Complete Inhibition Of Tumor Growth Research Articles

Photothermal induced chemo-immunological synergistic therapy for anaplastic thyroid carcinoma treatment

It is still of great challenge for anaplastic thyroid carcinoma (ATC) treatment since ATC is an extremely destructive and lethal thyroid malignancy. Herein, a kind of multifunctional nanoplatform (denoted as PDA-dsCpG-DOX NP) has been prepared and applied for tri-modal ATC therapy including photothermal therapy (PTT), chemotherapy, and immunotherapy. Polydopamine nanoparticle (PDA NP) serves as a photothermal agent to generate PTT, as well as a carrier of doxorubicin (DOX) and immunotherapeutic DNA (i.e., oligonucleotide comprising unmethylated cytosine-phosphate-guanine dinucleotide (CpG ODN)). The high temperature induced by PTT results in the release of CpG ODNs for immunotherapy and the liberation of DOX for chemotherapy. PDA-dsCpG-DOX NPs exhibit a phenomenal collaborative therapy effect contrasted with a corresponding single therapy or dual-modal treatment. By use of mice bearing C643 thyroid tumors as a model, PDA-dsCpG-DOX NPs have been demonstrated to realize nearly complete tumor growth suppression with good biocompatibility. Hence, PDA-dsCpG-DOX NPs introduce a synergistic scheme for ATC treatment and foreground the congregate effects of tri-modal therapy.

Polymer theranostics with multiple stimuli-based activation of photodynamic therapy and tumor imaging.

Background: Efficient theranostic strategies concurrently bring and use both the therapeutic and diagnostic features, serving as a cutting-edge tool to combat advanced cancers. Goals of the Investigation: Here, we develop stimuli-sensitive theranostics consisting of tailored copolymers forming micellar conjugates carrying pyropheophorbide-a (PyF) attached by pH-sensitive hydrazone bonds, thus enabling the tumor microenvironment-sensitive activation of the photodynamic therapy (PDT) effect, fluorescence or phosphorescence. Results: The nanomedicines show superior anti-tumor PDT efficacy and huge tumor-imaging potential, while reducing their accumulation, and potentially side effects, in the liver and spleen. The developed theranostics exhibit clear selective tumor accumulation at high levels in the mouse sarcoma S180 tumor model with almost no PyF found in the healthy tissues after 48 h. Once in the tumor, illumination at λexc = 420 nm reaches the therapeutic effect due to the 1O2 generation. Indeed, an almost complete inhibition of tumor growth is observed up to 18 days after the treatment. Conclusion: The clear benefit of the specific PyF release and activation in the acidic tumor environment for the targeted delivery and tissue distribution dynamics was proved. Conjugates carrying pyropheophorbide-a (PyF) attached by pH-sensitive hydrazone bonds showed their excellent antitumor PDT effect and its applicability as advanced theranostics at very low dose of PyF.

Disease-driven engineering of peptide-targeted DM1 loaded liposomal nanoparticles for enhanced efficacy in treating multiple myeloma by exploring DM1 prodrug chemistry

Here, we report a CD138 receptor targeting liposomal formulation (TNP[Prodrug-4]) that achieved efficacious tumor growth inhibition in treating multiple myeloma by overcoming the dose limiting severe toxicity issues of a highly potent drug, Mertansine (DM1). Despite the promising potential to treat various cancers, due to poor solubility and pharmacokinetic profile, DM1's translation to the clinic has been unsatisfactory. We hypothesized that the optimal prodrug chemistry would promote efficient loading of the prodrug into targeted nanoparticles and achieve controlled release following endocytosis by the cancer cells, consequently, accomplish the most potent tumor growth inhibition. We evaluated four functional linker chemistries for synthesizing DM1-Prodrug molecules and evaluated their stability and cancer cell toxicity in vitro. It was determined that the phosphodiester moiety, as part of nanoparticle formulations, demonstrated most favorable characteristics with an IC50 of ∼16 nM. Nanoparticle formulations of Prodrug-4 enabled its administration at 8-fold higher dosage of equivalent free drug while remaining below maximum tolerated dose. Importantly, TNP[Prodrug-4] achieved near complete inhibition of tumor growth (∼99% by day 10) compared to control, without displaying noticeable systemic toxicity. TNP[Prodrug-4] promises a formulation that could potentially make DM1 treatment available for wider clinical applications with a long-term goal for better patient outcomes.

A Light-Responsive Injectable Hydrogel with Remodeling Tumor Microenvironment for Light-Activated Chemodynamic Therapy.

Chemodynamic therapy (CDT) based on Fenton-like reaction is often limited by the tumor microenvironment (TME), which has insufficient hydrogen peroxide, and single CDT treatment is often less efficacious. To overcome these limitations, a hydrogel-based system is designed to enhance the redox stress (EOH) by loading the composite nanomaterial Cu-Hemin-Au, into the agarose hydrogels. The hydrogels can reach the tumor site upon intratumoral injection, and then coagulate and stay for extended period. Once irradiated with near-infrared light, the Cu-Hemin-Au act as a photothermal agent to convert the light energy into heat, and the EOH gradually heated up and softened, releasing the Cu-Hemin-Au residing in it to achieve photothermal therapy (PTT). Benefiting from the glucose oxidase (GOx)-like activity of the Au nanoparticles, glucose in the tumor cells is largely consumed, and hydrogen peroxide (H2 O2 ) is generated in situ, and then Cu-Hemin-Au react with sufficient H2 O2 to generate a large amount of reactive oxygen species, which promote the complete inhibition of tumor growth in mice during the treatment cycle. The hydrogel system for the synergistic enhancement of oxidative stress achieves good PTT/CDT synergy, providing a novel inspiration for the next generation of hydrogels for application in antitumor therapy.

The Novel Bispecific Innate Cell Engager (ICE®) AFM28 Efficiently Directs Allogeneic NK Cells to CD123+ Leukemic Stem- and Progenitor Cells in AML

Introduction: Acute myeloid leukemia (AML) is a common hematologic malignancy characterized by expansion of undifferentiated myeloid cells in the bone marrow (BM) and peripheral blood. Although AML can be treated with curative intent, residual leukemic stem cells (LSCs) are frequently reservoirs for relapse, which is associated with a poor prognosis. Moreover, especially elderly patients are often not eligible for intensive treatment regimen. Therefore, there is a high need for novel treatment strategies, which are capable of eliminating LSCs in order to induce long-term remissions, suppress measurable residual disease (MRD) and prevent relapse. A promising emerging approach is the adoptive transfer of allogeneic natural killer (NK) cells, which was shown to be clinically active and could induce complete remissions in relapsed/refractory (R/R) AML. Innate Cell Engagers (ICE®) may further augment the activity of NK cell therapies, in particular against LSCs and hold the potential to increase frequency and durability of responses. The tetravalent bispecific CD123/CD16A ICE®, AFM28, directs the cytotoxic activity of CD16A-expressing NK cells towards CD123-expressing (CD123+) AML blasts and LSCs. In this study, we evaluated the efficacy of AFM28 in pre-clinical models of AML. Methods: Antibody-dependent cell-mediated cytotoxicity (ADCC) data were obtained by a 4h calcein release assay using AML cell lines and primary human NK cells (E:T ratio 2.5:1) in the presence of titrated antibodies. CD64 and CD123 expression levels on AML cell lines were evaluated by flow cytometry. Intracellular FACS was used to detect STAT5/pSTAT5. Proliferation of TF-1 cells in the presence of IL-3 or GM-CSF was measured via CellTiter Glo Assay (Promega). Using primary BM samples of n=6 AML patients and n=4 healthy donors, ADCC assays and colony-forming unit (CFU) assays were performed. BM-MNC (ADCC assay) or enriched CD34+ cells (CFU assay) and allogeneic NK cells (E:T ratio 1:1) were treated with AFM28 or an Fc-enhanced anti-CD123 antibody comparator at different concentrations of AFM28 (0-500 or 0/10/100/1000 pM, respectively) for 24h. Subsequently, ADCC assays were analyzed by flow cytometry and CFU assays were evaluated after 7-14 days. Systemic anti-tumor activity of AFM28 in vivo was assessed using in-life imaging (BLI) in a T cell-depleted huFcgR-C57BL6 transgenic mouse model against intravenously administered luciferase-transfected murine AML cells expressing human CD123 as tumor antigen. Results: Pre-clinical in vitro models using a panel of AML cell lines showed efficacious ADCC against CD123+ tumor cells by allogeneic NK cells in the presence of AFM28. ADCC induction by AFM28 was independent of leukemic cell mutational profiles and even targeted cells with low levels of CD123 surface expression. Of note, expression of the high-affinity IgG receptor CD64 on AML cell lines completely abolished ADCC induction by an Fc-enhanced anti-CD123 antibody whereas AFM28 also showed high ADCC efficacy against all tested CD64+ cell lines. In addition, AFM28 exerted an NK cell-independent inhibitory effect on the IL-3-induced phosphorylation of STAT5 and abolished proliferation of CD123+ TF-1 cells, indicating an antagonistic effect on IL-3R signaling. In ex vivo models using primary AML patient cells, AFM28 demonstrated strong ADCC mediated by allogeneic NK cells. CFU assays of patient-derived AML BM samples performed subsequent to incubation with allogeneic NK cells with and without AFM28 showed significantly reduced numbers of outgrowing colonies in AFM28 treated samples, indicating that AML LSCs and progenitor cells were eliminated. In the in vivo efficacy study with huFcgR-C57BL6 mice, treatment with AFM28 at 15 mg/kg b.w. led to complete inhibition of tumor growth in 5/5 animals throughout a treatment period of 42 days. In contrast, all untreated control mice (6/6) developed systemic disease. Conclusion: AFM28 induced highly potent and selective lysis of CD123+ leukemic cells, including LSCs and progenitor cells, by allogeneic NK cells in vitro and in vivo. The capacity to eradicate LSCs holds the promise for durable responses and the potential for long-term remissions in patients with R/R AML. AFM28 is currently being prepared for a first-in-human clinical investigation as monotherapy and in combination with allogeneic NK cells.

IKS03, a Next Generation CD19-Targeted Antibody Drug Conjugate, Shows Potent Activity in Preclinical Models of Aggressive B-Cell Lymphomas

Diffuse large B-cell lymphoma (DLBCL) is a common subtype of non-Hodgkin lymphoma (NHL). Although many patients respond to frontline chemoimmunotherapy, and treatment options for patients with relapsed or refractory DLBCL have improved with the success of CAR T cell therapies, clinical outcome for many patients is still suboptimal. CD19 is widely expressed in DLBCL tumor cells, with normal tissue expression limited to B-cells, making it an attractive target for targeted therapies including antibody drug conjugates (ADC). IKS03 is a CD19-targeting ADC with a PBD dimer pro-drug payload that induces DNA crosslinking and blocks DNA replication ultimately leading to cell death. Following tumor selective binding and uptake, IKS03 requires intracellular lysosomal processing of beta-glucuronidase protecting groups to fully activate the payload which minimizes systemic release of the PBD dimer in human plasma. IKS03 is derived from a human antibody that binds with nanomolar affinity to human and cynomolgus monkey CD19. It is generated by site-specific bioconjugation yielding a homogeneous conjugate with a drug to antibody ratio of 2. In vitro, IKS03 induces specific and dose dependent cell killing of diverse CD19+ lymphoma cell lines with sub-nanomolar EC50 values. Conversely, in vitro cytotoxicity of the conjugate against normal human primary peripheral blood mononuclear cells was attenuated with an EC50 of approximately 1 μM, similar to a non-PBMC binding ADC bearing the same PBD pro-drug. The unconjugated payload more potently reduced PBMC viability with a sub-nanomolar EC50. This is consistent with low non-specific payload release from the conjugate underscoring the improved properties of the PBD pro-drug design. Pharmacokinetics of IKS03 was assessed in a repeat dose toxicity study in monkeys and the plasma concentrations of total ADC and total antibody were determined by LC-MS/MS across the 3-week dosing interval. Overall mean half-life for total ADC was approximately 8 days and clearance rate was slow (mean 0.2 mL/h/kg). Total ADC peak concentration, total plasma exposure and half-life were comparable to that of total antibody demonstrating favorable ADC stability in plasma with limited systemic payload release. In vivo efficacy was evaluated in CD19-expressing preclinical human xenograft models representing different subtypes of B-NHL. Granta-519 cells contain the CCND1 t(11;14) translocation often present in mantle cell lymphoma (MCL). IKS03 was highly active against Granta-519 xenografts in SCID mice with complete tumor regressions observed with a single dose of 0.1 mg/kg. Efficacy was further evaluated in CD19-positive, low passage DLBCL patient-derived xenograft (PDX) models of known genomic profiles. IKS03 was highly active yielding complete tumor regressions following a single dose of 0.3 mg/kg in a refractory lymphoma Bcl2+ PDX model derived from a patient with no response to prior CHOP treatment. Post tumor collection, this patient was also refractory to EPOCH chemotherapy. A second xenograft model was derived from a treatment naïve high-grade 'triple hit' lymphoma patient (BCL2+; BCL6+; MYC+; XP01 amplified). Post tumor collection, this patient was refractory to R-CHOP therapy with disease progression within 2 months. Tumors rapidly progressed in this model, yet single dose treatment with IKS03 resulted in significant tumor growth inhibition at 0.3 mg/kg and complete tumor regressions at 1 mg/kg. Likewise, in a pre-treated MYC-amplified GCB lymphoma model, a single IKS03 dose of 0.3 mg/kg inhibited tumor growth and 1 mg/kg yielded complete inhibition of tumor growth. In conclusion, IKS03 is highly effective in preclinical MCL and DLBCL xenograft models that contain some of the genetic alterations commonly found in relapsed/refractory NHL patients in need of treatment. Preclinical data further demonstrates that IKS03's advanced ADC design results in specific target-dependent in vitro activity, excellent plasma stability and allows for effective targeting of B-cell tumors while sparing normal CD19-negative cells.

Development of a Potent DNA Damaging Agent LP-284 for Treatment of Mantle Cell Lymphoma

Introduction: Mantle cell lymphoma (MCL) is a rare and aggressive type of non-Hodgkin lymphoma that is characterized by the hallmark t(11;14) translocation and subsequent deregulation of expression of the Cyclin D1 (CCND1) gene. Immunochemotherapy followed by autologous stem cell transplantation and/or maintenance therapy remains the most common frontline choice for eligible individuals. Additionally, the introduction of bruton's tyrosine kinase inhibitors (BTKi), anti-CD19 chimeric antigen receptor T-cell therapy, and targeted therapies have advanced the treatment of r/r (relapsed/refractory) MCL. Unfortunately, not all patients are eligible for these therapies and durable remission is rare. Alternative therapies with novel mechanisms of action and increased efficacy are urgently needed for MCL patients. LP-284 is a novel small molecule and DNA damaging agent being developed for the treatment of MCL. The DNA lesions induced by LP-284 are repaired by transcription-coupled DNA repair and can trigger apoptosis in tumors with DNA damage repair deficiencies. As LP-284 DNA lesions block transcription, we hypothesize that LP-284 directly starves transcriptionally addicted tumors such as those with actively transcribing onco-fusion genes such as CCND1. We previously reported that LP-284 shows nanomolar potency across 6 MCL cell lines, including cells resistant to BTKi, bortezomib (Btz.), and venetoclax. Here we report LP-284's efficacy in MCL xenograft mouse models and its ability to induce ATM and subsequent gamma-H2AX phosphorylation, transcriptomic response in MCL cell lines, and preliminary toxicological profile in dogs. Methods and Results: In JEKO1-derived MCL xenograft mice, LP-284 (2 or 4 mg/kg; i.v.) was tested to evaluate its survival benefits and efficacy compared to ibrutinib (Ibr.; 50mg/kg; oral) or Btz. (1 mg/kg; i.p.). LP-284 significantly (p< 0.001; n=7 or 8 per group) increased the overall survival of these mice from 20 days in the vehicle control group to 41 days (LP-284, 2 mg/kg) and 48 days (LP-284, 4 mg/kg). Additionally, LP-284 at either concentration showed significantly increased tumor growth inhibition (TGI) compared to Ibr. or Btz. At day 17 post treatment, 113% TGI was observed in the 4 mg/kg LP-284 arm and 63% TGI was observed in the 2 mg/kg LP-284 arm, whereas Ibr. or Btz. only resulted in 8% and 22% TGI, respectively. We also tested if LP-284 (4 mg/kg) could rescue tumors that grew after treatment of Ibr. or Btz. Our data showed that LP-284 treatment resulted in near complete TGI in tumors pretreated by and grew resistant to Ibr. or Btz. No tumor regrowth nor animal death was observed 15 days after the last dose of LP-284. Additionally, we performed western blot and RNAseq on JEKO1 and REC1 cells treated by LP-284 or the vehicle for 24 hours. LP-284 treatment resulted in increased phosphorylation of ATM and gamma-H2AX in JEKO1, indicating the formation of double-strand DNA breaks. Differential gene expression analysis indicated that LP-284 reduced the expression of CCND1 by ~2 fold and there was a significant down-regulation of the MYC pathway targets. The toxicological profile of LP-284 was evaluated in a non-GLP repeat-dose toxicity study in dogs. LP-284 was administered i.v. once a day on days 1, 8, and 15 and at 0.3, 0.6, or 1.2 mg/kg/doses. Adverse effects in body weight, clinical pathology and pathology were only observed at ≥0.6 mg/kg/dose. LP-284-related changes in hematology were noted at ≥0.3 mg/kg/dose. At the microscopic level, 0.3 mg/kg/dose LP-284-related hematological changes were considered non-adverse based on the small magnitude and no associated changes in clinical hematology compared with the control animals. Therefore, under the condition of this study, the No Observed Adverse Effect Level (NOAEL) was determined to be 0.3 mg/kg/dose. Conclusions: Taken together, our results demonstrated that LP-284 has significantly increased efficacy over Ibr., Btz., and vehicle treated MCL animal models. In addition, Ibr./Btz. resistance could be overcome by LP-284. We also demonstrated that LP-284 reduced the expression of the onco-fusion gene CCND1 and the MYC pathway, which are both genes and pathways implicated in MCL. LP-284's strong potency in in vivo MCL tumor models, novel mechanism of action, and the progress in toxicological studies are in favor of further development of LP-284 as a novel MCL therapy.

VIP943 Is a Novel CD123 Antibody Drug Conjugate with in Vitro and In Vivo Efficacy in Acute Myeloid Leukemia (AML) Models

Background: Current treatment options (cytarabine with anthracyclines) for patients with acute myeloid leukemia (AML) are often associated with severe and barely tolerable toxicities. In patients who tolerate induction therapy, the complete remission rate is 60%-80%; however, 50% of patients relapse due to the presence of leukemic stem cells (LSC). Antibody-drug conjugates (ADCs) are putatively designed to deliver potent drugs directly to cancer cells while sparing normal tissue; however, first-generation ADCs are associated with severe bone marrow and liver toxicity. VIP943 is a CD123-targeting ADC which uses a kinesin spindle protein inhibitor (KSPi) as a new payload class and a unique legumain linker designed to overcome liabilities of currently approved ADC therapies. The payload is delivered with a drug-antibody ratio of 6. It consists of the KSPi warhead which is modified with a CellTrapperTM moiety so it accumulates within the CD123-targeted tumor cell and cannot enter normal cells. Non-human primate toxicology studies demonstrated a highly favorable and differentiated safety profile for VIP943 (Kirchhoff et al, Cancers 2020). Here we characterize VIP943 in various patient-derived AML models. Methods: For co-localization experiments, MOLM-13 AML cell line was stained with labelled anti-CD123 antibody or isotype control and CytoPainter LysoGreen indicator reagent and analyzed by imaging flow cytometry. AML primary patient mononuclear cells from blood and bone marrow were treated with VIP943 for 24, 48, and 72 hours. Flow cytometry immunophenotyping for AML blasts and CD34+/CD38- LSCs was used to analyze these populations. An in vivo patient-derived AML PDX mouse model was treated with VIP943 (5 mg/kg IV every 7 days) or in combination with 5-azacytidine (2.5 mg/kg SC days 1-5 x 3) and venetoclax (50 mg/kg PO days 1-5 x 3). Cytokine release syndrome was analyzed comparing VIP943 to isotype control ADC, anti-CD123 antibody, and positive comparator antibodies by in vitro cytokine release assays (CRA) in two different assay formats using whole blood and isolated mononuclear cells from 10 healthy donors. Flow cytometric lymphocyte and leukemic immunophenotyping was performed from blood of non-human primates. A leukemia cell line panel drug screen with VIP943 was evaluated and baseline genomic features were correlated with response. Results: Co-localization studies confirm specific internalization and efficient intracellular trafficking to the lysosome of VIP943. In vitro treatment with VIP943 (66 nM, continuous exposure) delivers blast reductions of 37% after a 24h incubation with VIP943, 45% after 48h incubation and 83% after an 72h incubation. In the CD34+/CD38- cell population, a reduction of 71% was achieved. In a patient-derived AML model, VIP943 with 5-azacytidine and venetoclax led to complete tumor growth inhibition after 21 days showing superiority of this triplet therapy (complete regression: 8/9 mice) with high tolerability (maximum body weight loss -4.8 %). The immunotoxicological evaluation in both CRA formats and with the tested antibody concentrations (100µg, 10µg, 1µg) exerts no induction of cytokines by VIP943 compared to controls. Immunophenotyping demonstrates no impact of VIP943 treatment on lymphocytic subpopulations, whereas the CD123-positive basophils are reduced after 24h followed by an almost complete recovery. VIP943 IC50 ranges from 0.1nM-300nM in a leukemia cell line panel screen and features associated with sensitivity are investigated. Conclusions: VIP943 is a next-generation ADC with a differentiated safety profile from currently approved ADCs, including lack of in vitro cytokine release. In vitro and in vivo studies using patient-derived AML cells show VIP943 has favorable monotherapy and combination efficacy including targeting of leukemic stem cells, which drive relapse. These findings warrant evaluating VIP943 in clinical trials.

Targeted degradation of PCNA outperforms stoichiometric inhibition to result in programed cell death

Biodegraders are targeted protein degradation constructs composed of mini-proteins/peptides linked to E3 ligase receptors. We gained deeper insights into their utility by studying Con1-SPOP, a biodegrader against proliferating cell nuclear antigen (PCNA), an oncology target. Con1-SPOP proved pharmacologically superior to its stoichiometric (non-degrading) inhibitor equivalent (Con1-SPOPmut) as it had more potent anti-proliferative effects and uniquely induced DNA damage, cell apoptosis, and necrosis. Proteomics showed that PCNA degradation gave impaired mitotic division and mitochondria dysfunction, effects not seen with the stoichiometric inhibitor. We further showed that doxycycline-induced Con1-SPOP achieved complete tumor growth inhibition invivo. Intracellular delivery of mRNA encoding Con1-SPOP via lipid nanoparticles (LNPs) depleted endogenous PCNA within hours of application with nanomolar potency. Our results demonstrate the utility of biodegraders as biological tools and highlight target degradation as a more efficacious approach versus stoichiometric inhibition. Once invivo delivery is optimized, biodegraders may be leveraged as an exciting therapeutic modality.

Modular Smart Molecules for PSMA-Targeted Chemotherapy.

New targeted chemotherapeutics are urgently needed to minimize off-target toxicity and reduce the high-mortality rate associated with metastatic prostate cancer. Herein, we report on the modular synthesis, pharmacokinetics, and efficacy of two small-molecule-drug conjugates (SMDC) targeted to prostate-specific membrane antigen (PSMA) incorporating either: (i) a cathepsin-B-cleavable valine-citrulline (Val-Cit), or (ii) an acid-cleavable phosphoramidate linker. Crucial components used in the design of the conjugates include: (i) CTT1298, a nanomolar affinity ligand that binds irreversibly to PSMA and has proven in past studies to rapidly internalize and shuttle payloads into PSMA-expressing prostate cancer cells, (ii) MMAE, a known potent cytotoxic payload, and (iii) an albumin-binder, proven to improve residence time of drug conjugates. At dose of 0.8 mg/kg (∼250 nmol/kg), the two SMDCs showed significant efficacy in a PSMA(+) PC3-PIP mouse model of human prostate cancer compared with controls, without inducing systemic toxicity. Though localization of the SMDCs was observed in tissues apart from the tumor, release of MMAE was observed predominantly in tumor tissue, at levels that were 2-3 orders of magnitude higher than non-target tissues. Furthermore, SMDC2, which incorporated a novel pH-responsive phosporamidate linker, demonstrated significantly improved efficacy over SMDC1 that has a Val-Cit linker, with a 100% survival over 90 days and 4 out of 8 mice showing complete tumor growth inhibition after 6 weekly doses of 0.8 mg/kg (244 nmol/kg). Our findings demonstrate the potential of irreversible PSMA inhibitors combined with pH-responsive linkers as a way to specifically deliver chemotherapeutic drugs to prostate cancer tumors with minimal toxicity.

Tumor microenvironment regulation - enhanced radio - immunotherapy.

Radiotherapy (RT) is frequently utilized for cancer treatment in clinical practice and has been proved to have immune stimulation potency in recent years. However, its inhibitory effect on tumor growth, especially on tumor metastasis, is still limited by many factors, including the complex tumor microenvironment (TME). Therefore, the TME - regulating SiO2@MnO2 nanoparticles (SM NPs) were prepared and applied to the combination of RT and immunotherapy. In a bilateral animal model, SM NPs not only enhanced the inhibitory effect of RT on primary tumor growth, but also strengthened the abscopal effect to inhibit the growth of distant untreated tumors. As for the distant untreated tumor, 40% of mice showed complete inhibition of tumor growth and 40% showed a suppressed tumor growth. Moreover, SM NPs showed modulation functions for TME through inducing the increase in intracellular levels of oxygen and reactive oxygen species after their reaction with hydrogen peroxide and the main antioxidative agent glutathione in TME. Lastly, SM NPs also effectively induced the increase in the amounts of cytokines secreted by macrophage - like cells, indicating modulation functions for immune responses. This work highlighted a potential strategy of simultaneously inhibiting tumor growth and metastasis through the regulation of TME and immune responses by SM NPs - enhanced radio - immunotherapy.

Abstract ND11: Discovery of GDC-6036, a clinical stage treatment for KRAS G12C-positive cancers

Abstract Mutations in KRAS are the most common oncogenic driver mutations in human cancers. The KRAS G12C mutation is one of the most prevalent KRAS mutations, present in approximately 12% of non-small cell lung cancer, 4% of colorectal cancer, and up to 4% of other cancer types. The recent discovery and development of covalent KRAS G12C inhibitors provides an opportunity to inhibit what has historically been considered to be an “undruggable” target. GDC-6036 is an orally bioavailable, highly potent and selective KRAS G12C inhibitor, with a median IC50 in the sub-nanomolar range and greater than 18,000-fold selectivity for G12C versus non-G12C cell lines. GDC-6036 demonstrates greater potency and selectivity compared with other KRAS G12C inhibitors in vitro, and complete tumor growth inhibition in multiple KRAS G12C-positive cell lines and in xenograft mouse models. We will highlight the research program that led to the discovery and optimization of GDC-6036, which is currently in clinical development. Citation Format: Hans Purkey. Discovery of GDC-6036, a clinical stage treatment for KRAS G12C-positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND11.

Abstract 6281: Preclinical development of a bispecific antibody-trap selectively targeting CD47 and CD24 for cancer immunotherapy

Abstract Limited treatment beneficial rate and market saturation of the first generation of immune checkpoint inhibitors (Such as PD-(L)1 inhibitors) have turned the attention of pharmaceutical industry to the next generation of innate immune checkpoints such as CD47/SIRPα and CD24/Siglec-10. Various cancers of lung, breast, colon, prostate, ovarian, stomach, pancreas, myeloid and lymphoid tissues are known to highly express CD47 and/or CD24, which confer cancer cells means of immune-evasion via induction of “Don’t eat me” signals. Precisely and simultaneously neutralizing CD47 and CD24 signaling on tumors, when coupled with an IgG1 antibody’s potent phagocytosis and cytotoxicity activity, may serve as potent immunotherapy for multiple cancer types. We have developed a humanized CD24 neutralizing antibody which has been revealed to neutralize CD24-Siglec-10 interaction and completely eliminate established colon cancer in syngeneic mouse models via proposed mechanisms of antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and subsequent tumor-specific immunity as a result of macrophage activation. Using this mAb as backbone, we developed an CD24 mAb-CD47 trap by conjugating the SIRPα decoy receptor to the N-terminal of the antibody heavy chain. Initial in vitro studies indicated that this mAb-trap has simultaneous two-target binding as well as blocking activities. More promisingly, eight administrations of the mAb-trap at 3mg/kg dose in mice bearing triple-negative breast cancer resulted in a complete tumor growth inhibition. Furthermore, in a Herceptin-resistant xenograft tumor model, the mAb-trap is also efficacious and resulted in over 60% of tumor growth inhibition when administrated at 7.2mg/kg. Taken together, our data show that precise neutralization of CD47 and CD24 signaling on tumors using IgG1 antibody may serve as potent monotherapy for cancers. Citation Format: Wenzhi Tian, Song Li, Dianze Chen, Dandan Liu, Huiqin Guo, Chunmei Yang, Li Zhang, Wei Zhang, Xiaoping Tu, Liang Peng, Gui Zhao, Ruliang Zhang, Fan Zhang. Preclinical development of a bispecific antibody-trap selectively targeting CD47 and CD24 for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6281.

Abstract 5216: Optimizing antitumor efficacy of granzyme B human fusion constructs targeting the Fn14 antigen in solid tumors. Pre IND pharmacokinetics, schedule, dose optimization and MTD studies

Abstract We previously demonstrated that a human fusion protein construct targeting the Fn14 receptor and containing the cytotoxic granzyme B (GrB) payload (GrB-Fc-IT4) displays impressive in vitro and in vivo cytotoxic effects. Comprehensive mechanism of action studies show that GrB-Fc-IT4 effectively induces cell death in vitro against a broad selection of tumor types with high specificity. The intracellular pathway includes irreversible activation of the caspase cascade and independent mitochondrial depolarization leading to an intense apoptotic cellular damage. This activation occurs quickly and efficiently. Pharmacokinetic studies in mice showed that GrB-Fc-IT4 was cleared bi-exponentially from plasma with a rapid initial clearance (t ½alpha = 0.36 hours) followed by a prolonged terminal-phase plasma half-life (t 1/2beta = 35 hours) similar to that of IgGs. Based on our pharmacokinetic study, we employed a QODX5 therapy schedule and demonstrated impressive antitumor activity against several tumor models including PDX-lung, A459 (NSCLC) and MDA-MB231(TNBC) tumor models. In the present study we compared the in vivo antitumor efficacy of GrB-Fc-IT4 against A549 (NSCLC) tumor cell line, using two different dosing schedules (QODx5 vs QWx5). There was a clear dose and schedule dependence and that treating mice bearing A549 tumors with 32mg/kg/dose using a QOWx5 schedule induced a complete tumor growth inhibition (7/10 tumors regressed) with 3/10 tumors showing no growth up to 50 days after the last dose. Preliminary results suggest that GrB-Fc-IT4 may reduce the formation of spontaneous A549 metastasis into lungs. Further studies are planned to examine the potential of the fusion construct to prevent spontaneous metastatic spread in lung tumor models. Although the fusion construct cross-reacts with the murine Fn14 antigen, maximum tolerated dose studies have confirmed that GrB-Fc-IT4 is a safe product, showing no evidence of toxicity (weight loss) in mice treated with up to 500mg/kg. This suggests that the therapeutic index (TI) for this class of agents is &amp;gt;4. Our data suggest that GrB-Fc-IT4 is a novel class of antitumor agents with a unique mechanism of action. This agent seems to be an exceptionally safe and effective drug and appears to be an ideal candidate for advancing to clinical trials. Research conducted, in part, by Clayton Foundation for Research. Citation Format: Ana Alvarez De Cienfuegos, Lawrence H. Cheung, Khalid A. Mohamedali, Mihai Gagea, Michael G. Rosenblum. Optimizing antitumor efficacy of granzyme B human fusion constructs targeting the Fn14 antigen in solid tumors. Pre IND pharmacokinetics, schedule, dose optimization and MTD studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5216.

Near-Infrared II Plasmonic Phototheranostics with Glutathione Depletion for Multimodal Imaging-Guided Hypoxia-Tolerant Chemodynamic-Photocatalytic-Photothermal Cancer Therapy Triggered by a Single Laser.

Tumor microenvironment (TME)-activatable phototheranostics is highly desirable in cancer management but still remains challenging for clinical applications owing to the lack of multifunctional theranostic agents and the limited tissue penetration depth. Reported here is an "all-in-one" phototheranostic platform based on near-infrared II (NIR-II) dual-plasmonic Au@Cu2-x Se core-shell nanocrystals (dpGCS NCs) for combined photoacoustic (PA)/photothermal (PT) imaging-guided chemodynamic therapy (CDT)/photocatalytic therapy (PCT)/photothermal therapy (PTT) all triggered by a single NIR-II laser. The dpGCS NCs feature excellent NIR-II plasmonic and PT properties, which guarantee their capabilities of NIR-II PA and PT imaging for real-time visual observation of tumor size and location during cancer treatment. Additionally, the TME-activated in situ •OH production via dpGCS NC-catalyzed Fenton-like reaction is further enhanced by the NIR-II irradiation, while photoexcited plasmonic hole-induced formation of extra •OH is also evidenced for PCT. Both in vitro and in vivo experiments confirm remarkable therapeutic efficacy of the present phototheranostic platform under NIR-II laser through the CDT/PCT/PTT trimodal combination therapy, achieving complete inhibition of tumor growth in tumor-bearing mice after administration of dpGCS NCs plus a single NIR-II laser irradiation. This work provides a distinctive paradigm for the development of NIR-II phototheranostic platforms for imaging-guided cancer therapy using a single laser.

ADG152, a Novel CD20xCD3 T Cell Engager Prodrug with Enhanced Therapeutic Index, Demonstrates Strong Anti-Tumor Activity with Improved Safety

Recent clinical data illustrate the effectiveness of CD20xCD3 T cell engagers (TCEs) that redirect the patient's endogenous T cells to eliminate CD20-positive tumor cells. While several of these products have demonstrated promising clinical activities in B-cell malignancies, their potential therapeutic utility is limited by cytokine release syndrome (CRS), even after strategies such as step-up dosing are implemented.ADG152 is a novel CD20xCD3 TCE prodrug engineered using Adagene's SAFEbody technology to minimize or eliminate CRS and on-target/off-tumor toxicities. The anti-CD20 arm of ADG152 has been engineered for enhanced binding to CD20 compared to other clinical stage or approved antibodies, while its anti-CD3 arm has modulated affinity for CD3 and is also masked by a conditionally activable peptide. In normal tissues and in circulation, the masking moiety on the anti-CD3 arm can function to block the binding of ADG152 to T cells; however, in an activable condition such as the tumor microenvironment where protease activity has been reported to be elevated, the masked antibody can be activated, enabling the activated ADG152 to simultaneously engage T cells and neighboring CD20-expressing tumor cells.In vitro studies showed that ADG152 has enhanced binding to human B cells and CD20-positive Raji tumor cells compared with the benchmark CD20xCD3 TCE plamotamab. On the other hand, ADG152 has significantly reduced binding to the human CD3 δ/ε protein dimer and no binding to human CD3+, CD4+, and CD8+ T cells isolated from PBMCs of normal human donors. Consistent with these results, ADG152 shows significantly decreased ability (more than 100-fold) compared with the benchmark and the unmasked parental molecule to activate CD8+ T cells and to induce T cell-mediated killing in the presence of tumor cells in vitro.ADG152 demonstrated strong anti-tumor effects in vivo. In a human PBMC-engrafted Raji xenograft mouse tumor model, dosing with ADG152 resulted in almost complete tumor growth inhibition at 1.5 mg/kg. In exploratory toxicology studies in cynomolgus monkeys, ADG152 resulted in significantly less cytokine release in monkey blood compared with benchmark, giving ~100-fold safety margin for ADG152 for cytokine induction (Figure). In addition, ADG152 was as effective as the benchmark at inducing B cell depletion from peripheral blood of cynomolgus monkeys. In summary, the preclinical characterization of ADG152 demonstrates that our SAFEbody platform can be used to engineer safe and potent bispecific T cell engagers with increased therapeutic index by allowing for strong anti-tumor activities in mice at doses with minimal cytokine release in monkeys, thereby supporting its advancement to clinical development either as a single agent or in combination with other therapies for the treatment of CD20-expressing B cell malignancies. [Display omitted] DisclosuresCai: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Nguyen: Sparcbio, LLC: Ended employment in the past 24 months; Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Zheng: Janssen Pharmaceuticals: Ended employment in the past 24 months; Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Shi: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Liu: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Li: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Du: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Frankel: Cytovia Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company; Adagene Inc.: Consultancy, Current equity holder in publicly-traded company; Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months; IMV: Consultancy; Precision Biosciences: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Sutro: Membership on an entity's Board of Directors or advisory committees; Immunai: Consultancy, Membership on an entity's Board of Directors or advisory committees; Minerva Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Myeloid Therapeutics: Consultancy; RAPT Therapeutics: Consultancy; Syros: Consultancy. Luo: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Xu: Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Adagene Inc.: Current Employment, Current equity holder in publicly-traded company.

Library Screening to Identify Highly-Effective Autophagy Inhibitors for Improving Photothermal Cancer Therapy.

The small molecular inhibitor-associated downregulation of autophagy can remarkably enhance the efficiency of photothermal cancer therapy. To identify a more effective autophagy inhibitor, we screened a library of 20 compounds and found chloroquine, hydroxychloroquine, dauricine, and daurisoline were more efficient than the others to improve the photothermal killing of cancer cells. Interestingly, the four agents all disturb the autophagosome formation and fusion process, indicating it is a promising target to enhance cancer therapeutic efficiency. Among the four agents, daurisoline was identified to be the most efficient one. It reduced the viability of cancer cells treated by low-energy photothermal therapy from 86.27% to 32.92%. Finally, the combination treatment mediated by nanodrugs loaded with daurisoline and indocyanine green was more efficient than the individual modalities, resulting in complete inhibition of tumor growth. The study gives new inspiration to autophagy modulation-associated photothermal therapy and other therapeutic modalities for cancer treatment.

In Situ Generated and Amplified Oxidative Stress with Metallo‐Nanodrug Assembly for Metastatic Cancer Therapy with High Specificity and Efficacy

AbstractCombination of anti‐metastasis antagonists with cytotoxic agents has shown clinical activity, yet is constrained with unconditional activation and low tumor selectivity. Here, a chemokine receptor‐targeted metallo‐nanodrug is reported for in situ generated and hyperthermia‐enhanced oxidative stress and metastatic pathway blockade with tumor specificity to treat breast cancer metastasis. The self‐assembled metallo‐nanodrugs trigger reactive oxygen species (ROS) generation in situ with tumor stimuli, self‐amplify ROS level upon intrinsic photothermal heating, and accurately block chemokine receptor CXCR4 to suppress tumor cell anti‐apoptosis, migration, and invasion. CXCR4‐targeted metallo‐nanodrugs plus laser irradiation induced complete inhibition of tumor growth in 80% of mice, and caused 92.8% reduction of the number of gross metastatic lung nodules in murine breast carcinoma models. The present work provides an intelligent and bioactivatable platform for precision nanomedicine of metastatic breast cancer.

Dendrimeric nanosystem consistently circumvents heterogeneous drug response and resistance in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer with no efficacious treatment. The application of nanomedicine is expected to bring new hope to PDAC treatment. In this study, we report a novel supramolecular dendrimeric nanosystem carrying the anticancer drug doxorubicin, which demonstrated potent anticancer activity, markedly overcoming the heterogeneity of drug response and resistance of primary cultured tumor cells derived from PDAC patients. This dendrimer nanodrug was constructed with a fluorinated amphiphilic dendrimer, which self-assembled into micelles nanostructure and encapsulated doxorubicin with high loading. Because of the fine nanosize, stable formulation and acid-promoted drug release, this dendrimeric nanosystem effectively accumulated in tumor, with deep penetration in tumor tissue and rapid drug uptake/release profile in cells, ultimately resulting in potent anticancer activity and complete suppression of tumor growth in patient-derived xenografts. Most importantly, this dendrimer nanodrug generated uniform and effective response when treating 35 primary pancreatic cancer cell lines issued from patient samples as a robust platform for preclinical drug efficacy testing. In addition, this dendrimer nanodrug formulation was devoid of adverse effects and showed excellent tolerability. Given all these uniquely advantageous features, this simple and convenient dendrimer nanodrug holds great promise as a potential candidate to treat the deadly PDAC.

Mild Magnetic Hyperthermia-Activated Innate Immunity for Liver Cancer Therapy.

Magnetic hyperthermia therapy (MHT) is noninvasive and features excellent tissue penetration for deep-seated tumors, but unfortunately, it suffers the low therapeutic efficacy due to the limited magneto-thermal efficiency and insufficient intratumor accumulation of conventional intravenous-injected magnetic nanoparticles, which are actually mostly sequestered by the mononuclear phagocyte system, especially the liver. Such a disadvantageous characteristic of preferential liver uptake is here exploited, for the first time as far as we know, to treat orthotopic liver cancer by mild MHT using specially designed composite magnetic nanoparticles. A kind of core-shell-structured and Zn2+-doped Zn-CoFe2O4@Zn-MnFe2O4 superparamagnetic nanoparticles (ZCMF) has been synthesized which exhibits excellent and highly controllable magnetic hyperthermia performance owing to an exchange-coupled magnetism between the core and shell, and Zn2+ doping. The controllable mild MHT at 43-44 °C based on ZCMF demonstrates almost complete inhibition of liver cancer cell proliferation and tumor growth, which is associated with the suppression of heat shock protein 70 (HSP70) expression. More importantly, the mild MHT-treated liver cancer cells are capable of activating natural killer (NK) cells by dramatically upregulating the expression of UL16-binding proteins (ULBPs), ligands of natural killer group 2 member D (NKG2D). As a result, the growth of both xenograft tumors and orthotopic liver tumors were almost completely suppressed under mild MHT via induced NK-cell-related antitumor immunity in vivo. This work not only evidences the great potential of mild MHT but also reveals the underlying immunity activation mechanism in liver cancer treatment by mild MHT.