Development of an orally-administrable tumor vasculature-targeting therapeutic using annexin A1-binding D-peptides

We previously reported that IF7 peptide, which binds to the annexin A1 (ANXA1) N-terminus, functions as a tumor vasculature-targeted drug delivery vehicle after intravenous injection. To enhance IF7 stability in vivo, we undertook mirror-image peptide phage display using a synthetic D-peptide representing the ANXA1 N-terminus as target. We then identified peptide sequences, synthesized them as D-amino acids, and designated the resulting peptide dTIT7, which we showed bound to the ANXA1 N-terminus. Whole body imaging of mouse brain tumor models injected with near infrared fluorescent IRDye-conjugated dTIT7 showed fluorescent signals in brain and kidney. Furthermore, orally-administered dTIT7/geldanamycin (GA) conjugates suppressed brain tumor growth. Ours is a proof-of-concept experiment showing that ANXA1-binding D-peptide can be developed as an orally-administrable tumor vasculature-targeted therapeutic.


Introduction 45
It is widely accepted that vasculature surfaces are heterogenous and express varying tissue-

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Thus we hypothesized that an IF7-conjugated drug would overcome the blood-brain-barrier (BBB) to 58 eradicate brain tumors. Indeed, intravenous injection of the IF7-conjugated anti-tumor agent SN-38 59 into model mice harboring brain tumors efficiently reduced the size of brain tumor at low dosage, 60 which apparently invoke host immune reaction against brain tumor leading into complete remission 61 of brain tumor [6].

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We conjugated IF7 to SN-38 through an esterase-cleavable linker, allowing SN-38 to be freed 64 from the peptide once it reached the tumor vasculature. IF7 peptide itself was also susceptible to 4 undertook mirror-image phage library screening taking an advantage of the fact that IF7 binds to 69 chemically synthesized ANXA1 N-terminal domain (1-15 residues plus additional cysteine at 16), 70 designated as MC16 [6]. This phage library screening identified the peptide dTIT7, which represents 71 an ANXA1-binding D-type peptide. We then conjugated it to geldanamycin (GA) through an 72 uncleavable linker to generate GA-dTIT7. We present proof-of-concept data showing that orally-73 administered GA-dTIT7 suppresses brain tumor growth in mice.         Japan. Experiments of brain tumor model mouse were conducted when tumor size determined as 7 photon number was between 1 x10^4 and 1x10^7. When brain tumor grew more than 1x10^7, the 145 mouse was euthanized by placing the animal under saturated isoflurane gas (1~2 mL isoflurane in 250 146 mL chamber) followed by cervical dislocation. No animal died before meeting the criteria for 147 euthanasia.

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Generation of brain tumor model mice. C6-Luc cells (4.8 x10 4 in 4 µl PBS) were injected 150 into C57BL/6 mouse brain striatum using a stereotaxic frame as described [11]. Seven days later,       Table 1). We designated TITWPTM as TIT7 and a synthetic peptide of TIT7 210 composed of D-amino acids as dTIT7.

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with a Kd of 8.5 nM ( Fig. 2A). We then assessed specificity of TIT7 binding to MC16 in a binding 218 10 assay using mutant forms of MC16. That analysis indicated that dTIT7 binding affinity to MC16 219 mutants F7A, K9A and W11A was significantly lower than to WT MC16 (Fig. 2B).

Fig. 2 228
To confirm that dTIT7 and MC16 interact in solution, we analyzed a mixture of both peptides 229 using NMR spectroscopy (Fig. 3A). We observed that the spectrum of the mixture was similar but 230 differed in key ways from the sum of respective peptides. Most prominently, a distinct peak in the 231 mixture spectrum emerged at 0.73 ppm (Fig. 3A, black arrow) and was absent in the summed 232 spectrum. Concomitantly, we observed relative broadening of many peaks of the mixture spectrum.

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Note that a split in the peak at 1.08 ppm (Fig. 3A, red arrow) was relatively shallower in the mixture.

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Peak broadening has been attributed to shortened transverse relaxation time (T2) [13], which was 235 indeed the case for the peak at 1.08 ppm (Fig. 3B). Moreover, T2 shortening is typically associated 236 with an increase in molecular weight [13]. Overall, these results indicate an association between 237 dTIT7 and MC16 in solution, at least at equilibrium.

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We then generated a computer-simulated docking pose of dTIT7 with L-MC16 (Fig. 3C). To  IRdye-dTIT7 targeted brain tumor and kidney and remained detectable in these locations for up to 6 251 days after injection.

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In the same model, we also tested in vivo tumor vasculature-targeting of additional IRDye-253 conjugated peptides identified in our mirror-image phage library screen, namely d-LRF7, dSPT7, 254 dMPT7 and dLLS7, using whole body imaging. That analysis revealed signals in brain, kidney and 255 other organs (Fig. 4B). These results suggest that D-peptide sequences deduced in our screen targeted 256 primarily the brain tumor and kidney vasculature.   In our previous study we found that intravenously-injected GA-IF7 at 6.5 µmoles/kg suppressed 267 growth of melanoma, lung carcinoma, prostate cancer, and breast cancer models in the mouse [5].

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When we injected GA-dTIT7 at 6.5 µmoles/kg intravenously to the tumor-bearing mice in the same 269 manner as we have done for GA-IF7, GA-dTIT7 did not suppress tumor growth (data not shown).

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Since it is known that the GA analogue 17-DMAG, which is a part of GA-dTIT7 (Fig. 5), is orally-

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Next, we tested the therapeutic effect of orally-administered GA-dTIT7 on brain tumors in vivo.

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We had previously shown that IF7-SN38 overcame the BBB and suppressed brain tumor growth in tumor growth in GA-dTIT7-treated mice, while tumors continued to grow in control mice that had 292 received 1 mg GA-C (the molar equivalent of GA-dTIT7) daily for 5 days (Fig. 6D). Comparable 293 analysis using C6-Luc brain tumor models in nude mice revealed tumor growth suppression by GA-294 dTIT7 but not control GA-C (Fig. 6E). These results showed, as a proof-of-concept, that orally-295 administered GA-dTIT7 suppresses brain tumors in vivo in mice.

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Compared with other organ systems, FDG-PET imaging of the brain presents unique challenges 329 because of high background glucose metabolism in normal gray matter [21]. We consider that D-330 peptides identified here warrant further testing in imaging of brain tumors.

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Although we had anticipated that intravenously-injected GA-dTIT7 would exhibit anti-tumor 333 activity in vivo, we did not observe therapeutic activity of dTIT7-conjugated drugs following 334 intravenous injection, suggesting that either higher dosages of GA-dTIT7 or different drug 335 formulation may be required. Relevant to the latter, detergents significantly alter IF7-SN38 that GA-dTIT7 (Fig. 5) is orally administrable and suppressed tumor growth in mouse brain tumor 345 models (Fig. 6 DE). We were able to test oral administration of GA-dTIT7 as this compound exhibits 346 cytotoxic activity (Fig 6A). GA-dTIT7 should be stable in vivo, as GA is linked to dTIT7 through an 347 esterase-resistant linker and dTIT7 is expected to be resistant to digestive proteases. Although the 348 efficacy of GA-dTIT7 gut-to-blood transport was low here (Fig. 6C), future studies should address 349 how to improve this efficacy. Additional modification of GA-dTIT7 to enhance ANXA1-binding and