Tandem repeat cancer targeting peptides for molecular ligation or engineering and their use in cancer theranostics

Abstract

An isolated cancer-targeting peptide comprising at least two copies of the amino acid sequence PFLP (SEQ ID NO: 1) or PELF (SEQ ID NO: 2). A pharmaceutical composition for treating cancer is also disclosed. The composition comprises the isolated cancer-targeting peptide and an anti-cancer agent. A bispecific anti-cancer antibody comprising the isolated cancer-targeting peptide and an antigen-binding peptide that stimulates T cell activity is also disclosed. A method for treating cancer by administering a pharmaceutical composition or a bispecific anti-cancer antibody is provided. A method for diagnosing cancer by administering a radionuclide-labeled cancer-targeting peptide to an individual and imaging the location of the radionuclide is also provided.

Description

[Background technology]

[0001] Cancer recurrence is a major clinical challenge. Cancer stem cells, which exist as a subpopulation in tumors, are particularly resistant to chemotherapy and radiation. See Lee et al., 2015, FASEB J.29:Supplement 629.18. After conventional chemotherapy, an elevated proportion of cancer stem cells in tumors is an important predictor of cancer recurrence. See Lee et al.

[0002] The 78 kDa glucose-regulated protein (GRP78), a member of the HSP70 protein family, has been reported to be present on the surface of various cancer cells but not on normal cells. See Wang et al., 2016, Biomaterials 94:31-44 and Liu et al., 2013, Clin. Cancer Res. 19:6802-11. GRP78 has also been implicated in both cancer cell drug resistance and stem-like cell behavior and has further been shown to be a targetable cell surface receptor. See Bachelder, 2018. Therefore, GRP78 is an attractive target for anticancer therapies that should reduce damage to normal cells and reduce recurrence.

[0003] Cancer targeting peptides (CTPs) that specifically bind to GRP78 have previously been identified. See Wang et al. CTPs were found to interact with the peptide-binding domain (PBD) of GRP78 in a linear peptide structure. These CTPs, when conjugated to the chemotherapeutic agent doxorubicin, were shown to enhance the antitumor efficacy of the agent and target both cancer cells and stem cells, thereby reducing the rate of cancer recurrence. See Liu et al.

[0004] There is a need to develop CTPs with higher affinity for cancer stem cells and CTP-based anticancer therapies. Summary of the Invention

[0005] To meet this need, an isolated cancer targeting peptide is disclosed, which comprises at least two copies of the amino acid sequence PFLP (SEQ ID NO: 1) or PFLF (SEQ ID NO: 2).

[0006] Also disclosed herein is a pharmaceutical composition for treating cancer, the composition comprising an isolated cancer targeting peptide and an anti-cancer agent.

[0007] Additionally, bispecific anti-cancer antibodies are disclosed, which comprise an isolated cancer-targeting peptide and an antigen-binding peptide that stimulates T cell activity.

[0008] Additionally, methods of treating cancer by administering pharmaceutical compositions or bispecific anti-cancer antibodies are both within the scope of the present invention.

[0009] Another method for diagnosing cancer is disclosed, which method is performed by administering to an individual a radionuclide-labeled cancer targeting peptide comprising at least two copies of the amino acid sequence PFLP (SEQ ID NO: 1) or PFLF (SEQ ID NO: 2), and subjecting the individual to an imaging technique to determine the location and amount of the radionuclide-labeled cancer targeting peptide.

[0010] The details of several embodiments of the invention are set forth both in the description that follows and in the drawings. All features, objects, and advantages of the invention will become apparent from the description and drawings, as well as the appended claims. The following description refers to the accompanying drawings. [Brief explanation of the drawings]

[0011] [Figure 1] 1 is a bar graph showing the percent injected dose per gram (%ID / g) of each of the four indicated radiolabeled peptides as determined by positron emission tomography (PET) analysis after injection into tumor-bearing mice. The peptide sequences are shown in Table 1 below. [Figure 2]1 is a bar graph showing the relative signal ratios of tumor tissue sections from tumor-bearing mice injected with radiolabeled peptides P6, F4P6, P13, or F4P13. [Figure 3] Figure 1. Diagram of bispecific anti-cancer antibody constructs of the invention. α-CD3 = anti-CD3 scFv, Fc(K) = heavy chain with knob dimerization sequence, Fc(H) = heavy chain with hole dimerization sequence, F4P6-TR-CTP = F4P6 tandem repeat cancer targeting peptide that binds to GRP-78. [Figure 4A] 1 is a bar graph showing percent lysis (% Lysis) of TOV21G ovarian cancer cells after 40 hours of incubation with PBMC effector cells in the absence (PBMC) or presence (Ctrl-BsAb and F4P6-BsAb) of the indicated concentrations of bispecific antibodies. [Figure 4B] 1 is a bar graph showing the percent lysis (% Lysis) of NCI-N87 gastric cancer cells after 90 hours of incubation with PBMC effector cells in the absence (PBMC) or presence (Ctrl-BsAb and F4P6-BsAb) of the indicated concentrations of bispecific antibodies. [Figure 5] Schematic representation of anticancer micellar nanocomplexes (MNCs). EGCG = epigallocatechin-3-O-gallate; oEGCG = oligomerized epigallocatechin-3-O-gallate; PEG = poly(ethylene glycol); TR-CTP = tandem repeat cancer-targeting peptide. [Figure 6] 1 is a bar graph showing percent survival of BT474 human breast cancer cells incubated with vehicle, MNC-Herceptin, or F4P6-MNC-Herceptin, as indicated. [Figure 7] Plot of tumor volume versus days after injection of mice with N87 tumor cells. Mice were injected with the indicated treatment once a week for 4 weeks. *p=0.041. DETAILED DESCRIPTION OF THE INVENTION

[0012] As summarized above, an isolated cancer targeting peptide is provided that includes at least two copies of the amino acid sequence PFLP (SEQ ID NO: 1).

[0013] The two copies of PFLP in the cancer targeting peptide can overlap each other. For example, the cancer targeting peptide can be RPFLPFLPY (SEQ ID NO: 5) and RPFLPFLPYRPFLPFLPY (SEQ ID NO: 6).

[0014] Another isolated cancer targeting peptide includes at least two copies of PFLF (SEQ ID NO: 2). Examples of this peptide include RPFLFPFLFY (SEQ ID NO: 7) and RPFLFPFLFYRPFLFPFLFY (SEQ ID NO: 8).

[0015] The above-mentioned cancer targeting peptides can specifically bind to GRP-78 on cancer cells. In other words, no additional amino acids are required. Therefore, the present invention encompasses any of the above-mentioned cancer targeting peptides that do not contain the sequence RLLDT (SEQ ID NO: 15).

[0016] Also within the scope of the present invention is a pharmaceutical composition for treating cancer comprising any of the above-described isolated cancer targeting peptides and an anti-cancer agent.

[0017] In certain compositions, the anti-cancer agent is a monoclonal antibody, for example, anti-Her2 / neu, anti-PD-1, anti-PD-L1, or anti-CTLA4.

[0018] In other compositions, the anti-cancer agent is a chemotherapeutic agent, such as doxorubicin, vincristine, vinorelbine, paclitaxel, or irinotecan.

[0019] Additionally, the anticancer agent in the pharmaceutical composition may be a radioisotope, e.g. 90 Y, 125 I, 188 Re, 68 Ga, 111 In, or 131It may include I. In certain instances, the radioisotope is chelated by a chelator that is attached to the cancer targeting peptide.

[0020] Returning to pharmaceutical compositions for cancer therapy, particular examples include micellar nanocomplexes (MNCs) having a core encapsulating an anti-cancer drug and a shell comprising an isolated cancer targeting peptide.

[0021] The core can be, for example, a monoclonal antibody conjugated with oligomeric epigallocatechin-3-O-gallate (EGCG). In certain compositions, the monoclonal antibody is an anti-Her2 / neu antibody, such as trastuzumab.

[0022] The shell can be formed from a complex of EGCG and polyethylene glycol (PEG), with the cancer targeting peptide, for example, attached to the PEG. The shell can further comprise a PEG / EGCG complex without the cancer targeting peptide.

[0023] Alternatively, the shell can be a liposome formed from distearoylphosphatidylcholine, cholesterol, and PEG-distearoylphosphoethanolamine. Polymers such as poly(lactic-co-glycolic acid) and polyvinyl chloride can also be used as components of the shell.

[0024] The scope of the present invention also encompasses bispecific anti-cancer antibodies comprising any of the above-described isolated cancer-targeting peptides and an antigen-binding peptide that stimulates T cell activity. Exemplary antigen-binding peptides specifically bind to CD3, PD-1, CTLA-4, LAG-3, TIM-3, TIGIT, VISTA, B7-H3, OX40, GITR, ICOS, or 41BB. The antigen-binding peptide can be, for example, a single-chain Fv (scFv) or a single-domain antibody. In an exemplary bispecific anti-cancer antibody, the antigen-binding peptide is an anti-CD3 scFv. A specific example of a bispecific anti-cancer antibody is a heterodimer consisting of SEQ ID NO: 12 and SEQ ID NO: 14.

[0025] Methods for treating cancer utilizing the cancer-targeting properties of the aforementioned pharmaceutical compositions and bispecific anti-cancer antibodies are provided.

[0026] For example, one method for treating cancer is to administer the above-mentioned pharmaceutical composition containing a cancer-targeting peptide and an anticancer drug to a cancer patient. In a specific method, the treatment is performed by administering MNCs having a core of oligomeric EGCG conjugated with trastuzumab and a shell containing (i) a cancer-targeting peptide bound to a PEG-EGCG conjugate and (ii) a PEG-EGCG conjugate lacking the peptide.

[0027] Another method of treating cancer is carried out by administering the bispecific anti-cancer antibody to a cancer patient. In one example, the bispecific anti-cancer antibody is a heterodimer consisting of SEQ ID NO: 12 and SEQ ID NO: 14.

[0028] Cancers that can be treated by the above methods include, but are not limited to, breast cancer, hepatocellular carcinoma, prostate cancer, lung cancer, ovarian cancer, renal cancer, uterine cancer, cervical cancer, melanoma, embryonal carcinoma, leukemia, and osteosarcoma.

[0029] The foregoing is a method of diagnosing cancer using a radionuclide-labeled cancer targeting peptide comprising at least two copies of the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2. The radionuclide-labeled cancer targeting peptide can have the amino acid sequence of SEQ ID NO: 5, 6, 7, or 8.

[0030] To achieve this method, a radionuclide-labeled cancer targeting peptide is administered, for example, injected, to an individual suspected of having cancer. The individual is then subjected to an imaging technique, such as positron emission tomography, to quantify the amount of radionuclide-labeled cancer targeting peptide accumulated in various body tissues. If the amount of radionuclide-labeled cancer targeting peptide accumulated in a localized area of ​​tissue is greater than the background level in adjacent areas of tissue, cancer is diagnosed.

[0031] In certain methods, the cancer targeting peptide having the amino acid sequence of SEQ ID NO: 6 is 68 It is labeled with Ga and administered to an individual.

[0032] Without further elaboration, it is believed that one skilled in the art can, based on the disclosure herein, utilize the present disclosure to its fullest extent. Accordingly, the following specific embodiments are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications and patent documents cited herein are hereby incorporated by reference in their entirety.

[0033] Example Example 1: Tandem repeat cancer targeting peptide (TR-CTP) TR-CTPs were designed containing at least one tandem repeat of the binding motif, PFLX1 (where X1 is P or F), found in the CTP previously described in U.S. Patent No. 8,846,623. The peptide sequences are shown in Table 1 below, with the repeat sequence underlined and in bold. Note that in F4P6-TR-CTP and F5P6-TR-CTP, the repeat sequences overlap by one amino acid.

[0034] Without being bound by theory, it is believed that the increased length of TR-CTP, compared to CTP, prevents steric hindrance when incorporated into a bispecific antibody. Furthermore, the repetition of the binding motif should increase binding affinity. Furthermore, the novel TR-CTP with the repeats described herein should be suitable for conjugation at its N- or C-terminus. [Table 1]

[0035] Example 2: Binding affinity of N-terminally extended TR-CTP Biotin-labeled TR-CTP and CTP were synthesized (Biotools Co., Ltd., Taiwan), and the kinetics of binding to GRP78 was evaluated by surface plasmon resonance. TR-CTP and CTP were extended at their N-termini with five amino acids (GGGGS; SEQ ID NO: 9). The N-termini were labeled with one biotin molecule per TR-CTP / CTP via an aminohexanoic acid bond.

[0036] Two negative control peptides in which the L-Leu residues in the sequences of F4P6-TR-CTP and F4P13-TR-CTP were replaced with D-Leu residues (dF4P6 and dF4P13, respectively) were also biotin-labeled.

[0037] Streptavidin was immobilized on a sensor chip (CM5; GE Healthcare) with a matrix of carboxymethylated dextran covalently bound to a gold surface using standard amine coupling techniques according to routine procedures. Biotin-labeled TR-CTP was immobilized on the chip by flowing it over the chip at a flow rate of 5 μl / min using HBS-P+ (GE Healthcare) as the running buffer.

[0038] Next, the binding affinity of various concentrations (approximately 0.1-15 μM) of the peptide-binding domain of GRP78 (GRP78-PBD, amino acids 421-639) to chip-bound TR-CTP and CTP was analyzed using a BIACORE™ T200 instrument (GE Healthcare). The sensor chip was regenerated by washing the chip surface with glycine buffer (10 mM; pH 11.5) for 30 seconds. After two chip regeneration cycles, followed by a 120-second wash with running buffer, the chip was reused. The results are shown in Table 2 below. [Table 2]

[0039] The dissociation constants (K ) of the P6 series peptides (PFLP repeats), P6-CTP (1 copy), F4P6-TR-CTP (2 copies), and F5P6-TR-CTP (4 copies) were D ) are 1.9x10 -6 M, 1.2x10 -6 M, and 1.0x10 -6 The increase in binding affinity, i.e., K D The reduction in β correlated with an increase in the copy number of the binding sequence present in the TR-CTP peptide.

[0040] Similar results were observed for the P13 series peptides (repeat sequence PFLF). K values ​​for P13-CTP (1 copy), F4P13-TR-CTP (2 copies), and F5P13-TR-CTP (4 copies) were D The value is 2.7x10 -6 M, 2.1x10 -6 M, and 6.1x10 -7 Among all the peptides tested, F5P13-TR-CTP showed the highest binding affinity to the peptide-binding domain of GRP78.

[0041] Dissociation rate (K off ) reflects the binding stability of the TR-CTP / GRP78-PBD complex. off The smaller the value, the slower the dissociation rate, i.e., the greater the stability of the complex. As shown in Table 2, the K values ​​for P6-CTP, F4P6-TR-CTP, and F5P6-TR-CTP off The values ​​are 3.0x10 -3 S -1 , 2.8x10 -3 S -1 , and 9.1x10 -4 S -1 The K values ​​of P13-CTP, F4P13-TR-CTP, and F5P13-TR-CTP were off The values ​​are 3.4x10 -3 S -1 , 1.7x10 -3 S -1 , and 1.2x10 -3 S-1 A general correlation was observed between the number of repeats in TR-CTP and the dissociation rate.

[0042] As expected, the two D-Leu substituted negative control peptides, i.e., dF4P6 and dF4P13, showed negligible binding to GRP78-PBD, and their K D Values ​​could not be determined, see last two rows of Table 2.

[0043] Example 3: Binding affinity of C-terminally extended TR-CTP The P13 series peptides from Example 1 above were also biotinylated after extending their C-termini with the sequence GGGGSK (SEQ ID NO: 10). Biotin was conjugated to the C-terminal lysine residue using standard techniques. The binding affinity of GRP78-PBD to the sensor chip-bound peptides was measured as described above in Example 2. The results are shown in Table 3 below. [Table 3]

[0044] The binding affinity of GRP78-PBD to C-terminally biotinylated peptides was similar to that to their N-terminally labeled counterparts. Furthermore, the binding affinity (K ) of F4P13-TR-CTP and MF4P13-TR-CTP-cBiotin (both of which contain two copies of PFLF) with C-terminal biotin was significantly higher than that of PFLF. D ) and dissociation rate (K off ) were all better than these values ​​of P13-CTP-cBiotin (which has only one copy). The peptide MF4P13-TR-CTP has the same amino acid sequence as F4P13-TR-CTP with an additional methionine at its N-terminus.

[0045] Example 4. In vivo tumor targeting with TR-CTP The ability of TR-CTP to target tumor cells in vivo was tested in N87 tumor-bearing mice, a HER2-positive gastric cancer xenograft model. N87 tumors were established in NOD SCID gamma (NSG) mice using standard protocols. Briefly, NSG mice were subcutaneously inoculated with 3x10 6-HT1C16 per animal. 6 Tumors were established by injecting N87 cells. Tumors were allowed to grow to 100-200 mm before the following treatments were performed. 3 It grew to.

[0046] Four peptides, namely, F4P13-TR-CTP, P13-CTP, F4P6-TR-CTP, and P6-CTP, were purified using standard techniques. 68 Briefly, dodecanetetraacetic acid polyethylene glycol (DOTA-PEG3350) was conjugated to each peptide to form DOTA-CTP-PEG3350 (Mission Biotech, Taipei, Taiwan). Each DOTA-CTP-PEG3350 was diluted in 0.1 M sodium acetate buffer (pH 5.5) and then diluted with HCl. 68 It was mixed with GaCl3 (itG, Germany) and incubated at 95°C for 10 minutes. 68 The Ga-DOTA-CTP-PEG3350 was used directly without further purification.

[0047] More specifically, a 250 μCi dose of the four aforementioned 68 Each Ga-labeled peptide was injected intravenously into four NSG mice separately. Fifteen minutes after injection, each mouse was scanned for 15 minutes using a nanoScan PET / CT (Mediso Pacific) to acquire static microPET images of the tumor. Quantification of the images can be seen in Figure 1. The results showed that the percent injected dose per gram (%ID / g) in the tumor after administration of F4P13-TR-CTP and F4P6-TR-CTP was significantly higher than that of P13-CTP and P6-CTP, respectively. The presence of tandem repeats of the binding sequences, i.e., PFLF and PFLP, clearly improved tumor targeting of CTP.

[0048] PET studies were confirmed by autoradiographic examination of frozen sectioned tumor tissues removed from injected mice. 68 The distribution pattern of the Ga-labeled peptide was heterogeneous throughout the tumor. Quantification of the radiographic signal in tumor sections is shown in Figure 2. 68 Ga-F4P6-TR-CTP and 68 The radiographic signals of Ga-F4P13-TR-CTP injected mice were, respectively: 68 Ga-P6-CTP and 68 Ga-P13-CTP-injected mice, indicating that the tandem repeat peptide, i.e., TR-CTP, can better target cancer than CTP. 68 compared with the radiographic signal from Ga-P6-CTP set as 1. 68 Ga-P13-CTP, 68 Ga-F4P6-TR-CTP, and 68 Ga-F4P13-TR-CTP were 1.8, 2.3, and 11, respectively.

[0049] Example 5. Bispecific antibodies The suitability of TR-CTP for immunotherapy was evaluated by genetically engineering a bispecific antibody-like protein and utilizing the so-called "knob-and-hole" technique for highly efficient heterodimer formation. See, e.g., U.S. Patent No. 8,961,971. Briefly, F4P6-TR-CTP was fused separately to the C-terminus of an Fc hole peptide and the C-terminus of an Fc knob peptide. An anti-CD3 scFv was fused to the N-terminus of the Fc hole peptide. This bispecific antibody-like construct, designated F4P6-BsAb, is shown schematically in Figure 3. In this example, the F4P6-TR-CTP Fc hole fusion has the amino acid sequence of SEQ ID NO: 12, and the F4P6-TR-CTP Fc knob fusion has the amino acid sequence of SEQ ID NO: 14. The control antibody, i.e., Ctrl-BsAb, lacked the F4P6-TR-CTP sequence.

[0050] After expression and purification of F4P6-BsAb, the kinetics of GRP78-PDB binding to it was assessed by surface plasmon resonance as described above in Example 1, except that F4P6-BsAb was directly coupled to a CM5 sensor chip by amine coupling. The results showed that GRP78-PDB bound to F4P6-BsAb at a binding affinity of 1.1x10 -6 K of M D value, and 9.4x10 -4 S -1 K off values ​​that were close to those of GRP78-PBD for the isolated peptide F4P6-TR-CTP (see Table 2).

[0051] Example 6. Cancer cell killing induced by F4P6-BsAb The ability of the bispecific antibody-like construct F4P6-BsAb to induce cell killing by peripheral blood mononuclear cells (PBMCs) was tested on TOV21G ovarian cancer target cells and N87 gastric cancer target cells.

[0052] Target cells were plated at 2 x 10 per well of a 96-well electronic microtiter plate ("E-plate"; ACEA Biosciences, Inc.). 4 Cells were seeded at 100 μg / ml and allowed to attach for 2 hours. PBMC effector cells, PBMC effector cells plus F4P6-BsAb, and PBMC effector cells plus Ctrl-BsAb lacking the P4P6-TC-CTP sequence were added to a final effector / target ratio of 10:1. The concentrations of F4P6-BsAb and Ctrl-BsAb were 12.5 nM. Data were collected and quantified using the xCELLigence Real-Time Cell Analysis System ("RTCA"; ACEA Biosciences, Inc.) according to the manufacturer's instructions. The results are shown in Figure 4A and Figure 4B.

[0053] In the presence of F4P6-BsAb, PBMCs mediated lysis of 22% and 15% of TOV21G cells (Fig. 4A ) and N87 cells (Fig. 4B ), respectively, whereas no cell lysis was detectable in the absence of F4P6-BsAb or in the presence of Ctrl-BsAb.

[0054] Example 7. TR-CTP micelle nanocomplex Micellar nanocomplexes (MNCs) having (i) a core formed from the anti-cancer monoclonal antibody trastuzumab (Herceptin®; anti-HER-2 / neu mAb) and oligomerized epigallocatechin-3-O-gallate (oEGCG), and (ii) a shell formed from poly(ethylene glycol)-EGCG (PEG-EGCG), are known to have better tumor selectivity, more potent cancer cell growth inhibitory activity, and a longer blood half-life than free Herceptin. See, e.g., Chung et al., Nat. Nano-technol. 9:907-12.

[0055] MNCs were prepared to test the ability of TR-CTP to improve the efficacy of MNCs. F4P6-TR-CTP was conjugated to PEG-EGCG to obtain F4P6-TR-CTP-PEG-EGCG as described below. F4P6-TR-CTP was PEGylated using CHO-PEG-NHS by the addition of N,N-diisopropylethylamine in dimethylformamide. F4P6-TR-CTP-PEG-EGCG was synthesized via a Bayer reaction between the aldehyde (CHO) group of PEGylated F4P6-TR-CTP and the nucleophilic ring of EGCG. The resulting product was dialyzed (molecular weight cutoff = 3500) and lyophilized to obtain F4P6-TR-CTP-PEG-EGCG. See Chung et al.

[0056] Herceptin / oEGCG cores were prepared as previously described (see Chung et al.). MNCs were prepared by mixing the Herceptin / oEGCG cores with PEG-EGCG to form MNC-Herceptin or with F4P6-TR-CTP-PEG-EGCG to form F4P6-MNC-Herceptin. The MNCs are illustrated in Figure 5.

[0057] Example 8. In vitro cancer cell killing by TR-CTP-MNC The ability of F4P6-TR-CTP-MNC-Herceptin to kill cancer cells was compared to that of MNC-Herceptin in HER-2 / neu. Briefly, BT-474 human breast cancer cells overexpressing HER2 / neu were cultured at 1x10 per well of a 96-well E-plate. 4 Cells were seeded at 1000 x 1000 cells per well and cultured for 1 day. Wells were treated with vehicle, Herceptin alone, MNC-Herceptin, or F4P6-TR-CTP-MNC-Herceptin at amounts equivalent to 0.5 mg / ml and 1.25 mg / ml of Herceptin. After 3 days, cell viability was assessed using RTCA as described in Example 6 above. The results, expressed as percent survival, are shown in Figure 6.

[0058] At the concentrations tested, Herceptin alone did not inhibit BT-474 cell viability compared to vehicle control. MNC-Herceptin reduced cell viability by 30-40% at both concentrations tested, compared to 100% viability of vehicle-treated cells.

[0059] F4P6-MNC-Herceptin was more effective in killing BT-474 breast cancer cells than Herceptin alone or MNC-Herceptin. Indeed, treatment of cells with F4P6-MNC-Herceptin at a concentration equivalent to 1.25 mg / ml of Herceptin surprisingly reduced their survival to only 1% of vehicle control. TR-CTP clearly enhanced the in vitro cell killing activity of MNC-Herceptin.

[0060] Example 9. In vivo cancer cell killing by TR-CTP-MNC Because Herceptin inhibits tumor growth, we sought to determine whether TR-CTP-MNC-Herceptin enhances the anticancer activity of Herceptin in vivo. N87 tumors were established as described in Example 4 above. 100-200 mm 3 Mice bearing tumors of size 1.0 mm were intravenously injected with 125 μg of free Herceptin, MNC-Herceptin, or F4P6-MNC-Herceptin once a week for 4 weeks. The results are shown in FIG.

[0061] F4P6-MNC-Herceptin inhibited tumor growth to a greater extent than free Herceptin. Indeed, 61 days after tumor cell injection, the tumor size of F4P6-MNC-Herceptin-treated mice was significantly smaller than that of the Herceptin-treated group. See Figure 7. The tumor growth inhibition mediated by F4P6-MNC-Herceptin was slightly greater than that mediated by MNC-Herceptin.

[0062] Other embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0063] From the foregoing, those skilled in the art can readily ascertain the essential features of the present disclosure, and can make various changes and modifications to the present invention to adapt it to various uses and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are also within the scope of the following claims.

Claims

1. An isolated cancer targeting peptide consisting of the amino acid sequence RPFLFPFLFY (SEQ ID NO: 7) or RPFLFPFLFYRPFLFPFLFY (SEQ ID NO: 8).

2. 10. A pharmaceutical composition for treating cancer, comprising the isolated cancer targeting peptide of claim 1 and an anticancer agent.

3. 3. The pharmaceutical composition of claim 2, further comprising a micellar nanocomplex having a core encapsulating said anticancer drug and a shell comprising said isolated cancer targeting peptide.

4. 4. The pharmaceutical composition of claim 3, wherein the isolated cancer targeting peptide is conjugated to polyethylene glycol.

5. 5. The pharmaceutical composition of claim 4, wherein the anti-cancer agent is a therapeutic monoclonal antibody selected from anti-Her2 / neu, anti-PD-1, anti-PD-L1, and anti-CTLA4.

6. 6. The pharmaceutical composition of claim 5, further comprising epigallocatechin-3-O-gallate, wherein the anti-cancer agent is an anti-Her2 / neu monoclonal antibody.

7. 5. The pharmaceutical composition of claim 4, wherein the anticancer drug is doxorubicin, vincristine, vinorelbine, paclitaxel, or irinotecan.

8. The pharmaceutical composition of claim 2 , wherein the anticancer agent comprises a radioisotope.

9. The radioisotope is ９０ Y. １２５ I, ６８ Ga, １８８ Re, １１１ In, or １３１ The pharmaceutical composition of claim 8, wherein the compound is I.

10. 10. A bispecific anti-cancer antibody comprising the isolated cancer targeting peptide of claim 1 and an antigen-binding peptide that stimulates T cell activity.

11. 11. The bispecific anti-cancer antibody of claim 10, wherein the antigen is selected from the group consisting of CD3, PD-1, CTLA-4, LAG-3, TIM-3, TIGIT, VISTA, B7-H3, OX40, GITR, ICOS, and 41BB.

12. 1. Use of a cancer targeting peptide and an anti-cancer agent in the manufacture of a medicament for the treatment of cancer, wherein the cancer targeting peptide consists of the amino acid sequence RPFLFPFLFY (SEQ ID NO: 7) or RPFLFPFLFYRPFLFPFLFY (SEQ ID NO: 8), and the anti-cancer agent is a therapeutic monoclonal antibody selected from anti-Her2 / neu, anti-PD-1, anti-PD-L1, and anti-CTLA4, or doxorubicin, vincristine, vinorelbine, paclitaxel, or irinotecan.

13. 1. Use of a cancer targeting peptide, a micelle nanocomplex, epigallocatechin-3-O-gallate, and an anti-Her2 / neu monoclonal antibody in the manufacture of a medicament for the treatment of cancer, comprising: the cancer targeting peptide consists of the amino acid sequence of RPFLFPFLFY (SEQ ID NO: 7) or RPFLFPFLFYRPFLFPFLFY (SEQ ID NO: 8) and is conjugated to polyethylene glycol; the micelle nanocomplex having a core encapsulating the anti-Her2 / neu monoclonal antibody and a shell comprising the cancer targeting peptide; use.

14. 1. Use of a cancer targeting peptide and a radioisotope in the manufacture of a medicament for the treatment of cancer, wherein the cancer targeting peptide consists of the amino acid sequence of RPFLFPFLFY (SEQ ID NO: 7) or RPFLFPFLFYRPFLFPFLFY (SEQ ID NO: 8), and the radioisotope is ９０ Y. １２５ I, ６８ Ga, １８８ Re, １１１ In, or １３１ I am using.

15. 11. Use of the bispecific antibody of claim 10 in the manufacture of a medicament for the treatment of cancer.

16. 1. A composition comprising a radionuclide-labeled cancer targeting peptide for use in a method of aiding in the diagnosis of cancer in an individual, comprising: In said use, said composition is administered to said individual, and said individual is subjected to an imaging technique to determine the location and amount of said radionuclide-labeled cancer targeting peptide in said individual; A composition wherein said radionuclide-labeled cancer targeting peptide consists of the amino acid sequence RPFLFPFLFY (SEQ ID NO: 7) or RPFLFPFLFYRPFLFPFLFY (SEQ ID NO: 8).

17. The radionuclide ６８ 17. The composition of claim 16, wherein the imaging technique is positron-enhanced tomography.

Images