Enhanced Immunogenic Cell Death Based On HPMA Copolymers For Anticancer Therapy

Primary tumors may initially respond to chemotherapy,but residual cancer cells can cause lethal tumor recurrence and metastasis after termination of the treatments.An ideal chemotherapy regimen requires（1）a direct killing effect on the primary tumor and（2）a long-term immune surveillance effect on potential residual and metastatic tumors.However,the effective combination of chemotherapy and immunotherapy is barely reported.In the past,apoptosis was hypothesized to be poorly immunogenic,but Guido Kroemer et al.found that immunogenic cell death（ICD）would occur when tumor cells were treated with specific apoptosis inducers.Tumor cells that undergoing ICD upregulate"eat me"and"danger"signals.The"eat me"signal,such as the exposure of calreticulin（CRT）on the cell surface promotes the phagocytosis of tumor cells by dendritic cells and the presentation of tumor-associated antigens.On the other hand,the extracellular release of"danger"signals inculuding adenosine triphosphate（ATP）and high mobility group box-1 protein（HMGB1）can recruit dendritic cells to the tumor site,promote dendritic cell maturation and trigger antigen-specific T cell response.Therefore,ICD can harness the immune system to recognize and eliminate residual tumors.This strategy that can greatly improve the treatment outcome of tumor chemotherapy.However,at present,most antitumor drugs only show cytotoxicity-dependent antitumor activity,and only 5.3%of FDA-approved antitumor drugs can induce ICD.Many antitumor agents,such as cisplatin,can only induce the release of ATP and HMGB1 from tumor cells,but fail to induce the exposure of CRT.Thus,ICD cannot be successfully provoked.In addition,a few drugs that can induce ICD（such as anthracyclines）also have poor tumor accumulation（less than 1%intravenously injected drugs）.Based on this,we wonder whether a drug delivery system could be constructed to complement the deficiency in the ICD stimulation process,transforming non-immunogenic drugs into immunogenic ones,increasing tumor retention and activating antitumor immune response.Endoplasmic reticulum（ER）stress is closely related to the survival and development of tumor.Abnormal high ER stress can induce ICD production and activate antitumor immune response.Cardiac glycosides such as digoxin have been found to indirectly generate ER stress by interacting with Na⁺/K⁺-ATPase on the cell membrane and promoting Ca²⁺influx,thus inducing CRT exposure and amplifying ICD.However,the combination of cardiac glucoside with drug delivery systems has not been reported.Water soluble N-（2-hydroxypropyl）methacrylamide（HPMA）copolymer has been studied as drug carrier for decades due to good biocompatibility.It can take advantage of the damaged lymphatic circulation to remain in the tumor sites.In contrast to free drugs,some HPMA conjugates also exhibited immune stimulation ability.These results suggested that HPMA polymer backbone might amplify the immunogenicity of drug.However,the underline mechanisms remain to be elucidated.Therefore,in the first chapter of this thesis,digoxin,an exceptionally efficient CRT exposure inducer,was used in combination with cisplatin to stimulate ICD.In order to further increase the tumor retention and enhance the immunomodulatory effect,cisplatin was conjugated to HPMA copolymer to obtain P-Cis.Firstly,the cytotoxicity of P-Cis was investigated by MTT assay,and it was found that the cytotoxicity of P-Cis was significantly improved compared with Cisplatin.After intratumoral administration,ICP-MS was used to measure the drug retention in tumors.The results demonstrated that the intratumoral retention of P-Cis was 7-fold higher than that of Cisplatin（P<0.05）.Confocal microscopy and flow cytometry were used to investigate the exposure of CRT on the cell surface.The results revealed that Cisplatin and P-Cis alone failed to induce CRT exposure,but a large number of CRT could be observed on the cell surface after addition of Dig（Cisplatin+Dig and P-Cis+Dig）.The concentration of ATP in the supernatant of treated cells was quantitatively determined by chemiluminescence method.The positively charged HPMA polymer backbone was able to induce the extracellular release of a large amount of ATP.Compared with Cisplatin,ATP release in P-Cis group was significantly increased,which was 10.9-fold higher than that in Cisplatin group and 2.2-fold higher than that in polymer backbone group,respectively（P<0.05）.After the combination of Dig,P-Cis+Dig could induce even more ATP release,which was 1.2-fold higher than that of P-Cis（ATP in tumor microenvironment can be used as an immune adjuvant to promote ICD,P<0.05）.In in vivo antitumor efficacy assay,compared with the weak tumor inhibition ability of Cisplatin+Dig,both P-Cis and P-Cis+Dig can significantly inhibit the tumor growth（tumor inhibition rate:more than 95%,P<0.05）.The primary tumor at the inoculation site almost disappeared after treatment.Furthermore,the legs of mice were dissected and stained with hematoxylin-eosin（H&E）,and it was found that there were residual tumor infiltrating into the muscle tissue of mice in P-Cis group,while no significant residual tumor tissue was observed in P-Cis+Dig group.These results suggested that P-Cis+Dig had good antitumor effect（tumors disappeared completely in 9 out of 10 mice）.To investigate the antitumor immune response of this combination strategy in vivo,T cells in tumor sites were stained and analyzed.The infiltration of CD3⁺T lymphocytes in P-Cis+Dig group was 24.8-fold and2.8-fold higher than that in Cisplatin+Dig group and P-Cis group,respectively（P<0.05）.The ratio of cytotoxic T lymphocytes（CTL）and immunosuppressive regulatory T lymphocytes(T_reg)in tumors was determined,and the ratio of CD8⁺CTL/Treg in P-Cis+Dig group was also significantly higher than that in P-Cis group.Finally,in order to investigate the anti-metastasis effect of P-Cis+Dig group in vivo,mouse models with bilateral melanoma and pulmonary metastatic melanoma were established,respectively.The results showed that P-Cis+Dig could significantly inhibit the growth of not only primary tumor,but also distant tumor via the provoked anti-tumor immune response（tumor inhibition rate:80.8%,P<0.05）.Furthermore,tumor cells succumbing to P-Cis+Dig served as a vaccine to block the metastasis of tumor cells in the blood to the lungs.The metastatic nodules in the lungs of mice in the P-Cis+Dig group were significantly reduced when compared with Control group and P-Cis group.Therefore,the lung weight of P-Cis+Dig group was close to that of normal mice.All these results indicated that the combination strategy reported here could transform non-immunogenic drugs into immunogenic.Tumor cells killed by this strategy further stimulated anticancer immune response,which provided new ideas for anticancer chemotherapy.In the first chapter,Dig was used to promote CRT exposure.However,Dig has three disadvantages:1)the effect of Dig on inducing CRT exposure is indirect;2)narrow therapeutic window and 3)poor tumor accumulation.Doxorubicin,an anthracycline,can cause ER stress and induce CRT exposure while passing through the ER.Nevertheless,doxorubicin prefers to accumulate in nuclei instead of ER.We hypothesized that doxorubicin might be able to achieve more efficient CRT exposure and enhanced ICD if it was targeted to ER.Therefore,in the second chapter of this thesis,we attempted to construct an ER targeted drug delivery system that can induce CRT exposure more effectively and enhance ICD for stronger antitumor effect.At present,there are few studies on ER targeted drug delivery systems.In general,ER targeting sequences or small molecules that interacted with specific sites of ER were used for ER targeting.Based on this,we hypothesized that if the ER targeting sequence was conjugated to doxorubicin,the obtained ER targeted doxorubicin derivatives might realize greater ER stress and more CRT exposure.Therefore,five ER targeted doxorubicin derivatives were prepared.Three short ER targeting peptides（KKAA,KKKEK,KDEL）were conjugated to doxorubicin,respectively.Two small molecules（p-toluene sulfonyl and butyryl）which were reported to interact with specific sites in ER were also grafted to doxorubicin,respectively.Firstly,the ER targeting ability of doxorubicin and its derivatives were investigated.The results showed that among all the derivatives,p-toluene sulfonyl modified doxorubicin derivative（Phe-DOX）had the strongest ER targeting ability.Its colocation coefficient with ER was 0.94,which was 18.8-fold higher than that of doxorubicin（P<0.05）.Phe-DOX also exhibited the strongest ability to induce CRT exposure.In CRT exposure assay,Phe-DOX exhibited the most potent efficacy.Specifically,Phe-DOX induced much more CRT exposure（75.4%CRT positive）,compared with doxorubicin（28.3%）.Thus,we chose Phe-DOX in the following experiment.Furthermore,we conjugated Phe-Dox into HPMA copolymer to obtain P-Phe-DOX（doxorubicin conjugated HPMA copolymer,P-DOX）.This conjugation was based on the hydrazone bond formation between ketone carbonyl group in doxorubicin derivative and the hydrazine group in HPMA copolymer precursor（P-NHNH₂）.After conjugation,we found that the ER targeted P-Phe-Dox selectively accumulated in ER and induced CRT exposure more effectively than unmodified P-DOX（2.1-fold higher,P<0.05）.However,the cytotoxicity of P-Phe-DOX was weak,which might be attributed to that the action site of doxorubicin is nucleus rather than ER.Thus,cytotoxic P-DOX was added to further complement ICD because cell death is necessary for activating anticancer immune response during ICD.In in vitro cytotoxicity and apoptosis study,P-Phe-Dox+P-DOX group not only showed a significant synergistic effect（combination index<1）but also induced more apoptotic tumor cells（89.3%）,which was 4.1-fold higher and 2.4-fold higher than that of P-Phe-DOX and P-DOX,respectively.In addition,combination group also showed the strongest ability to induce the extracellular release of ATP and HMGB1,compared with unmodified P-DOX or ER targeted P-Phe-DOX alone.In in vivo anticancer study,it also had a stronger tumor inhibition rate of 71.1%（P-DOX 38.8%,P-PHE-DOX 31.8%）.Analysis of immune cell types in tumor tissues showed that the combination strategy induced more cytotoxic CD8⁺T cell infiltration.Moreover,the ratio of CD8⁺CTL/T_reg representing the anticancer immune response potency was measured.The CD8⁺CTL/T_reg ratio of P-Phe-Dox+P-DOX was 3.6-fold,3.2-fold and 1.64-fold higher than that of P-Phe-DOX,P-DOX and P-DOX+Dig,respectively.These results suggested that targeting ER led to higher ER stress and better antitumor effect in vivo.This strategy provided a new strategy for antitumor therapy.But it was worth noting that although we combined P-Phe-DOX and P-DOX,residual tumors were observed after treatment.Therefore,we further analyzed the programmed cell death protein 1 ligand（PD-L1）expression on tumor cells and found that the expression of PD-L1 was up-regulated in tumor cells after chemotherapy.This result implied that part of the tumor cells avoided the attack of the immune system and survived.Thus,in the third chapter of this thesis,we attempted to reduce the expression of PD-L1 and reawaken the tumoricidal ability of antitumor immune response.Traditional PD-1 monoclonal antibody binds to PD-1 on T cells to block PD-1/PD-L1 pathway and restarts the tumoricidal ability of immune cells.However,it has several disadvantages including serious immune-related adverse reactions,high cost,inconvenient transportation and storage.The small molecule BET bromodomain inhibitor JQ1 has been reported as a potential candidate to inhibit PD-L1 expression,but the poor pharmacokinetic hinders its in vivo efficacy.Erythrocyte membrane camouflage technic is reported to be biocompatible and effective in prolonging in vivo circulation.Therefore,we not only loaded JQ1 into PLGA nanoparticles,but also used erythrocyte membrane camouflage to construct RNP@JQ1.The dynamic diameter and zeta potential of RNP@JQ1 was around 80nm and-20 m V,respectively.The nanoparticles wrapped with erythrocyte membrane had similar characteristic proteins in SDS-PAGE based protein separation assay,compared with the erythrocyte membrane.TEM images showed that RNP@JQ1 had a regular spherical morphology.In in vivo experiments,compared with PEG-modified nanoparticles（PNP@JQ1）,RNP@JQ1 had a longer in vivo circulation and more efficiently inhibited the expression of PD-L1 in tumor tissues.We further investigated whether the addition of RNP@JQ1 could enhance the anticancer efficacy of P-Phe-DOX+P-DOX.The results showed that the addition of RNP@JQ1 could further increase the tumor inhibition rate to 85.2%（the tumor inhibition rate of P-Phe-DOX+P-DOX group was 71.1%）.Analysis of immune cell types in tumor tissues showed that the addition of RNP@JQ1 failed to induce more CD8⁺T lymphocytes into tumor tissue,meaning that RNP@JQ1 alone could not activate antitumor immune response.However,the expression of PD-L1 in tumor tissues was significantly reduced by RNP@JQ1,suggesting that RNP@JQ1improved the antitumor efficacy by down-regulating PD-L1 expression and subsequently unleashing the tumor killing ability of the antitumor immune response.In summary,we proposed three strategies in this thesis to improve therapeutic efficacy of HPMA copolymer drug delivery system by enhancing immunogenic cell death:（1）In the first part,we used Dig to indirectly interact with the ER and induce subsequent CRT exposure.Cisplatin which is a non-immunogenic cell death inducer was conjugated to HPMA copolymer for further enhancement.P-Cis+Dig combination could transform dying tumor cells into tumor vaccines to further provoke a long-term immune response to eliminate the residual tumor and prevent recurrence and metastasis.（2）In the second part,we constructed a drug delivery system that could directly target the ER and induce CRT exposure more effectively.The results showed that the combination of P-Phe-DOX+P-DOX could synergically kill tumor cells and had a stronger tumor inhibition efficacy in vivo.（3）In addition,we also found that after chemotherapy,tumor cells up-regulated the expression of PD-L1 to avoid immune surveillance.Therefore,in the third part,RNP@JQ1 was used to reduce the expression of PD-L1.PD-L1 inhibition amplified antitumor immune response induced by P-Phe-DOX+P-DOX and achieved a stronger tumor inhibition.We provided a generalizable framework of complementing CRT exposure deficiency of chemotherapeutic and combining immune checkpoint blockade to combat cancer.