ID NO:2.

8. The therapeutic fusion protein of claim 5, wherein the V.sub.L domain of the antibody against CD3 comprises the sequence set forth in SEQ ID NO:3.

9. The therapeutic fusion protein of claim 5, wherein the first amino acid linker and the second amino acid linkers consist of at least one glycine residue and at least one serine residue.

10. The therapeutic fusion protein of claim 5, wherein the amino acid sequence of the therapeutic fusion protein comprises the sequence set forth in SEQ ID NO:4 or SEQ ID NO:5.

11. A method of treating a subject having glioma, the method comprising: administering to the subject a glioma-targeting therapeutic fusion protein, wherein the glioma-targeting therapeutic fusion protein comprises: a truncated form of chlorotoxin being 13-18 amino acid residues comprising a sequence with at least 75% homology to SEQ ID NO:1, and an anti-glioma therapeutic agent, wherein the anti-glioma therapeutic agent is linked to the truncated form of chlorotoxin.

12. The method of claim 11, wherein the truncated form of chlorotoxin consists of the sequence set forth in SEQ ID NO:1.

13. The method of claim 11, wherein the anti-glioma therapeutic agent comprises a V.sub.H domain and a V.sub.L domain of an antibody against CD3.

14. The method of claim 13, wherein the amino acid sequence of the glioma-targeting therapeutic fusion protein comprises the sequence set forth in SEQ ID NO:4 or SEQ ID NO:5.

15. A method of producing a glioma-targeting therapeutic fusion protein, the method comprising: providing the polypeptide of claim 1; providing an anti-glioma therapeutic agent; and linking the polypeptide to the anti-glioma therapeutic agent to produce the glioma-targeting therapeutic fusion protein.

16. The method of claim 15, wherein the C-terminus of the polypeptide is linked to the anti-glioma therapeutic agent.

17. The method of claim 15, wherein the step of linking anti-glioma therapeutic agent to the polypeptide comprises linking the anti-glioma therapeutic agent and the polypeptide with an amino acid linker sequence.

18. The method of claim 15, wherein the anti-glioma therapeutic agent comprises a V.sub.H domain and a V.sub.L domain of an antibody against CD3.

19. The method of claim 15, wherein the amino acid sequence of the glioma-targeting therapeutic fusion protein comprises the sequence set forth in SEQ ID NO:4 or SEQ ID NO:5.

20. A plasmid for expressing the therapeutic fusion protein of claim 10, wherein the sequence of the plasmid comprises SEQ ID NO:6.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0018] FIGS. 1A and 1B depict, in accordance with certain embodiments, the design of the therapeutic fusion protein (FIG. 1A) and its purported interaction with glioblastoma cells (FIG. 1B).

[0019] FIGS. 2A-2C depict, in accordance with certain embodiments, the design and expression of ACDClxΔ15.

[0020] FIGS. 3A-3D depict, in accordance with certain embodiments, analysis of GBM binding via immunocytochemistry.

[0021] FIGS. 4A-4D depict, in accordance with certain embodiments, analysis of ACDClxΔ15-GMB binding via flow cytometry.

[0022] FIGS. 5A-5C depict, in accordance with certain embodiments, analysis of GBM-T-cell interactions via flow cytometry and FACS.

DETAILED DESCRIPTION OF THE INVENTION

[0023] Detailed aspects and applications of the invention are described below in the drawings and detailed description of the invention. Unless specifically noted, it is intended that the words and phrases in the specification and the claims be given their plain, ordinary, and accustomed meaning to those of ordinary skill in the applicable arts.

[0024] In the following description, and for the purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the various aspects of the invention. It will be understood, however, by those skilled in the relevant arts, that the present invention may be practiced without these specific details. It should be noted that there are many different and alternative configurations, devices, and technologies to which the disclosed inventions may be applied. The full scope of the inventions is not limited to the examples that are described below.

[0025] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a step” includes reference to one or more of such steps.

[0026] As used herein, the term “chlorotoxin-like peptide” refers to a peptide with at least 60% sequence identity to chlorotoxin (UniProt KB P45639.1) comprising at least three di-sulfide bridges. A list of representative chlorotoxin-like peptides may be found in Table 1, reproduced from Dardevet et al, Toxins (Basel). 2015, 7(4): 1079-1101, and in FIG. 4 of Ali et al., Toxins. 2016, 8(2):36. The contents of Dardevet et al., and Ali et al., are incorporated by reference herein.

TABLE-US-00001 TABLE 1 Primary sequence alignments of chlorotoxin-like protein. Alignments were performed by using @TOME V2. Percentage sequence of identity is given as compared to chlorotoxin by using @TOME V2. Disulfide Bridge pattern is given when known. Cysteine residues involved in disulfide bridges appear in blue in the table and are numbered in order of appearance. Disulfide bridge Toxin Primary sequence Length Identity pattern Species Chlorotoxin MC.sub.1MPC.sub.2FTTDHQMARKC.sub.3DDC.sub.4C.sub.5G- 36 AA 100% C.sub.1-C.sub.4, Leiurus GK-GRGKC.sub.6YGPQC.sub.7LC.sub.8-R C.sub.2-C.sub.6, quinquestriatus (SEQ ID NO. 7) C.sub.3-C.sub.7, quinquestriatus C.sub.5-C.sub.8 I.sub.1 MC.sub.1MPC.sub.2FTTRPDMAQQC.sub.3RAC.sub.4C.sub.5K- 36 AA  71% C.sub.1-C.sub.4, Buthus eupeus GR-GK--C.sub.6FGPQC.sub.7LC.sub.8GYD- C.sub.2-C.sub.6, (SEQ ID NO. 8) C.sub.3-C.sub.7, C.sub.5-C.sub.8 I.sub.3 MC.sub.1MPC.sub.2FTTDHQTARRC.sub.3RDC.sub.4C.sub.5G- 36 AA  82% C.sub.1-C.sub.4, Buthus eupeus GR-GR-KC.sub.6FG-QC.sub.7LC.sub.8GYD- C.sub.2-C.sub.6, (SEQ ID NO. 9) C.sub.3-C.sub.7, C.sub.5-C.sub.8 I.sub.4 MC.sub.1MPC.sub.2FTTDHNMAKKC.sub.3RDC.sub.4C.sub.5G- 35 AA  82% C.sub.1-C.sub.4, Buthus eupeus GN---GKC.sub.6FGPQC.sub.7LC.sub.8NR C.sub.2-C.sub.6, (SEQ ID NO. 10) C.sub.3-C.sub.7, C.sub.5-C.sub.8 I.sub.5 MC.sub.1MPC.sub.2FTTDPNMANKC.sub.3RDC.sub.4C.sub.5G- 35 AA  79% C.sub.1-C.sub.4, Buthus eupeus GG-KK--C.sub.6FGPQC.sub.7LC.sub.8NR- C.sub.2-C.sub.6, (SEQ ID NO. 11) C.sub.3-C.sub.7, C.sub.5-C.sub.8 I.sub.5A MC.sub.1MPC.sub.2FTTDPNMAKKC.sub.3RDC.sub.4C.sub.5G- 35 AA  79% C.sub.1-C.sub.4, Buthus eupeus GN-GK--C.sub.6FGPQC.sub.7LC.sub.8NR- C.sub.2-C.sub.6, (SEQ ID NO. 12) C.sub.3-C.sub.7, C.sub.5-C.sub.8 Bs-8 RC.sub.1KPC.sub.2FTTDPQMSKKC.sub.3ADC.sub.4C.sub.5G-GK- 35 AA  80% C.sub.1-C.sub.4, Buthus GKGKC.sub.6YGPQC.sub.7LC.sub.8---- C.sub.2-C.sub.6, sindicus (SEQ ID NO. 13) C.sub.3-C.sub.7, C.sub.5-C.sub.8 Lqh-8/6 RC.sub.1SPC.sub.2FTTDQQMTKKC.sub.3YDC.sub.4C.sub.5G-GK- 38 AA  72% C.sub.1-C.sub.4, Leiurus GKGKC.sub.6YGPQC.sub.7IC.sub.8APY- C.sub.2-C.sub.6, quinquestriatus (SEQ ID NO. 14) C.sub.3-C.sub.7, hebraeus C.sub.5-C.sub.8 PBITx1 RC.sub.1KPC.sub.2FTTDPQMSKKC.sub.3ADX.sub.4C.sub.5G-GX-- 25 AA  64% Parabuthus KX schlechteri (SEQ ID NO. 15) Bs-14 -C.sub.1GPC.sub.2FTKDPETEKKC.sub.3ATC.sub.4C.sub.5G-GI- 36 AA  61% C.sub.1-C.sub.4, Buthus GR--C.sub.6FGPQC.sub.7LC.sub.8NRGY C.sub.2-C.sub.6, sindicus (SEQ ID NO. 16) C.sub.3-C.sub.7, C.sub.5-C.sub.8 Neurotoxin P2 -C.sub.1GPC.sub.2FTTDPYTESKC.sub.3ATC.sub.4C.sub.5G-GR- 35 AA  70% C.sub.1-C.sub.4, Androctonus GK--C.sub.6VGPQC.sub.7LC.sub.8NRI- C.sub.2-C.sub.6, mauretanicus (SEQ ID NO. 17) C.sub.3-C.sub.7, mauretanicus C.sub.5-C.sub.8 AaCtx MC.sub.1IPC.sub.2FTTNPNMAAKC.sub.3NAC.sub.4C.sub.5G- 34 AA  61% C.sub.1-C.sub.4, Androctonus SRRGS--C.sub.6RGPQC.sub.7IC.sub.8---- C.sub.2-C.sub.6, australis (SEQ ID NO. 18) C.sub.3-C.sub.7, C.sub.5-C.sub.8 GaTx1 -C.sub.1GPC.sub.2FTTDHQMEQKC.sub.3AEC.sub.4C.sub.5G-GI- 34 AA  79% C.sub.1-C.sub.4, Leiurus GK--C.sub.6YGPQC.sub.7LC.sub.8NR---- C.sub.2-C.sub.6, quinquestriatus (SEQ ID NO. 19) C.sub.3-C.sub.7, hebraeus C.sub.5-C.sub.8 BmKCT -C.sub.1GPC.sub.2FTTDANMARKC.sub.3REC.sub.4C.sub.5G-GI- 35 AA  76% C.sub.1-C.sub.4, Buthus GK--C.sub.6FGPQC.sub.7LC.sub.8NRI- C.sub.2-C.sub.6, martensii (SEQ ID NO. 20) C.sub.3-C.sub.7, C.sub.5-C.sub.8 Bm12-b -C.sub.1GPC.sub.2FTTDANMARKC.sub.3REC.sub.4C.sub.5G-GN- 35 AA  76% C.sub.1-C.sub.4, Buthus GK--C.sub.6FGPQC.sub.7LC.sub.8NRE- C.sub.2-C.sub.6, martensii (SEQ ID NO. 21) C.sub.3-C.sub.7, C.sub.5-C.sub.8 Lepidopteran RC.sub.1GPC.sub.2FTTDPQTQAKC.sub.3SEC.sub.4C.sub.5G-RK- 37 AA  63% C.sub.1-C.sub.4, Buthus tamulus GG-VC.sub.6KGPQC.sub.7IC.sub.8GIQ- C.sub.2-C.sub.6, (SEQ ID NO. 22) C.sub.3-C.sub.7, C.sub.5-C.sub.8 BtITx3 RC.sub.1PPC.sub.2FTTNPNMEADC.sub.3RKC.sub.4C.sub.5G-GR-- 35 AA  53% C.sub.1-C.sub.4, Buthus tamulus GY-C.sub.6ASYQC.sub.7IC.sub.8PG---- C.sub.2-C.sub.6, (SEQ ID NO. 23) C.sub.3-C.sub.7, C.sub.5-C.sub.8 GaTx2 --VSC.sub.1--------EDC.sub.2PDHC.sub.3STQK- 29 AA  38% C.sub.1-C.sub.4, Leiurus ARAKC.sub.4DNDKC.sub.5VC.sub.6-EPI C.sub.2-C.sub.5, quinquestriatus (SEQ ID NO. 24) C.sub.3-C.sub.6 hebraeus

[0027] Immunotherapies such as the bispecific T cell engager represent the most promising of new, experimental treatment strategies for glioblastoma in part due to their unique ability to stimulate T cell responses against cancer. Although capable of surpassing many of the hurdles standing in the way of effective GBM treatment, these immunotherapies have been largely ineffective due to the lack of a universal target for GBM. Chlorotoxin, a scorpion-derived peptide, has been shown to overcome this through its unique ability to bind human glioma cells without off-target toxicity to other tissues.

[0028] Disclosed herein is a truncated form of chlorotoxin and chlorotoxin-like protein that are useful for glioma immunotherapy, in particular the treatment of glioblastoma. Nearly 20 years ago, researchers studying chlorotoxin, a 36-amino acid peptide found in the venom of the deathstalker scorpion (L. quinquestriatus), reported its unique ability to selectively bind glioma cells but not healthy tissue. This affinity for glioma increases in proportion to the grade of the tumor, with chlorotoxin binding 100% of grade IV glioma (GBM) cells (31/31 samples positive). Importantly, chlorotoxin also displayed high selectivity for GBM, as it was found to not bind samples of various healthy human tissues. Together, these qualities make the peptide an interesting candidate for use in GBM treatment.

[0029] Owing to its selectivity for glioma cells and lack of toxicity to humans, chlorotoxin has found relevance as a supplementary tool within the current standard of care. In the clinical setting, a modified chlorotoxin molecule attached to a Cy5.5 infrared dye, coined Tumor Paint, has been utilized to enhance visualization of the tumor during surgery in mice and is awaiting clinical trials in humans. Despite intensive research and promising clinical applications, chlorotoxin's binding target on glioma cells remains unknown. Multiple potential targets have been reported, including matrix metalloproteinase 2 (MMP-2), annexin A2, Nrp1, and glioma-specific chloride channels, yet none have demonstrated evidence of direct interaction with chlorotoxin. Regardless, it is clear that chlorotoxin possesses incredible therapeutic potential as a targeting molecule for GBM.

[0030] Eight of chlorotoxin's 36 amino acids are cysteine residues, which form four disulfide bonds (DSBs) in a highly compact inhibitor-cysteine knot (ISK) motif. These DSBs posed problems during the expression of ACDClx, a chlorotoxin-conjugated immunotherapeutic protein and the subject of U.S. Pat. No. 10,745,478, in both Nicotiana benthamiana and Escherichia coli, as in both systems DSB formation outside of its natural context resulted in largely insoluble, aggregated protein. In E. coli, DSB formation during recombinant expression has been previously achieved by selectively expressing the gene of interest within the periplasm; however, periplasm targeted ACDClx remained insoluble in solution. Recently, researchers studying chlorotoxin as part of a peptide-drug conjugate (PDC) reported that chlorotoxin was metabolized in the GBM tumor microenvironment to form peptide fragments that retained their GBM-binding capabilities. Interestingly, many of these fragments all retained a C-terminal arginine residue thought to be responsible for GBM binding.

[0031] For the first time, a truncated form of chlorotoxin comprising 13-18 amino acid residues was found to be sufficient for specific binding to glioma cells, specifically glioblastoma cells. This truncated form of chlorotoxin was selected for minimizing disulfide bonds and thus only comprises the first three cysteines of a chlorotoxin or chlorotoxin-like protein, as the first disulfide bond in chlorotoxin and chlorotoxin-like proteins is formed between the first and fourth cysteine residues. Thus, the polypeptide with specific binding to glioma cells has 13-18 contiguous residues of the amino acid sequence of chlorotoxin and chlorotoxin-like protein. In some aspects, the truncated form comprises the first 13-18 contiguous amino acid residues of chlorotoxin or chlorotoxin-like protein. This deletion reduces the number of cysteine residues present within the peptide from eight to two, thereby allowing for the formation of only one possible DSB within the truncated chlorotoxin molecule. In particular embodiments, the polypeptide with specific binding to glioma cells comprises a sequence with at least 75%, at least 80%, or at least 90% sequence identity to SEQ ID NO:1. In some aspects, such a polypeptide comprises no more than three cysteine residues. Thus, in particular embodiments, the polypeptide of 13-18 amino acid residues comprises one cysteine residue, two cysteine residues, or three cysteine residues.

[0032] Accordingly, also described herein is a method of targeting a therapeutic agent to glioma cells. The method comprises linking the therapeutic agent to the above-described polypeptide with specific binding to glioma cells to produce a fusion protein; and administering the fusion protein to a subject, wherein the subject has glioma cells. In some embodiments, the polypeptide with specific binding to glioma cells has an amino acid sequence comprises a sequence with at least 75%, at least 80%, or at least 90% sequence identity to SEQ ID NO:1. The amino acid sequence of the polypeptide preferably comprises less than three cysteine residues. In particular embodiments, the polypeptide with specific binding to glioma cells has an amino acid sequence consisting of SEQ ID NO:1 (also referred to herein as “CltxΔ15”). In some aspects, the C-terminus of the polypeptide with specific binding to glioma cells is linked to the therapeutic agent, for example with an amino acid linker. In certain embodiments, the amino acid linker consists of at least one glycine residue and at least one serine residue. In particular implementations, the method targets the therapeutic agent to glioblastoma cells.

[0033] Also described herein is a therapeutic fusion protein comprising the polypeptide with specific binding to glioma cells and a V.sub.H domain and a V.sub.L domain of an antibody against CD3. This therapeutic fusion protein is designed based on the bispecific T cell engager (BiTE™) model, which is a method originally proposed in 1985 to promote T cell-mediated destruction of target cells. BiTE proteins comprise two single-chain variable fragments (scFvs) of antibodies which are tethered together by a short, inert linker to form a fusion protein. The therapeutic fusion protein described herein comprises the polypeptide with specific binding to glioma cells in place of one of the scFvs, while the other scFvs comprise the V H domain and the V L domain of an antibody against CD3, for example, an antibody against CD3c, where binding leads to structural changes and eventual signal transduction to induce sustained activation of T cells when the target cell is also bound by its complementary antibody. Accordingly, the therapeutic fusion protein further comprises a first amino acid linker sequence that links the C-terminus of the V H domain the antibody against CD3 to the N-terminus of V L domains of the antibody against CD3 and a second amino acid linker sequence that links the C-terminus of the polypeptide with specific binding to glioma cells to the N-terminus of the V.sub.H domain of an antibody against CD3. This method of inducing T cell activation is notable in that it does not require recognition by the major histocompatibility complex (MHC), allowing for T cell-mediated elimination of target cells regardless of TCR specificity. Additionally, it overcomes the problem of MHC-downregulation by cells such as GBM which are known to do so in order to evade the immune response.

[0034] In some aspects, the V.sub.H domain of the antibody against CD3 comprises the sequence set forth in SEQ ID NO:2 or a sequence having at least 50% identity, at least 60% identity, at least 70% identity, at least 80% identity, at least 90% identity, or at least 95% identity to SEQ ID NO:2. In some aspects, the V H domain of the antibody against CD3 comprises the sequence set forth in SEQ ID NO:3 or a sequence having at least 50% identity, at least 60% identity, at least 70% identity, at least 80% identity, at least 90% identity, or at least 95% identity to SEQ ID NO:3. In some embodiments, the first amino acid linker and the second amino acid linkers consist of at least one glycine residue and at least one serine residue. In a particular embodiment, the amino acid sequence of the therapeutic fusion protein comprises the sequence set forth in SEQ ID NO:4. In a certain embodiment, the amino acid sequence of the therapeutic fusion protein is the sequence set forth in SEQ ID NO:5 (also referred to herein as “ACDClxΔ15”).

[0035] As demonstrated in the Examples, the truncated form of chlorotoxin allow for more efficient production of ACDClxΔ15 while still retaining its ability to bind GBM cells. When GBM cells and NIH-3T3 mouse embryonic fibroblast cells were incubated with ACDClxΔ15, which was then probed for its N-terminal histidine-tag with fluorescent antibodies. Confocal microscopy of these samples revealed that ACDClxΔ15 did bind GBM cells but did not bind healthy fibroblast cells (FIGS. 3A and 3C). These results were verified using flow cytometry to measure the number of cells that were positive for ACDClxΔ15. As expected, ACDClxΔ15 further demonstrated its ability to bind GBM cells in vitro, with high levels of fluorescence detected for anti-6xHis-PE among cells incubated with ACDClxΔ15 when compared to control samples (FIGS. 4A and 4B). These results also show that CltxΔ15 is capable of selectively binding GBM cells without off-target toxicity when part of a BiTE-like fusion protein The scFVs from an antibody against CD3c was also shown to bind to T cells. Thus, the therapeutic fusion protein described herein is able to bring CD8+ T cells to glioblastoma cells as shown in FIG. 1B. Accordingly, a method of treating as subject having glioma and a method of producing a glioma-targeting therapeutic fusion protein are also described.

[0036] In some aspects, the method of treating a subject having glioma comprises administering to the subject a glioma-targeting therapeutic fusion protein, wherein the glioma-targeting therapeutic fusion protein comprises a truncated form of chlorotoxin that comprises a sequence with at least 75% homology to SEQ ID NO:1 linked to an anti-glioma therapeutic agent. The truncated form of chlorotoxin that may be linked to an anti-glioma therapeutic agent via an amino acid linker, for example, an amino acid linker consisting of at least one glycine residue and at least one serine residue. In some aspects, the truncated form of chlorotoxin is 13-18 amino acid residues in length. In particular implementations, the truncated form of chlorotoxin consists of the sequence set forth in SEQ ID NO:1. In some aspects, the anti-glioma therapeutic agent comprises a V.sub.H domain and a V.sub.L domain of an antibody against CD3. In particular implementations, the V.sub.H domain of the antibody against CD3 comprises the sequence set forth in SEQ ID NO. 2 and the V.sub.L domain of the antibody against CD3 comprises the sequence set forth in SEQ ID NO. 3. In such embodiments, a first amino acid linker sequence links the C-terminus of the V.sub.H domain the antibody against CD3 to the N-terminus of V.sub.L domains of the antibody against CD3 and a second amino acid linker sequence links the C-terminus of the truncated form of chlorotoxin to the N-terminus of the V.sub.H domain of an antibody against CD3. In certain embodiments, the method of treating a subject having glioma comprises administering to the subject a glioma-targeting therapeutic fusion protein having the sequence set forth in SEQ ID NO:4 or SEQ ID NO:5.

[0037] The method of producing a glioma-targeting therapeutic fusion protein comprises providing a polypeptide of 13-18 amino acids residues comprising a sequence with at least 75% identity to SEQ ID NO:1; providing an anti-glioma therapeutic agent; and linking the polypeptide to the anti-glioma therapeutic agent to produce the glioma-targeting therapeutic fusion protein. In some embodiments, the C-terminus of the polypeptide is linked to the anti-glioma therapeutic agent. In certain implementations, the step of linking anti-glioma therapeutic agent to the polypeptide comprises linking the anti-glioma therapeutic agent and the polypeptide with an amino acid linker sequence. In some aspects, the amino acid linker sequence consists of at least one glycine residue and at least one serine residue. In certain embodiments, the anti-glioma therapeutic agent comprises a V.sub.H domain and a V.sub.L domain of an antibody against CD3. For example, the amino acid sequence of the glioma-targeting therapeutic fusion protein comprises the sequence set forth in SEQ ID NO:4 or SEQ ID NO:5.

[0038] An expression plasmid for expressing the therapeutic peptide or the glioma-targeting therapeutic fusion protein is also described. The nucleotide sequence of the expression plasmid is set forth in SEQ ID NO:6. In some embodiments, the nucleotide sequence of the expression plasmid is a sequence having at least 70% identity, at least 80% identity, at least 90% identity, or at least 95% identity to SEQ ID NO:6.

Examples

[0039] The present invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures, are incorporated herein by reference in their entirety for all purposes.

I. Design and Production of ACDClxΔ15

[0040] The amino acid sequence of ACDClxΔ15 was composed from previously published sequences of the VH and VL scFvs of the mouse anti-CD3 ε mAb 2C11 and a truncated chlorotoxin molecule which was obtained through deletion of 22 amino acids from the C-terminus of full-length chlorotoxin. These sequences were joined by a (Gly.sub.4 Ser).sub.3 linker and an 8x histidine tag (His-tag) was included at the N-terminus.

[0041] In order to produce ACDClxΔ15, the gene encoding its components was cloned into a plasmid capable of transforming electrocompetent E. coli cells (FIG. 2A). The gene encoding ACDClxΔ15 was cloned into an ampicillin resistant pET-11 a plasmid, and the resulting vector (pTM1031, nucleotide sequence set forth in SEQ ID NO:6) was used to transform electrocompetent BL21 (DE3) E. coli cells by electroporation.

[0042] Transformed cells recovered in SOC medium (CSH Protocols, 2006) for 1 hr at 37° C. before growing overnight on agar plates treated with ampicillin. Antibiotic selection and PCR screening confirmed cell colonies positive for pTM1031; positive colonies were then grown in 1 L cultures and induced with 0.3 mM IPTG after 4 hrs of shaking. Cultures were then centrifuged at 6000×g and 4° C. for 20 min (Beckman JA-17 rotor #369691). Pelleted cells were resuspended in extraction buffer (150 mM NaCl, 50 mM Tris pH 8, and 2 mM EDTA in PBS) and freeze-thawed twice to lyse cells. Resuspended cells were vortexed and incubated at 30° C. on a shaker before adding 0.5% PMSF and 20 mg lysozyme. In 45 min increments, the following were added: 0.5% Triton X-100, followed by 0.18% DNAse I and 1 M MgSO4. Samples receiving s-sulfonation to prevent DSB formation were denatured and s-sulfonated overnight at 4° C. in buffer containing 6 M guanidine, 200 mM sodium sulfite, 150 mM sodium tetrathionate dihydrate, 50 mM Tris pH 8, and 150 mM NaCl.

[0043] All samples were purified via metal-affinity chromatography by incubating overnight at 4° C. with Roche cOmplete™ nickel affinity resin. Purified protein was eluted through the addition of 1 M imidazole to the column. Concentration of the resulting eluent was measured on an ND-1000 NanoDrop spectrophotometer. S-sulfonated samples were then refolded by diluting 1:100 v/v overnight at 4° C. in oxidizing buffer (0.5 M arginine, 2 mM oxidized glutathione, 100 mM Tris base, and 2 mM EDTA in PBS). Samples were re-concentrated by centrifugation in Millipore Amicon Ultra-15 centrifugal filter tubes with a 3.5 kDa MW cutoff (GE Healthcare, Pittsburgh, PA) for 15 min at 4° C. in a Beckman-Coulter JS-5.3 swinging-bucket rotor before being exchanged into PBS on a Sephadex G-25 desalting column. Mock-purified protein refers to samples collected from BL21 (DE3) E. coli cells that were not transformed with the pTM1031 plasmid. These samples were treated identically to those produced from transformed cells as described above.

II. SDS-PAGE and Immunoblotting

[0044] Following induction with IPTG, cells were harvested and ACDClxΔ15 was present in insoluble inclusion bodies within the total cell lysate of transformed cells but not in untransformed cells, as detected by Western blot (FIG. 2B).

[0045] To verify the presence and purity of ACDClxΔ15 following purification, samples were mixed with 6x Laemmli buffer (4% SDS, 20% glycerol, 0.004% bromophenol blue, 0.125 M Tris-HCl, and 10% DTT in ddH.sub.2O), boiled for 5 min at 100° C. and briefly centrifuged before loading onto 4-20% Mini-PROTEAN TGX stain-free polyacrylamide gels (Bio-Rad) alongside Bio-Rad Precision Plus dual-color protein standards. Gels were run at 150 V for 40 min, or until the sample buffer reached the bottom of the gel.

[0046] Following SDS-PAGE, gels were imaged for total protein via UV transillumination before being transferred to nitrocellulose membranes on a Trans-Blot® SD semi-dry transfer cell at 15 V for 15 min. Membranes were blocked in PB ST+2.5% non-fat milk (PBST-M) solution for 1 hr at RT and washed with PBST for 15 min before staining with mouse anti-His primary Ab (Sigma H1029) for 30 min. Membranes were again washed with PBST for 15 mins before donkey anti-mouse HRP secondary Ab (Jackson ImmunoResearch #715-305-150) for 30 min. Following a final 15 min wash in PBST, membranes were incubated with HRP substrate for 1 min and exposed onto X-ray film for 30 sec in the dark.

[0047] Purification and subsequent concentration resulted in a 24% yield of ACDClxΔ15 (FIG. 2C). Any proteins present in untransformed samples (mock-purified protein) were not detected by anti-His antibody and therefore were not expected to be capable of binding nickel resin for purification.

III. Immunocytochemistry

[0048] For the first test of CltxΔ15's ability to bind GBM cells as part of a BiTE-like molecule, ACDClxΔ15 was incubated with GL261-LucNeo mouse glioblastoma cells and stained with antibodies which fluoresce when excited with lasers at a wavelength of 488 nm. GL261-LucNeo mouse glioblastoma cells and NIH-3T3 mouse embryonic fibroblasts (ATCC), generously provided by Dr. Adrienne C. Scheck at Barrow Neurological Institute and Dr. Debra P. Baluch at Arizona State University, respectively, were grown in culture media (DMEM+10% FBS/F12+1% PSG) at 37° C. to approximately 70% confluency. Cells were passaged and split 1:6 before seeding on four coverslips per cell line in separate wells of two 6-well plates. Cells were grown to approximately 90% confluency on coverslips before fixing in 1 mL 2% paraformaldehyde. Coverslips were washed with 1 mL PBS+5% nonfat milk (PBS-M) before adding 1 mL of 1 μM ACDClxΔ15 in PBS for 1 hr on the rocker at 4° C. Cells were washed with 1 mL PBS-M 3x for 5 min each on a rocker at RT. Mouse anti-6xHis primary antibody was added to all coverslips except secondary control at a 1:500 dilution in 1 mL PBS-M and incubated on rocker at 4° C. overnight. Primary antibody was removed before adding 1 mL of secondary antibody (donkey anti-mouse IgG H&L Alexa Fluor® 488, Abcam ab150109) and incubating in the dark at 4° C. on a rocker for 1 hr. Secondary antibody was removed and coverslips were washed with PBS-M 3x for 5 min each on rocker. Antibody wash was removed, and cells were incubated with DAPI nuclear stain for 30 min before mounting and sealing coverslips on microscope slides. Slides were imaged on a Leica SP8 White Light Laser Confocal microscope using a 488 nm wavelength laser.

[0049] When incubated with ACDClxΔ15 and visualized with fluorescent confocal microscopy, GBM cells displayed bright green fluorescence compared to mouse embryonic fibroblast cells (NIH-3T3) also incubated with ACDClxΔ15 (FIGS. 3A and 3B). Control samples receiving either mock protein, primary and secondary Ab only, or ACDClxΔ15 and secondary Ab alone displayed negligible levels of fluorescence (FIGS. 3C and 3D). As a quantitative method to measure ACDClxΔ15-GBM binding, GL261-LucNeo cells were incubated with PBS, mock protein, anti-6xHis-PE, or ACDClxΔ15 and analyzed using flow cytometry. A distinct peak was observed for samples treated with ACDClxΔ15 but not those receiving controls (FIG. 4A). Dot plots of all events recorded per sample showed similar results, with clear increases in cells positive for anti-6xHis-PE observed compared to controls (FIG. 4B). After gating for positive cells based on these negative controls, the percent of positive cells was calculated and found to be higher in samples treated with ACDClxΔ15, with 35.5% of cells displaying fluorescence compared to 0.92%, 0.59%, and 0.59% for samples treated with PBS, anti-6xHis, and mock protein respectively (FIG. 4C). Median fluorescent intensity of positive events was likewise higher in ACDClxΔ15 samples than in controls (FIG. 4D). Together, these results indicate that ACDClxΔ15 is capable of binding murine GBM cells, but not other mouse tissue types, in vitro.

IV. Analyzing GBM Binding Via Flow Cytometry

[0050] After testing its ability to bind mouse GBM cells specifically via fluorescence microscopy and flow cytometry, ACDClxΔ15 was then co-incubated with GBM cells and splenocytes to determine whether or not it was capable of both binding and activating T cells in the presence of GBM. To achieve this, flow cytometry was again utilized to measure fluorescent signals of co-incubated GBM and T cells treated with specific antibodies that would detect the presence of important markers of T cell binding and activation. Specifically, T cells were stained for the presence of CD4 and CD8 and incubated with GBM cells stained for beta tubulin and binding events were determined by the presence of GBM and either CD4 or CD8 probes in single recorded events.

[0051] GL261-LucNeo cells were grown in culture media (DMEM+10% FBS+1% pen/strep) to approximately 90% confluency. Cells were harvested and washed with FACS buffer (1% bovine serum albumin and 0.1% sodium azide in PBS) and aliquoted into a 96-well round bottom plate (approx. 1.0×10.sup.6 cells/well). Cells were washed 2× with FACS buffer by centrifugation for 5 min at 1200 rpm on a Thermoscientific Bioliner rotor (#75003667) before being incubated with either 5 μM ACDClxΔ15, mock-purified protein, or PBS for 30 min at 4° C. Anti-6xHis-PE antibody was added to all samples except one PBS-only control at a 1:200 dilution in FACS buffer and incubated for 30 min at 4° C. Samples were again washed as before and resuspended in 1 mL FACS buffer before running on a BD LSRFortessa™ flow cytometer. Results were analyzed using FlowJo™ v10.8 software (BD Life Sciences).

[0052] Samples of GBM and T cells co-incubated with or without ACDClxΔ15 displayed prominent peaks when probed with anti-beta tubulin, anti-CD4, and anti-CD8, which act as markers for these cells (FIG. 5A). The sample which received ACDClxΔ15 showed a small signal peak for its corresponding anti-His detection while all other samples displayed negligible fluorescence.

V. Analyzing GBM-T Cell Interactions Via Flow Cytometry and FACS

[0053] Following flow cytometry, cells were sorted using FACS to isolate events that contained GBM cells, CD8+T cells, and ACDClxΔ15.

[0054] GL261-LucNeo cells were grown and prepared for flow cytometry as described in Example IV and aliquoted into 15 mL Falcon™ tubes. Spleens were harvested from wild-type mice and splenocytes were isolated by straining spleens into RPMI media. Red blood cells were lysed by centrifugation for 5 min at 1200 rpm and 4° C. before incubating in 1 mL Ack Lysis buffer for 2 min at RT. Cells were quenched with RPMI and centrifuged as above before resuspending in 8 mL final volume of RPMI on ice.

[0055] Splenocytes were counted and approximately 1.0×10.sup.6 cells were aliquoted into separate Falcon™ tubes and incubated with their respective treatments. Antibodies used in this experiment are listed in Table 2, and 1 mL ACDClxΔ15 was added to samples at 1 μM concentration. All incubations were performed for 30 min at 4° C. in the dark except for anti-CD69, which was incubated for 2 hrs. Two washes were performed between each incubation by centrifuging samples at 1200 rpm for 5 min at 4° C. and resuspending cells in 2 mL FACS buffer. Following the final wash, samples were resuspended in 1 mL FACS buffer and collected on a ThermoFischer Attune NxT flow cytometer before analyzing as described in Example IV.

TABLE-US-00002 TABLE 2 Fluorescent antibodies used for flow cytometry and FACS experiments. Antibody Fluorophore Catalog # anti-6xHis APC 362605 (BioLegend) anti-beta tubulin Alexa Fluor@488 A4-251-C100 (EXBIO) anti-CD4 V450 FP20224002 (MBL Internatioi anti-CD8a Brilliant Violet 711 100747 (BioLegend) anti-CD69 PE-Damie 594 104535 (BioLegend)

[0056] Samples obtained were run on a BD FACSAria™ IIu cell sorter in order to identify samples positive for both GBM and T cells. Samples in which GBM and splenocytes were co-incubated with ACDClxΔ15 and all five antibodies were sorted for events that were positive for anti-6xHis and either anti-CD4 or anti-CD8 to indicate GBM-T cell binding events. Resulting samples were plated onto coverslips and fixed before imaging on a Lecia SP8 confocal microscope.

[0057] These samples were imaged using confocal microscopy in order to visualize binding events between GBM and T cells (FIG. 5C). Fluorescence was detected for all three probed antibodies, and a CD8+ cell was observed in close proximity to a cell positive for anti-beta tubulin, the marker used here to identify GBM cells. However, isolated scans show that this anti-beta tubulin signal was also present on the CD8+ cell.

VI. Analysis of T Cell Activation by ACDClxΔ15

[0058] Calcium flux is an indicator of initial T cell activation. Calcium flux (as indicated by an increase in Fluo-4 AM fluorescence intensity using FITC excitation/emission filter) are measured immediately after mixing GL261-LucNeo mouse glioma cells with freshly isolated mouse splenocytes with (1) nothing added, (2) ACDClxΔ15 protein buffer (no ACDClxΔ15), (3) ACDClxΔ15 added, or (4) anti-CD3 antibody which acts as a positive control for T cell activation. Four equivalent samples are measured consecutively: Splenocytes+GL261 cells (nothing added); Splenocytes+GL261+protein buffer; Splenocytes+GL261+ACDClxΔ15, and Splenocytes+GL261+anti-CD3 antibody.

[0059] CD4+ and CD8+T cell activation are measured by CD69 expression 36 hours post-treatment. The treatment comprises: Media (negative control), 1 μM ACDClxΔ15, or anti-CD3 antibody (positive control). Treatments are incubated with freshly isolated mouse splenocytes (B6 background) and confluent GL261-LucNeo mouse GBM cells.