Patent Application
20200079858
The present relates to an antibody conjugate compound for detecting or treating muscle invasive bladder cancer.
Although armed antibodies (hereinafter antibody-conjugate [AC]) delivering molecules for the imaging or treatment of targeted tissues is now a prominent approach in medicine, enhancing AC cellular retention and tumor uptake is necessary to improve their effectiveness. The increased residence time inside cancer cells and overall tumor uptake can have important implications for improved tumor killing and detection. Small molecules and radionuclides transported into cells by ACs are sensitive to a variety of mechanisms that ultimately leads to poor accumulation. Thus, new strategies for intracellular delivery technologies are needed to improve AC tumor cell accumulation and potentially effectiveness for its intended application. For example, one potential target is bladder cancer which is one of the most prevalent cancers impacting adults worldwide. Patients often have local and distant disease at the time of initial diagnosis of the primary tumor. This is significant as patients with organ confined bladder cancer undergo different treatment regimens compared to patients with metastatic disease. Thus, targeted therapeutic approaches are an active area of research to more accurately stage pre-treatment bladder cancer.
The hydrophobic interiors of cellular membranes are barriers for ACs to efficiently access the intracellular environment, which limits controlled placement and accumulation of delivered molecular payloads such as chemotherapeutics and radioisotopes. Upon receptor-mediated internalization, current ACs are reliant on entrapment inside the endosomal-lysosomal trafficking pathway where cathepsin-mediated degradation is exploited for payload release. Unfortunately, this trafficking pathway often impedes the efficient intracellular accumulation of these payloads multifold. First, ACs may undergo increased recycling, which has been shown to be a limiting factor for tumor imaging and cytotoxic effectiveness. Second, upon degradation these payloads are released near the cell surface where they are actively exported by overexpressed membrane associated transport proteins. Three, cell surface receptors may be downregulated. In addition, delivering biological payloads (i.e. toxins) that recognize intracellular targets can be degraded and rendered inactive. Thus, a technology that i) would enable ACs to efficiently escape the endosomal-lysosomal pathway, and ii) subsequently route to alternative subcellular locations could greatly enhance payload placement and accumulation and, hence, effectiveness.
Technologies such as synthetic peptides or polymers coupled to antibody surface residues enabling ACs with endosome escape or subcellular destination routing controls are known in the art. In general, these strategies exploit cellular mammalian physiology such as pH-sensitive endosome membrane-destabilizing activities or nuclear and mitochondrial-specific localization sequences. First-generation peptides were developed from a class of agents known as cell-penetrating peptides (CPPs). 13 Although CPP-conjugated antibodies are remarkable for their ability to ‘penetrate’ membranes and accumulate payloads with high cellular accumulation, penetration is indiscriminate. In vivo, peptide-ACs suffer from increased accumulation in non-target tissue resulting in poor tumor targeting. This is most likely caused by a change in the overall AC net charge due to modification with large cationic/anionic peptides. An increase in net charge has been shown to increase AC plasma clearance or increase distribution in normal tissues.
While short peptides capable or penetrating plasma membranes or harboring nuclear localization signal (NLS) sequences represent an excellent platform for increasing mAb conjugate cellular accumulation, it is often at the expense of hindered specificity. As monoclonal antibodies (mAbs) including next-generation humanized and fully human antibodies are well established for the treatment of cancer, their excellent target-specific affinity and specificity provide mAbs with the potential to naturally extend into the clinic as diagnostic agents.
A recent advancement in ACs functionalization has been to empower ACs to achieve multi-selective targeting by attaching peptides that harbor compartment-localizing amino acids. In particular, the nuclear localization signal (NLS) sequence from SV-40 Large T-antigen has previously been incorporated into synthetic peptides and conjugated to proteins and demonstrated the ability to direct the transport of proteins into the nucleus. Although, the optimized NLS sequence is 25 amino acids long, the mAb 7G3 was conjugated to a 13-mer peptide (CGYGPKKKRKVGG) harboring a segment of the NLS (underlined) sufficient for nuclear translocation. An advantage of this short sequence is that it does not penetrate cells and allows mAbs to maintain cell selectivity. 7G3-NLS was used to deliver the radioisotope cargo indium-111 (111In) inside the nucleus. Molecular damage by 111In is due to its emissions of energetic Auger electrons. Because they travel only nanometer-micrometer distances they are more effective if delivered inside the nucleus. Unfortunately, cytotoxicity was not overwhelming relative to standard 111In-7G3 and the evidence suggested it was due to ineffective nuclear localization caused by entrapment in the endosomal-lysosomal and/or recycling pathways.
Therefore, there is still a need to be provided with an AC that can be used in detecting or treating cancer such as bladder cancer.
In accordance with the present description, there is now provided a conjugated anti-interleukin-5 receptor α-subunit (IL-5Rα) compound comprising cholic acid (ChAc) or a variant thereof, said ChAc conjugated to a non-cell penetrating peptide comprising a nuclear localization sequence (NLS) conjugated to an anti-interleukin-5 receptor α-subunit (IL-5Rα) compound.
In an embodiment, the anti-IL-5Rα compound is an antibody.
In another embodiment, the antibody is a monoclonal or polyclonal antibody.
In a further embodiment, the antibody is a mouse antibody, a goat antibody, a human antibody or a rabbit antibody.
In an embodiment, the antibody is a humanized antibody.
In another embodiment, the antibody comprises an epitope binding fragment selected from the group consisting of: Fv, F(ab′) and F(ab′)2.
In a further embodiment, the nuclear localization sequence is from SV40 large T antigen.
In another embodiment, the non-cell penetrating peptide comprises at least one spacer residue.
In an additional embodiment, the non-cell penetrating peptide comprises at least one cysteine for coupling to ChAc and the anti-IL-5Rα compound.
In a further embodiment, the non-cell penetrating peptide is as set forth in SEQ ID NO: 1.
In another embodiment, the compound of interest is an A14 antibody.
In a further embodiment, the ratio of ChAcNLS peptide conjugated per compound of interest is between 1 to 10 peptides per compound.
In another embodiment, the conjugated compound described herein further comprises a radionuclide attached thereto.
In an embodiment, the radionuclide is at least one of 47Sc, 51Cr, 52mMn, 55Co, 58Co, 52Fe, 56Ni, 57Ni, 61Cu, 62Cu, 64Cu, 67Cu, 66Ga, 68Ga, 67Ga, 72As, 77As, 89Zr, 90Y, 94mTc, 99mTc, 97Ru, 105Rh, 109Pd, 111Ag, 110In, 111In, 113mln, 114mln, 117mSn, 121Sn, 127Te, 142Pr, 143Pr, 149Pm, 151Pm, 149Tb, 153Sm, 157Gd, 161Tb, 166Ho, 165Dy, 169Er, 169Yb, 175Yb, 172Tm, 177Lu, 186Re, 188Re, 191Pt, 197Hg, 198Au, 199Au, 201Tl, 203Pb, 211At, 212Bi, 213Bi, 11C, 75Br, 76Br, 77Br, 82Br, 18F, 120I, 123I, 124I, 125I, 131I, 89Sr and 225Ac.
In a further embodiment, the radionuclide is 64Cu.
In another embodiment, the conjugated compound described herein further comprises a chemotherapeutic agent attached thereto.
In an embodiment, the chemotherapeutic agent is vinblastine or α-amanitin.
In an additional embodiment, the compound described herein is for treating bladder cancer.
In another embodiment, the compound described herein is for detecting bladder cancer.
In a further embodiment, the bladder cancer is muscle invasive bladder cancer (MIBC).
In another embodiment, the compound described herein is for detecting bladder cancer by PET imaging.
It is further provided herein the use of the conjugated anti-IL-5Rα compound as described herein for detecting and/or treating bladder cancer.
It is also provided the use of the conjugated anti-IL-5Rα compound as described herein in the manufacture of a medicament for treating bladder cancer.
It is also provided herein a method of detecting and/or treating bladder cancer in a subject, comprising the step of administering to a subject the conjugated anti-IL-5Rα compound as described herein.
In an embodiment, the subject is a mouse or a human.
It is also provided an anti-bladder cancer compound comprising cholic acid (ChAc) or a variant thereof, said ChAc conjugated to a non-cell penetrating peptide comprising a nuclear localization sequence (NLS) conjugated to an anti-interleukin-5 receptor α-subunit (IL-5Rα) compound, and conjugated to a chemotherapeutic agent attached thereto.
Reference will now be made to the accompanying drawings.
It is provided a novel design of compound-conjugates specific against rapidly internalizing receptors to link endosome escape and enhanced cellular uptake. More specifically, it is provided a conjugated compound comprising cholic acid conjugated to a non-cell penetrating peptide comprising a nuclear localization sequence (NLS) conjugated to the compound of interest.
It is provided a novel composite compound, cholic acid-NLS (ChAcNLS) that attaches stably to Abs. ChAcNLS utilizes biological mimicry of viruses for escaping endosome entrapment and routing to the nucleus without compromising for example Ab affinity and specificity. These independent actions are required for increasing Ab cellular accumulation.
It is provided herein in one embodiment an IL-5Rα-targeted 64Cu-labeled mAb modified with a novel compound that improves tumor targeting and sensitive visualization of variably expressing IL-5Rα-positive muscle invasive bladder cancer (MIBC). In an embodiment, the monoclonal antibody (mAb) A14 is modified with the compound ChAcNLS that enables mAbs to escape endosome entrapment and route to the nucleus in target cells. 64Cu-A14-ChAcNLS is able to increase cellular accumulation in target MIBC cells with high affinity and specificity. In addition, 64Cu-A14-ChAcNLS is able to visualize tumors in IL-5Rα-positive invasive bladder tumors below the threshold of detection using standard 64Cu-A14. The performance of 64Cu-A14-ChAcNLS establishes a rationale for the development of mAb conjugate PET agents that take charge of their intracellular trafficking to improve tumor imaging. 64Cu-A14-ChAcNLS also establishes an approach for IL-5Rα-targeted PET imaging. This PET tracer may impact the determination of MIBC during staging and improve therapy guidance.
It is thus described an AC coupled with cholic acid and coupled to a 13 amino acid peptide (SEQ ID NO: 1) containing a nuclear localization sequence (
In an embodiment, the antibody encompassed herein is a monoclonal or polyclonal antibody.
In another embodiment, the antibody is a mouse antibody, a goat antibody, a human antibody or a rabbit antibody, or a humanized antibody.
Also encompassed, the antibody might comprises an epitope binding fragment selected from the group consisting of: Fv, F(ab′), or F(ab′)2.
As showed herein, it is demonstrated that a ChAcNLS-modified AC has tumor targeting properties in vivo. It is also demonstrated the ability of A14-ChAcNLS to escape endosome entrapment and localize to the nucleus. PET imaging, biodistribution, and pharmacokinetic (PK) analysis were used to determine the impact on tumor and normal organ uptake, and hence, tumor targeting.
The interleukin-5 receptor α-subunit (IL-5Rα) is a target for PET imaging of muscle invasive bladder cancer (MIBC) using radiolabeled mAbs. Until recently, IL-5Rα had a limited role in cancer. However, IL-5Rα is preferentially overexpressed in invasive bladder cancer from a transcriptomic perspective (Lee et al., 2012, PLoS One, 7(9): e40267). Moreover, IL-5 treatment amplified components associated with cancer invasion such as enhanced cellular migration, expression of matrix metalloproteinases (MMPs), and the arrest of cellular proliferation. Using immunohistochemical analysis of primary bladder specimens obtained from 134 patients undergoing tumor resections or complete bladder removal, it was found that IL-5Rα protein was preferentially overexpressed in invasive bladder tumors relative to non-invasive tumors and healthy urothelial tissues. Furthermore, IL-5Rα has rapid internalization and re-expression dynamics, which were attractive for mAb targeting. To develop a potential PET imaging agent, the IL-5Rα-specific mAb A14 was conjugated to the chelator 2,2′-(7-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (NOTA-NHS) for complexation to copper-64 (64Cu; t½=12.7 h). 64Cu-A14 efficiently accumulated radioactivity inside target human IL-5Rα-positive invasive bladder cancer cell lines HT-1376 and HT-B9, which were grown bilaterally on the flanks of NOD/SCID mice. Immunohistochemical analysis of these xenografts revealed these tumor models were similar to the IL-5Rα expression heterogeneity observed in the patient tumors. Specifically, HT-1376 xenografts were comprised of >66% of IL-5Rα-positive tumor cells. In contrast, HT-B9 tumors were comprised of only 11% IL-5Rα-positive tumor cells that were present as small ‘island’ populations. Thus, these xenografts provided tumor models with high and low tumor cell densities for assessing IL-5Rα targeting. PET imaging at 48 h post-injection and examination by region-of-interest (ROI) analysis revealed that 64Cu-A14 can visualize HT-1376 tumors with high contrast. In comparison, 64Cu-A14 uptake in HT-B9 tumors was difficult to visualize due to reduced accumulation and hence, PET tumor signal.
It is described herein that ChAcNLS can be applied to 64Cu-A14 to improve its PET imaging of IL-5Rα-positive invasive bladder tumors by delivering 64Cu via a mechanism that increases intracellular accumulation in target cancer cells. Thus, PET imaging was used in the developed IL-5Rα tumor models to determine if 64Cu-A14-ChAcNLS improves specific tumor detection relative to 64Cu-A14. Fluorescence and genetic methods were used to demonstrate ChAcNLS mimics Calciviridae endosome escape followed by nuclear routing, and radioimmunoassays were used to show improved 64Cu cellular accumulation, and PET imaging and biodistribution analysis were also used to demonstrate 64Cu-A14-ChAcNLS provides increased contrast tumor images of HT-1376 and HT-B9 tumors at 24 h post-injection compared to 64Cu-A14. It is thus demonstrated that 64Cu-A14-ChAcNLS outperforms 64Cu-A14-NLS (no cholic acid) for detecting MIBC tumors.
Also encompassed herein, but not limited, are radionuclide conjugated to the compound described herein selected from 47Sc, 51Cr, 52mMn, 55Co, 58Co, 52Fe, 56Ni, 57Ni, 61Cu, 62Cu, 64Cu, 67Cu, 66Ga, 68Ga, 67Ga, 72As, 77As, 89Zr, 90Y, 94mTc, 99mTc, 97Ru, 105Rh, 109Pd, 111Ag, 110In, 111In, 113mln, 114mln, 117mSn, 121Sn, 127Te, 142Pr, 143Pr, 149Pm, 151Pm, 149Tb, 153Sm, 157Gd, 161Tb, 166Ho, 165Dy, 169Er, 169Yb, 175Yb, 172Tm, 177Lu, 186Re, 188Re, 191Pt, 197Hg, 198Au, 199Au, 201Tl, 203Pb, 211At, 212Bi, 213Bi, 11C, 75Br, 76Br, 77Br, 82Br, 18F, 120I, 123I, 124I, 125I, 131I, 89Sr and 225Ac. In addition, also encompassed are a chemotherapeutic conjugated as described herein, as for example vinblastine.
Accordingly, it was mechanistically validated that ChAcNLS enables A14 to escape endosome entrapment and localize to the nucleus in MIBC cells. A14-ChAcNLS treated HT-1376 cells contained increased intracellular antibody levels relative to cells treated with A14. Quantifying the overlap in fluorescence between the nuclear Hoechst stain and A14 determined a 33.9% colocalization for A14-ChAcNLS (
Enhanced nuclear and cellular accumulation coincided with events observed with Calciviridae liberation into the cytoplasm, namely the increased production of ceramide and intracellular vesicle disruption. A14-ChAcNLS treatment increased the amount of ceramide present by a factor of ˜3 (p<0.001) at 2 h over standard treatment with A14. The increased amount of ceramide remained increase by a factor of >2 at 6 h. By 16 h ceramide levels from cells treated with A14-ChAcNLS returned to levels observed from cells treated with A14. When HT-1376 cells transfected with GFP-Galectin-3, a marker for endosomal disruption, were treated with A14-ChAcNLS there were significant (p<0.05) increases in the number of intracellular fluorescent foci from 15 to 240 min. The foci per cell response in HT-1376 cells treated with A14-ChAcNLS was >4 times higher than in the control with a peak increase of ˜16-times higher at 60 min. There was also an increased amount of fluorescence specific for A14-ChAcNLS in the nucleus and cytoplasm relative to A14 in GFP-Galectin-3 transfected cells.
In virology cholic acid has been shown as an essential host element exploited by viruses to escape endosome entrapment. The increase in ceramide in endosomal membranes causes the formation of channels or lipid flip-flop sufficient for proteins to cross. Calciviridae family requires the presence of cholic acid for the activation of acid sphingomyelinase (ASM), which cleaves sphingomyelin to produce ceramide. This instability was the mechanism used by these viruses to escape endosome entrapment. However, it is not yet known how cholic acid engages with the viral particle. The peptide component of ChAcNLS harbors an optimized nuclear localization sequence derived from the simian virus (SV)-40 large T-antigen, and previously shown to translocate ACs into the nucleus.
HT-1376 cells incubated with A14-ChAcNLS contained a 55.4%±4.1% increase (p<0.0001) of ceramide at 2 h over control (
A second measure of endosome escape was to use a GFP-Galectin-3 assay. Galectin-3 is a lectin protein present diffusely throughout the cell cytosol that binds the β-galactoside sugars on the inner leaflet of endosomes and lysosomes. 35 When these vesicles are disrupted, the β-galactoside sugars are exposed to the cytosol allowing the cytosolic Galectin-3 to bind and aggregate on the inner leaflet. There were significant (p<0.0001) increases in the number of foci per cell by factors of >3-, 5-, 10-, 5-, and 5-fold at incubation time points of 15 min, 30 min, 1 h, 2 h, and 4 h, respectively. Moreover, there was visual colocalization between A14 and GFP-Galectin-3 in cells incubated with A14-ChAcNLS but not with A14 (
Nuclear localization by staining cells for A14 and Hoechst was evaluated and using confocal microscopy to visualize and determine the percentage of colocalized fluorescence. HT-1376 cells incubated with A14-ChAcNLS contained 33.9% colocalization (
The purity of 64Cu-A14, 64Cu-A14-NLS, and 64Cu-A14-ChAcNLS were A5% and ≥9% by ITLC and SDS-PAGE, respectively (
64Cu-A14-ChAcNLS showed nanomolar affinity for IL-5Rα. A14-ChAcNLS as a function of increasing concentrations of 64Cu-A14-ChAcNLS revealed specific binding approached saturation at concentrations of 3-5 nM in both HT-1376 and HT-B9 cells (
To establish ChAcNLS-mediated increase in cellular accumulation of A14 also increases the accumulation of 64Cu, HT-1376 and HT-B9 cell uptake experiments were performed followed by cell fractionation to dissect individual cellular subcompartments. 64Cu-A14-ChAcNLS increased total intracellular 64Cu accumulation compared to 64Cu-A14 and 64Cu-A14-NLS. In HT-1376 cells, 64Cu-A14-ChAcNLS increased intracellular 64Cu accumulation by factors of 9.4 and 3.2 over 64Cu-A14 and 64Cu-A14-NLS, respectively, at 24 h (p<0.0001;
64Cu-IgG-ChAcNLS had significantly (p<0.01) reduced nuclear and intracellular 64Cu accumulation relative to 64Cu-A14-ChAcNLS. (
It has been previously shown that peptide modification of ACs correlates with increased in vitro accumulation, but can also adversely affect PK and, hence, tumor targeting properties. For example, ACs functionalized with the cationic transcriptional activator protein (TAT) peptide from human immunodeficiency virus 1 have previously been investigated as tumor targeting agents. When radiolabeled TAT-single-chain Fv antibody fragments specific for the ED-B domain of fibronectin were injected into mice, biodistribution revealed tumor uptake was reduced nearly 3-fold. The TAT-modified AC was sequestered in the spleen and liver, which was due to an 80% uptake reduction in the blood. An intact IgG-TAT suffered from similar problems resulting in substandard tumor uptake.
To show the impact of ChAcNLS on the blood clearance profile of 64Cu-A14, blood sampling from 24 h to 96 h post injection was performed followed by liquid scintillation counting. Uptake was expressed in percent injected-dose/gram (% ID/g). The % ID/g in the blood from tumor bearing non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice injected with 64Cu-A14-ChAcNLS was reduced by approximately 40% relative to 64Cu-A14 at 24 h and 48 h (p<0.001;
When the behavior of a drug in the body is known in terms of transport and distribution in tissues, a validated PK model can be used. For novel drugs such as ChAcNLS-modified ACs, a non-compartmental model is more appropriate. The estimated half-life for 64Cu-A14-ChAcNLS was reduced by 45% (t1/2=54.4 h) relative to 64Cu-A14 (t1/2=99.5 h). These data show that there is also increased blood clearance 64Cu-A14-ChAcNLS relative to 64Cu-A14.
Even with decreased amounts in the blood, remarkably, 64Cu-A14-ChAcNLS is able to have comparable uptake relative to 64Cu-A14. In HT-1376 xenografts containing >66% of IL-5Rα-positive bladder tumor cells with strong and moderate expression, the 48 h tumor uptake for 64Cu-A14-ChAcNLS and 64Cu-A14 was 8.0±1.0% ID/g and 8.5%±1.0% ID/g, respectively. At 96 h the tumor uptakes for 64Cu-A14-ChAcNLS and 64Cu-A14 were 6.0%±0.4% ID/g and 6.9%±0.3% ID/g, respectively. Unlike the HT-1376 tumors, HT-B9 tumors are comprised of only ˜11% IL-5Rα-positive MIBC cells. The tumor uptake at 48 h for 64Cu-A14 and 64Cu-A14-ChAcNLS was 7.2%±2.8% ID/g and 6.9%±1.8% ID/g, respectively. At 96 h the tumor uptakes for 64Cu-A14-ChAcNLS and 64Cu-A14 were 3.2%±2.2% ID/g and 3.3%±1.2% ID/g, respectively. Thus, unlike previous peptide-modified ACs, ChAcNLS maintained good tumor uptake.
Distribution in normal organs revealed elevated kidney uptake with 64Cu-A14-ChAcNLS at 48 h (
64Cu-A14-
64Cu-A14-
64Cu-A14
64CuA14
0.0001†
†Significance in favor of 64Cu-A14
64Cu-A14-
64Cu-A14-
64Cu-A14
64CuA14
0.0026†
†Significance in favor of 64Cu-A14
To further evaluate tumor targeting performance, PET was performed and tissue distribution visualized. 64Cu-A14-ChAcNLS-mediated tumor imaging was highly specific as tumor uptake in both HT-1376 and HT-B9 tumors was reduced ˜2-fold upon pre-injection of excess unlabeled A14 (p<0.05). PET images at 24 h and 48 h in mice injected with 64Cu-A14-ChAcNLS without predosing of unlabeled A14 revealed strong uptake in HT-1376 tumors (
In comparison, the PET images in mice injected with 64Cu-A14 and 64Cu-A14-NLS revealed reduced uptake in HT-1376 tumors relative to 64Cu-A14-ChAcNLS (
For the HT-B9 tumors, only 64Cu-A14-ChAcNLS was able to provide PET images with sufficient tumor-contrast (
ROI analysis was used to quantify uptake in tumors and the adjacent muscle tissue to determine tumor/muscle ratios. HT-1376 tumor uptake for 64Cu-A14-ChAcNLS was comparable with 64Cu-A14 and was increased by 35% (p<0.05) relative to 64Cu-A14-NLS at 24 h (
For HT-B9 tumors, 64Cu-A14-ChAcNLS had comparable tumor uptake to 64Cu-A14 and was significantly (p≤0.01) increased relative to 64Cu-A14-NLS (
In the preclinical model of human IL-5Rα-positive MIBC, it is demonstrated that 64Cu-A14-ChAcNLS is able to increase the cellular accumulation of 64Cu via a mechanism that incorporates escape from the endosomal-lysosomal intracellular trafficking pathway coupled to nuclear localization. In vivo, 64Cu-A14-ChAcNLS achieved increased specific exposure to MIBC tumors at equal doses versus 64Cu-A14 and resulted in improved targeting of IL-5Rα-positive expressing MIBC tumors with high and low tumor cell densities. Importantly, this study reveals that the combination of increased cellular accumulation, faster clearance from the blood, and good tumor uptake shows that more payload can be delivered to a tumor cell per individual binding event. Thus, ChAcNLS is an effective conjugation moiety to improve AC tumor targeting. Moreover, ChAcNLS is a solution for ACs modified with peptides to deliver payloads to specific locations in the cell interior such as the nucleus. ChAcNLS did not provide increased tumor targeting for 64Cu-A14 through extremely rapid washout, which proportionally decreases tumor uptake as occurs for other peptide-modified ACs. Instead, 64Cu-A14-ChAcNLS displayed intermediate clearance and maintained high tumor uptake.
It is further encompassed herein the possibility of not only conjugated an antibody as described herein but also conjugating the antibody with a further drug, such as vinblastine and/or α-amanitin, which is used in combination with other chemotherapy drugs to treat bladder cancer.
Vinblastine is chemical analogue of vincristine. It binds tubulin, inhibiting the assembly of microtubules. Vinblastine is reported to be an effective component of certain chemotherapy regimens, particularly when used with bleomycin, and methotrexate, to treat a number of types of cancer, including Hodgkin's lymphoma, non-small cell lung cancer, bladder cancer, brain cancer, melanoma, and testicular cancer.
α-Amanitinis a cyclic peptide of eight amino acids, consisting of a selective inhibitor of RNA polymerase II and III and as showed a high antitumoral activity.
Accordingly, the conjugated compound described herein can be used for detecting or treating bladder cancer.
Accordingly, it is encompassed herein that the antibody-drug conjugates (ADCs) as described herein comprises a small molecule toxin such as for example and not limited to, microtubule disrupting agents (such as vinblastine, Monomethyl auristatin E or MMAE, DM1) and/or DNA alkylating agents.
For example, an antibody conjugated with ChAcNLS together with an attached chemotherapeutic molecule such as 4,4-difluoro-8-(4-carboxphenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY for short), which is a cytotoxic molecule used in photodynamic therapy applications in cancer, is encompassed.
For example, A14 is attached to vinblastine via its carbon-16 position, which has previously been attached to monoclonal antibodies (mAbs) for the development of ADCs. Encompassed herein is a formulation wherein for example A14-vinblastine is constructed using the crosslinkers sulfosuccinimidyl-4-N-malimidomethylcyclohexane-1-carboxylate (MCC) and the polyethylene glycol maleimide-containing spacer (SM(PEG)2).
As described herein, A14-vinblastine conjugates were designed to include carbon chains consisting of 2 or 10 carbons spaced between the MCC/SM(PEG)2 linker and vinblastine (see
The following formulations were developed and tested for cytotoxicity against IL-5Rx-positive HT-1376 MIBC cells.
As seen in
It is further described that the A14-vinlblastine (MCC conjugated) as encompassed herein was modified with ChAcNLS again using MCC as a crosslinker (see
As seen in
The present disclosure will be more readily understood by referring to the following examples which are given to illustrate embodiments rather than to limit its scope.
A14 was obtained and purified as previously described (Sun et al., 1996, Blood, 87: 83-92.
To evaluate conjugation, a reducing 12% polyacrylamide gel was loaded with A14, A14-NLS, A14-ChAcNLS, and protein standards (BioRad, Ontario, Canada). The gel was stained with Coomassie and protein standard retention factor (Rf) values obtained. The size of the A14-NLS and A14-ChAcNLS heavy and light chains were extrapolated by plotting the distance of migration against the Rf values. The numbers of ChAcNLS or NLS compounds per A14 were calculated by dividing the difference in molecular weight (MW) between the modified A14 conjugates and unmodified A14 by 1768.5 g/mol and 1418.8 g/mol, respectively.
A14 was first reacted with NOTA-NHS in a 5-to-1 NOTA-to-A14 ratio, purified, and then conjugated to NLS or ChAcNLS followed by purification. Radiolabeling efficiency was determined by autoradiography on both instant thin-layer chromatography strips (realized in 0.1 M sodium citrate, pH 5.5) and on a non-reducing 12% polyacrylamide gel was loaded with 64Cu-labeled conjugates (realized by SDS-PAGE).
HT-1376 cells were treated with 200 nmol/L of A14 or A14-ChAcNLS for 1 h at 37° C. followed by washing in ice cold PBS. Cells were then replenished with fresh antibody-free media and placed back at 37° C. for 1 h. Cells were then prepped for nuclear and antibody staining for evaluation by confocal microscopy as previously described (Beaudoin et al., 2016, Mol Pharm, 13: 1915-1926). For determination of ceramide levels, HT-1376 cells were treated with A14 or A14-ChAcNLS for increasing time points at 37° C. Cells were fixed and permeabilized. Cells were incubated with the anti-ceramide antibody conjugated to the fluorophore AlexaFluor 488 (Cedarlane, Ontario, Canada). Flow cytometric analysis measured the mean fluorescence intensity (MFI).
To explore endosome escape, HT-1376 cells were transfected with cDNA encoding for GFP-Galectin-3. Cells were then treated with 200 nM of A14 or A14-ChAcNLS for 1 h at 37° C. Cells were fixed and permeabilized as described above. Cells were evaluated by confocal microscopy evaluating GFP- and A14 (probed with anti-mFc AlexaFluor 647)-specific fluorescence.
When tumors were 65-100 mm3, mice (n=5) were intravenously injected with 64Cu-A14, 64Cu-A14-NLS, or 64Cu-A14-ChAcNLS (˜25 μg; ˜7 MBq; radiochemical purity ≥98%). Nicking of the saphenous vein and collection of blood was performed daily. Mice anesthetized under 2.5% isoflurane were then euthanized by CO2 inhalation at 48 h and 96 h post-injection. Major organs and tumors were excised, rinsed in saline, blotted dry, and placed in pre-weighted tubes and gamma counted. Radioactivity accumulation was corrected for decay and expressed as the injected dose per gram of tissue (% ID/g).
PET imaging studies were performed on 5 mice per group on a PET/CT Triumph™ scanner (Trifoil, Calif., USA) at 24 h and 48 h post-injection. PET scans were acquired for 30 and 45 min at 24 h and 48 h, respectively, with tumors near the center of the field of view, in double axial sampling mode to improve spatial resolution. The images were reconstructed using 20 iterations of an MLEM algorithm implementing a physical description of the detectors in the system matrix. A cylindrical phantom (24.8 ml) containing 5MBq of 64Cu at day 0 was used to obtain a calibration factor to convert the counts per seconds into kBq/mL, from which % ID/g values were derived from ROI drawings (n≥3) of the tumor, muscle, and heart using the AMIDE software.
While the present disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the including such departures from the present disclosure as come within known or customary practice within the art and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.
The present application claims benefit of U.S. Provisional Application No. 62/471,052 filed Mar. 14, 2017, the content of which is hereby incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2018/050303 | 3/14/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62471052 | Mar 2017 | US |
