The Dox-CBD-SA treatment significantly suppress tumor growth

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Medical researchers often use serum albumin (SA) as a drug carrier to deliver cytotoxic agents to tumors during biomedical drug delivery via passive targeting approaches.

To improve the targeting capacity of SA’s a team of scientists recently developed an approach to retain SA-drug conjugates in tumors by combining passive and active targeting mechanisms.

In the new study, Koichi Sasaki and colleagues in the Pritzker School of Molecular Engineering in the University of Chicago, U.S., recombinantly fused SA with a collagen-binding domain (CBD) of the von Willebrand factor protein.

The approach allowed binding within the tumor stroma after drug release, due to tumor-vascular permeability.

The work is now published on Science Advances.

The research team then conjugated doxorubicin (Dox; an anticancer drug) to the CBD-SA conjugate using a pH-sensitive linker.

The Dox-CBD-SA treatment significantly suppressed tumor growth compared to Dox-SA and aldoxorubicin treatment in a mouse model of breast cancer.

Notably, the new compound—Dox-CBD-SA, efficiently stimulated host anti-tumor immunity to completely eradicate established tumors in an animal model of MC38 colon carcinoma when the research team combined the compound with the anti-PD1 checkpoint inhibitor.

The compound DOX-CBD-SA decreased adverse side-effects compared to aldoxorubicin, confirming the bioengineered CBD-SA as a versatile, clinically relevant drug conjugate carrier protein with potential to treat solid tumors.

Serum albumin is the most abundant protein in blood and a number of small molecule compounds can be fused, conjugated with, or co-formulated with SA for improved drug delivery to disease lesions.

The exceptionally long plasma half-life and hydrophilicity (water loving nature) of SA contributed to improve the pharmacokinetics, safety and efficacy of the new drug compounds.

Notably, SA can passively target tumors via pathological permeability of the tumor vasculature, which is advantageous for cancer therapy.

Molecular engineering approaches have gained prominence in the field of cancer research to combine passive and active targeting during tumor drug delivery.

Engineering collagen-binding serum albumin (CBD-SA) as a drug conjugate carrier for cancer therapy
The binding interface between collagen type III and A3 domain of VWF. Crystal structure of the A3 domain of von Willebrand factor (CBD) in complex with type III collagen (PDB 4DMU). The Image was processed using UCSF chimera. Lysines are indicated as blue color. Credit: Science Advances, doi: 10.1126/sciadv.aaw6081

Sasaki and team had previously shown the targeted delivery of checkpoint inhibitor (CPI) antibodies and the cytokine interleukin-2 (IL-2) using a collagen binding domain (CBD) known as the A3 domain of von Willebrand factor (VWF).

The A3 domain of VWF has the highest affinity to collagen types I and III proteins.

This is since collagen is abnormally expressed and exposed to the bloodstream due to hyperpermeability of the tumor vasculature, forming a promising target for cancer drug delivery.

The bioengineered forms in the preceding study showed significantly stronger anti-tumor effects compared to their unmodified forms in mouse models of cancer.

Researchers had also suppressed treatment-related adverse events by fusing CBD to the drug during the tumor targeting strategy.

In the present work, Sasaki et al. hypothesized that CBD would be similarly compatible with SA-based drug delivery carriers to engineer an active serum albumin (SA) with tumor targeting potential.

The research team focused on Doxorubicin (Dox), a small-molecule anticancer drug approved by the US Food and Drug Administration (FDA) to treat a broad spectrum of cancer, where the molecule can internalize in proliferating cells through passive transmembrane diffusion to interfere with DNA functions and cause cell death.

However, its antitumor efficacy is not notable due to acquired drug resistance, bone marrow suppression and excessive inflammation among patient populations, leading to a poor therapeutic index.

To improve Dox efficacy, researchers often combine other chemotherapeutic agents, such as synergized treatment with CPI (checkpoint inhibitors) or Dox conjugated with SA for its release in the low pH (reportedly pH 6.5) tumor microenvironment in a mouse model.

Engineering collagen-binding serum albumin (CBD-SA) as a drug conjugate carrier for cancer therapy
Dox-CBD-SA shows comparable plasma pharmacokinetics with Dox-SA and higher tumor accumulation than aldoxorubicin and Dox-SA. (A) Aldoxorubicin, Dox-SA, or Dox-CBD-SA (5 mg/kg on a Dox basis) was administered to tumor-free FVB mice via tail vein injection. Blood plasma was collected at the indicated time points. Plasma concentration of Dox was measured by fluorescence (mean ± SEM; n = 4 for aldoxorubicin, n = 5 for Dox-SA and Dox-CBD-SA). (B) Plasma half-lives of Dox were calculated using two-phase exponential decay: MFI (t) = Ae−αt + Be−βt. t½, α, fast clearance half-life; t½, β, slow clearance half-life (mean ± SEM; n = 4 for aldoxorubicin, n = 5 for Dox-SA and Dox-CBD-SA). (C) MMTV-PyMT tumor-bearing mice were treated with aldoxorubicin, Dox-SA, or Dox-CBD-SA (4.16 mg/kg on a Dox basis). At the indicated time points, tumors were harvested, and the amount of Dox within the tumors was quantified (mean ± SEM; n = 5 for 2 hours, n = 7 for 24 hours per group). (D) DyLight 488–labeled SA (100 μg) or equimolar amounts of DyLight 488–labeled CBD-SA were injected intravenously to MMTV-PyMT tumor-bearing mice. One hour after injection, tumors were harvested and fluorescence was analyzed by confocal microscopy. Tissues were also stained with 4′,6-diamidino-2-phenylindole (DAPI) and anti-CD31 antibody. Scale bars, 100 μm. Representative images of three tumors each. Two experimental replicates. Statistical analyses were done using analysis of variance (ANOVA) with Tukey’s test. *P < 0.05; **P < 0.01; N.S., not significant. Credit: Science Advances, doi: 10.1126/sciadv.aaw6081

In the present work, Sasaki et al. designed a recombinant mouse SA by fusing the collagen binding domain of the VWF A3 domain (CBD-SA).

Then they conjugated aldoxorubicin (derivative of Dox) to CBD-SA using a pH-dependent cleavable hydrazone link, prior to experimental injection as the “Dox-CBD-SA” therapeutic agent.

The research team tested the engineered CBD-SA as a tumor-targeting drug carrier for improved antitumor efficacy with Dox, in the tumor microenvironment of a translational animal model.

The scientists first synthesized the new drug conjugates to target the tumor microenvironment and investigated the binding potential of CBD-SA to the recombinant collagen protein in vitro to show strong binding affinities to collagen types I and III.

They covalently conjugated aldoxorubicin to CBD-SA and conducted SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to observe the monomeric structure of purified DOX-SA and DOX-CBD-SA molecules.

They examined the release kinetics of Dox from conjugates under varying pH conditions to show maximum release at pH 5.0 and 6.5, consistent with previous reports on small chemical release kinetics in tumor microenvironments.

Engineering collagen-binding serum albumin (CBD-SA) as a drug conjugate carrier for cancer therapy
Dox-CBD-SA shows enhanced antitumor efficacy and infiltration of lymphocytes into tumor in the MMTV-PyMT breast cancer model. (A) MMTV-PyMT cells (5 × 105) were inoculated into FVB mice on day 0. Aldoxorubicin, Dox-SA, or Dox-CBD-SA (5 mg/kg on a Dox basis) was injected intravenously on day 7. Graphs depict tumor volume until the first mouse died (mean ± SEM). (B) Survival rate. (C to F) Individual tumor growth curves. CR indicates complete response frequency. Three experimental replicates. (G to L) MMTV-PyMT cells (5 × 105) were inoculated on day 0. Aldoxorubicin, Dox-SA, or Dox-CBD-SA (5 mg/kg on a Dox basis) was injected intravenously on day 7. Lymphocytes within tumors were extracted on day 14, followed by flow cytometric analysis. (G to I) Graphs depict the number of (G) CD45+CD8+CD3+ T cells, (H) CD45+CD4+CD3+ T cells, and (I) CD45+NK1.1+CD3− NK cells per tumor weight (in milligrams). Bars represent mean ± SEM. (J to L) Graph shows [CD45+CD8+CD3+ T cells per tumor weight (mg)] (J), [CD45+CD4+CD3+ T cells per tumor weight (mg)] (K), or [CD45+NK1.1+CD3− NK cells per tumor weight (mg)] (L) versus [tumor weight]. Two experimental replicates. Statistical analyses were done using (A, H, and I) ANOVA with Tukey’s test or (G) Kruskal-Wallis test followed by Dunn’s test or (B) log-rank (Mantel-Cox) test. *P < 0.05; **P < 0.01. Credit: Science Advances, doi: 10.1126/sciadv.aaw6081

Using cell culture studies the team detected the presence of Dox in the cytoplasm of MMTV-PyMT cells (mouse mammary tumor virus-polyomavirus middle T antigen) after 1-hour of incubation. After 24-hours of incubation with Dox-conjugates they noted the uptake of the liberated drug due to the acidic pH within the intracellular organelles of the cancer cell line. Cell viability tests in the lab verified all forms of Dox in the study to have comparable cytotoxicity to cause cancer cell death in vitro.

Inspired by in-lab pharmacokinetics and tumor accumulation studies, the research team tested the anti-tumor effects of Dox-CBD-SA in vivo in tumor-bearing mice.

For this, they injected the MMTV-PyMT orthotopic tumor-bearing mice with single intravenous injections of several Dox forms via the tail vein. The work showed that pre-conjugation of Dox with SA provided a higher therapeutic effect than the conjugation of aldoxorubicin with endogenous SA in situ.

Importantly, Dox-CBD-SA showed greater therapeutic potential compared to Dox-SA by extending the survival rate and inducing tumor remission in 2 of 12 mice. The data supported the superiority of the Dox carrier compared to the unmodified SA, relative to antitumor efficacy.

Engineering collagen-binding serum albumin (CBD-SA) as a drug conjugate carrier for cancer therapy
TOP: Dox-CBD-SA treatment shows reduced toxicity. Aldoxorubicin or Dox-CBD-SA (20 mg/kg on a Dox basis) was administered to tumor-free FVB mice via tail vein injection on day 0. (A to D) Plasma cytokine concentrations on day 3. (E) Red blood cell (RBC) counts on day 6. (F) White blood cell (WBC) counts on day 3. (G) Spleen weights on day 16. Data represent mean ± SEM. Two experimental replicates. Statistical analyses were done using ANOVA with Tukey’s test. *P < 0.05; **P < 0.01. BOTTOM: Dox-CBD-SA treatment completely eradicates established MC38 tumor in combination with anti-PD-1 CPI. MC38 cells (5 × 105) were inoculated on day 0. Mice were injected intravenously with aldoxorubicin or Dox-CBD-SA (5 mg/kg on a Dox basis) on days 6, 9, and 12. αPD-1 was also injected intraperitoneally on days 10 and 13. (A) The experimental schedule. (B) Graphs depict tumor volume until the first mouse died (mean ± SEM). (C) Survival rate. (D to G) Individual tumor growth curves. (H) On day 60, Dox-CBD-SA + αPD-1–treated survivors were rechallenged by subcutaneous injection of 5 × 105 MC38 cells. Naïve mice were also challenged with the same amounts of cells as a control group. The number of mice that developed palpable tumors is shown. Two experimental replicates. Statistical analyses were done using log-rank (Mantel-Cox) test for survival curves. *P < 0.05; **P < 0.01. Credit: Science Advances, doi: 10.1126/sciadv.aaw6081

Dox also induced immunogenic cell death (ICD) to stimulate tumor-infiltrating lymphocytes (TIL), which were biomarkers for favorable prognosis in multiple cancers.

Sasaki et al. therefore analyzed TILs after treatment with the new drug in the present work and recorded enhanced filtration of lymphocytes for antitumor effects.

The researchers observed lower toxicity of Dox-CBD-SA compared to aldoxorubicin due to its slow release under physiological pH.

For instance, aldoxorubicin increased the plasma concentration of inflammatory cytokines and decreased the red blood cell counts, white blood cell counts and hemoglobin concentration.

By contrast, the adverse impact of Dox-CBD-SA was mild. The work suggested a reduced toxicity spectrum during treatment after pre-conjugation of Dox with CBD-SA.

The research team further investigated if combining Dox-CBD-SA with CPI (checkpoint inhibitors) showed greater therapeutic effects compared to aldoxorubicin therapy with CPI. For this, they used the MC38 colon carcinoma model, which was immunogenic but not curable with Dox monotherapy alone.

They noted the new drug induced immunogenic cell death synergistically with the clinical CPI to achieve superior antitumor effects compared to aldoxorubicin and a clinical CPI.

In this way, Koichi Sasaki and colleagues developed a new therapeutic agent Dox-CBD-SA, which accumulated in tumors to activate host antitumor immunity.

Monotherapy of the new agent suppressed orthotropic MMTV-PyMT breast tumor growth in an animal model and prolonged their survival.

When combined with immune checkpoint inhibition, the drug completely eradicated tumors in an immunogenic MC38 mouse model. They conclude that the pre-conjugation of CBD-SA may hold potential for clinical translation during cancer therapy as an antitumor drug carrier.

DISCUSSION

Because small-molecule anticancer drugs broadly distribute to tissues and induce systemic side effects, modifications of drugs to improve their pharmacokinetics and biodistribution have been attempted. Nanoparticle-formulated (17) or SA-reactive (1819) Dox exhibits improved pharmacokinetics and accumulation within tumors based in part on their pathologically abnormal vasculature (5). However, this effect may not always be effective in human cancers because of their heterogeneity (29). Thus, drugs that are dependent on passive targeting alone may have room for improvement. Active targeting of tumor-specific or tumor-associated antigens for drug delivery is another therapeutic strategy. However, this intrinsically limits the applicable range of cancers and may also lead to acquired drug resistance due to antigen-selective cell targeting and killing, in which antigen may be lost by mutation (30). Here, we engineered CBD-SA to overcome these issues. Unlike other active targeting strategies, CBD-SA does not require the prior investigation of tumor-associated antigen expression because collagen is nearly ubiquitously expressed in tumors, and the CBD gains access to the tumor stroma via the abnormal blood vessel structure within the tumor microenvironment (6). Subsequently, the CBD-SA binds to exposed collagen (Figs. 1C and 2D; fig. S2) and converts the tumor stroma into a reservoir for chemotherapeutics. Dox conjugation to CBD-SA showed significantly higher accumulation of Dox within tumor tissue compared to aldoxorubicin and Dox-SA (Fig. 2C). After accumulation of Dox-CBD-SA within the tumor tissue, the hydrazone linkage, which can be cleaved under the slightly acidic conditions in the tumor microenvironment (Fig. 1E) (21), enables the sustained release of Dox from CBD-SA. At the same time, it is known that tumor cells uptake SA (1). Notably, CBD fusion did not alter the cellular uptake of SA (Fig. 1F), indicating that Dox-CBD-SA can also be delivered intracellularly as efficient as Dox-SA. Thus, part of the Dox release may occur in the tumor stroma, while the Dox-CBD-SA is still matrix-bound, and part may occur in the endolysosomal compartment following endocytosis. Incidentally, cancer stromal targeting (CAST) therapy using stroma-targeting antibodies has been proposed recently (31). The relatively low molecular mass of CBD-SA (88 kDa; fig. S1) may be a benefit compared to stroma-targeting antibodies in terms of diffusion into tumor tissues (32).

Regarding the payload of Dox, we conjugated about three Dox molecules per CBD-SA (Fig. 1D). The drug-to-protein ratio of antibody-drug conjugates is typically close to 4 (33). Considering that CBD-SA is 59% of the molecular weight of an immunoglobulin G (IgG) antibody, CBD-SA at a ratio of 3 achieved a high payload of Dox compared to typical antibody-drug conjugates. In addition, SA fusion increases the solubility of the CBD, which is important for hydrophobic drugs (4).

In terms of antitumor efficacy, Dox-CBD-SA significantly suppressed the growth of MMTV-PyMT breast cancer and extended the survival of mice compared to aldoxorubicin and Dox-SA (Fig. 3, A to F). Because Dox-CBD-SA showed the highest accumulation into tumor tissue in vivo, Dox-CBD-SA should induce tumor cell death more efficiently via inhibition of tumor cell proliferation. In addition to this effect, a single injection of Dox-CBD-SA brought a long-lasting therapeutic effect in spite of its faster plasma clearance half-life (Fig. 2, A and B). This could be explained by our observation that Dox-CBD-SA treatment induces a higher number and density of TILs compared to Dox-SA and aldoxorubicin treatments (Fig. 3, G to L). Therefore, the antitumor mechanism of action of Dox-CBD-SA may be not only direct cell killing but also the stimulation of host antitumor immunity. Since Dox-CBD-SA efficiently accumulates within tumors, it may induce ICD and tumor antigen exposure to the immune system more efficiently than aldoxorubicin and Dox-SA. As a consequence, Dox-CBD-SA synergistically eradicated MC38 colon carcinoma when administered in combination with αPD-1 (Fig. 5, B and G). Improved therapeutic efficacy of Dox-SA and Dox-CBD-SA in comparison with aldoxorubicin (Fig. 3, A to F) also indicates that preconjugation of Dox before injection would provide higher antitumor efficacy. In addition to rapid clearance from blood circulation, in situ conjugation of aldoxorubicin with other sulfhydryl compounds such as cysteine, glutathione, fibronectin, or α1-antitrypsin in plasma (18) is also a possible cause of inefficient therapeutic efficacy of aldoxorubicin.

Cardiac toxicity is a major drawback of Dox, which limits the lifetime cumulative dose of Dox (13). Histological analysis revealed that even Dox-CBD-SA administration (20 mg/kg) did not show any signs of cardiac damage (fig. S9). This suggests that Dox preconjugated with CBD-SA is less cardiotoxic than free Dox, which irreversibly damages cardiac tissue at a cumulative dose of 15 mg/kg in mouse (34). A cumulative dose of 15 mg/kg is nearly equivalent to the maximum cumulative dose in human (35). We hypothesize that unconjugated Dox could penetrate into and damage cardiac tissue, whereas its preconjugated form would not penetrate into tissues or release Dox under physiological pH (i.e., within nonmalignant tissue or blood), contributing to reduced adverse events.

In terms of the manufacturing process, we conjugated Dox using Traut’s reagent, which allows precise control of the drug conjugation ratio (36). This method has little risk to abrogate binding between the CBD and collagen since there are no lysine residues at the binding interface between the VWF A3 domain and collagen (fig. S5) (22). Moreover, SA contains approximately sevenfold the number of lysine residues as the CBD sequence (table S1), also suggesting the low risk of unfavorable conformational changes in the CBD due to conjugation. Traut’s reagent is also used for an ADC targeting CD70 (MDX-1203, Bristol-Myers Squibb) (37), indicating its translational applicability. As CBD-SA is produced with high yield [human embryonic kidney 293 (HEK293) cell culture (~70 to 100 mg/liter)], we propose that preconjugation of Dox to CBD-SA produces high antitumor efficacy with a simple and translatable production method.

As a potential limitation, CBD-SA might accumulate in undesirable sites in the body such as liver, kidney, and wounds, where collagens may be exposed via a fenestrated or leaky endothelium, although Dox would not be released from the CBD-SA if such locations are at neutral pH. At least, we did not observe pathological damage in the liver and kidney after Dox-CBD-SA administration (20 mg/kg) (fig. S9). As another limitation, chemical conjugation may decrease the half-life of SA in general. Methotrexate conjugation reportedly accelerated the clearance of methotrexate-SA conjugates from circulation in a drug-to-protein ratio–dependent manner (38). In this study, the half-lives of Dox-SA and Dox-CBD-SA were shorter than the reported half-life of native mouse SA [t½, β = 35 (hours)] (39). The reason why aldoxorubicin showed a relatively longer half-life than Dox-SA conjugates is probably that it reacted with endogenous SA at a 1:1 ratio due to the abundance of SA in circulation.

In conclusion, Dox-CBD-SA accumulated into tumors and activated host antitumor immunity. As a consequence, monotherapy of Dox-CBD-SA suppressed orthotopic MMTV-PyMT breast tumor growth and prolonged survival. Combination therapy of Dox-CBD-SA with immune checkpoint inhibition via αPD-1 completely eradicated tumors in the immunogenic MC38 model. CBD fusion provided an active targeting ability to SA, which is classically used as a passively targeted drug carrier, enabling effective drug delivery to tumors from the systemic circulation. CBD-SA is expected to be nonimmunogenic and biologically acceptable because it is composed of two proteins (VWF A3 domain and SA) that naturally exist in the blood. Furthermore, CBD-SA acts independently of tumor type–specific antigens and thus provides broad applicability to various types of solid tumors as a drug carrier. Therefore, CBD-SA may hold potential for clinical translation to cancer therapy as an antitumor drug carrier.

More information: Koichi Sasaki et al. Engineered collagen-binding serum albumin as a drug conjugate carrier for cancer therapy, Science Advances(2019). DOI: 10.1126/sciadv.aaw6081

Jun Ishihara et al. Targeted antibody and cytokine cancer immunotherapies through collagen affinity, Science Translational Medicine (2019). DOI: 10.1126/scitranslmed.aau3259

Robert C. Young et al. The Anthracycline Antineoplastic Drugs, New England Journal of Medicine (2010). DOI: 10.1056/NEJM198107163050305

Journal information: Science Advances , Science Translational Medicine , New England Journal of Medicine
Provided by Science X Network

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