Targeted Antitumor Activity Research Articles

Bacteriocins in Cancer Treatment: Mechanisms and Clinical Potentials.

Cancer poses a severe threat to human health. Although conventional chemotherapy remains a cornerstone of cancer treatment, its significant side effects and the growing issue of drug resistance necessitate the urgent search for more efficient and less toxic anticancer drugs. In recent years, bacteriocins, antimicrobial peptides of microbial origin, have garnered significant attention due to their targeted antitumor activity. This unique activity is mainly attributed to their cationic and amphiphilic nature, which enables bacteriocins to specifically kill tumor cells without harming normal cells. When involving non-membrane-disrupting mechanisms, such as apoptosis induction, cell cycle blockade, and metastasis inhibition, the core mechanism of action is achieved by disrupting cell membranes, which endows bacteriocins with low drug resistance and high selectivity. However, the susceptibility of bacteriocins to hydrolysis and hemolysis in vivo limits their clinical application. To overcome these challenges, structural optimization of bacteriocins or their combination with nanotechnology is proposed for future development. This review aims to study the mechanism of action and current research status of bacteriocins as anticancer treatments, thus providing new insights for their clinical development and application.

Abstract 5819: An anti-TROP2-CPT113 ADC, exhibits potent targeted antitumor activity and favorable pharmacokinetic properties

Abstract Trophoblast cell surface antigen 2 (TROP2) is highly expressed in a number of epithelial cancer types and is considered to be a poor prognostic marker. We have developed an anti-TROP2 antibody-drug conjugate, DXC1002, that contains a potent inhibitor of DNA topoisomerase I payload (CPT113) attached to the antibody mainly in the Fab region via a unique hydrophilic CrossConju™ linker. The anti-tumor activities of DXC1002 were studied in various human cancer cell lines and xenograft mouse models and pharmacokinetic profile of DXC1002 were evaluated in cynomolgus monkeys and rats. DXC1002 demonstrated potent anti-tumor activity in TROP2 tumor cells of various expression levels in vitro, and at low dose of 1 mg/kg for one injection, in both high and moderate Trop2 expression xenograft models. In plasma stability studies, wherein DXC1002 was incubated in the plasma of five different species for 168 hours (7 days) at 37 oC, over 90% ADC was intact, exhibiting good stability of the CrossConju™ linker. Pharmacokinetic studies of DXC1002 in monkeys (TROP2 cross-species) and rats (non-TROP2 cross-species) showed half-lives of about 61.27 and 236.77 hours, respectively. Systemic clearance in monkey and rat was 0.41-0.67 and 0.279-0.309 mL/h/kg, respectively. The longer half-lives and slower clearance observed in rats the results offered a more direct assessment of the linker stability as target-mediated clearance played no role in rats. In summary, with the conjugation of a DNA topoisomerase I inhibitor payload to Trop2 antibody via the CrossConju™ linker, DXC1002 demonstrates remarkable anti-tumor properties and pharmacokinetic profile, with a potential to be further validated in clinical applications. Citation Format: Huihui Guo, Xiangfei Kong, Yong Du, Zhicang Ye, Yongxiang Chen, Zhixiang Guo, Lingli Zhang, Lu Bai, Junxiang Jia, Yunxia Zheng, Wei Zheng, Jun Zheng, Wenjun Li, Yuanyuan Huang, Zhongliang Fan, Mengmeng Liu, Binbin Chen, Meng Dai, Juan Wang, Miaomiao Chen, Qingliang Yang, Robert Y. Zhao. An anti-TROP2-CPT113 ADC, exhibits potent targeted antitumor activity and favorable pharmacokinetic properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5819.

The endocytosis of nano-Pt into non-small cell lung cancer H1299 cells and intravital therapeutic effect in vivo

Lung cancer remains the most common fatal malignant disease, and the 5-year survival rate of patients with metastasis is merely 6%. In this research, the platinum nanocluster (short for nano-Pt) was used for optical imaging without the help of other fluorescent probes and possess targeted antitumor activity as well as low systemic toxicity. The endocytic pathway and distribution of nano-Pt in non-small cell lung cancer NSCLC H1299 cells was explored by the means of quantitative and qualitative tests. Furthermore, the targeting capability and antitumor efficiency of nano-Pt was detected by intravital imaging experiment and antitumor experiment. The research implies that nano-Pt entered H1299 cells dominatingly through macropinocytosis and clathrin-dependent endocytosis pathway, and has significant antitumor efficiency, targeting properties and reliable safety for mouse tumor, indicating this nano-Pt has great potential for clinical diagnosis and therapy of NSCLC H1299 cells.

Research on the synthesis of nanoparticles of betulinic acid and their targeting antitumor activity.

Betulinic acid (BA), a natural pentacyclic lupine-type triterpene, has shown its prominent efficiency on the selective antitumor activity. However, its poor water solubility and bioavailability have limited its application. Herein, targeting nanoparticles were prepared to improve BA-based liposome (BL)'s restricted chemotherapeutic efficacy. Multi-layers membranes from the cancer cells were added as highly penetrative targeting ligands to functionalize the BA-based liposomes. In vitro experiments including the MTT assay and the fluorescence imaging of live/dead staining were adopted to prove its great inhibition in the growth of tumor cells. And it manifests that the antitumor efficacy of BL coated with cell membranes (BLCM) achieves nearly 4.3 times as that of BL under the same conditions in the MTT experiments. In addition, the fluorescence imaging stained with DAPI-FITC was applied to prove the targeting positioning effects on the BLCM. In a nutshell, the nanomedicine has good targeting antitumor efficacy and has great potential in being applied for the personalized cancer clinical treatment.

A novel high doxorubicin-loaded Fe3O4@void@ZnO nanocomposite: pH-controlled drug release and targeted antitumor activity

A novel ferroferric oxide (Fe3O4)@void@zinc oxide (ZnO) nanocomposite was constructed through a template-assisted selective etching method. The experiment results showed that the magnetic saturation value of nanocomposite could reach to 44.07 emu g−1, indicating the favorable magnetic targeting function. The presence of the void endowed the nanocomposite with high drug doxorubicin (DOX) loading capacity (85.65 μg mg−1). The pH-sensitive drug release was realized due to the gating effect of ZnO, and the DOX release rates were 92.7%, 60.8% and 23.4% at the pH values of 5.0, 6.5 and 7.4, respectively (37 °C). Besides, the Fe3O4@void@ZnO-DOX exhibited desirable antitumor effects attributed to their cellular uptake and above advantages. Therefore, the Fe3O4@void@ZnO nanocomposite may serve as a promising drug carrier in cancer therapy and other biomedical applications.

Characteristics of Molecularly Engineered Anticancer Drug Conjugated Organic Nanomicelles for Site-Selective Cancer Cell Rupture and Growth Inhibition of Tumor Spheroids.

Site-selective uptake and specific biodistribution of chemotherapeutic drugs are essential prerequisites for targeted cancer therapy. Especially, antibody and peptide conjugated drugs have been attempted as localized therapeutic agents. However, the characteristics of drug conjugated nanosystems are less explored, which are limited with their toxicity, low therapeutic efficacy, complicated synthesis, and high costs. Herein, we report a biocompatible (about 95%) molecularly engineered anticancer drug conjugated nanomicelles (∼200 nm in size) for site-selective CD44 overexpressed cancer cell rupture and tumor growth inhibition. Microscopic analysis demonstrates the distinct visualization of organic-organic interfaces (∼5 nm), which are corroborated with spectroscopic measurements confirmed the conjugation of niclosamide drug with hyaluronic acid (NIC-HA). Uniformly distributed hemocompatible (about 99%) organic nanomicelles exhibit the cellular membrane and cytoplasmic targeting with significant cellular rupture (IC50 of 4 μM for MDA MB 231 cells) indicating their inherent targeting ability for cancer cells and cancer stem cells. An inclusive in vitro and in vivo analysis for targeted antitumor activity (HT1080 tumor xenograft model) of NIC-HA nanoconjugates (∼24.6% loading) exhibited promising cancer cell death and tumor growth inhibition (60%, p < 0.05) due to STAT-3 signaling pathway inhibition and induction of apoptosis in CD44-positive triple negative breast cancer cells.

Therapeutic efficacy and imaging assessment of the HER2-targeting chemotherapy drug ZHER2:V2-pemetrexed in lung adenocarcinoma Xenografts.

Chemotherapy has always been the first therapeutic option for patients with advanced non-small cell lung cancer (NSCLC) with untreatable oncogenic mutations. However, chemotherapy has demonstrated limited success and is associated with severe side effects. This research aimed to investigate the antitumor efficacy and cytotoxic safety of the conjugate ZHER2:V2-pemetrexed, a novel targeted chemotherapeutic drug. In this context, human epidermal growth factor receptor 2 (HER2) + A549 lung xenografts were treated using ZHER2:V2-pemetrexed, pemetrexed or physiological saline. Therapeutic efficacy was monitored by single photon emission computed tomography (SPECT) imaging using the 99mTc-labeled ZHER2:V2-pemetrexed conjugate and further confirmed by performing apoptosis assays using flow cytometry analysis and hematoxylin-eosin (H&E) staining. To evaluate the expression of HER2 in tumor tissues, immunohistochemistry was performed, accompanied by quantitative analysis using flow cytometry. A toxicological evaluation was also conducted. Imaging with 99mTc-ZHER2:V2-pemetrexed demonstrated that in HER2+ A549 models, ZHER2:V2-pemetrexed showed better antineoplastic effects than pemetrexed. Compared with pemetrexed, the results from the pathological and flow cytometry analyses also revealed that ZHER2:V2-pemetrexed exhibits high antitumor activity against A549 tumors, inducing necrosis, apoptosis and cell cycle arrest. In addition, the clinical signs of toxicity in the ZHER2:V2-pemetrexed treated group were reduced compared with those in the pemetrexed treated group. These data revealed that the ZHER2:V2-pemetrexed conjugate encompasses promising targeted antitumor activity against HER2-positive lung adenocarcinoma, with reduced side effects compared with pemetrexed. Thus, the ZHER2:V2-pemetrexed conjugate may serve as a novel molecular agent with tremendous clinical breakthrough potential in the diagnosis and treatment of HER2-positive lung adenocarcinoma.

Magnetic cationic liposomal nanocarriers for the efficient drug delivery of a curcumin-based vanadium complex with anticancer potential

In this work novel magnetic cationic liposomal nanoformulations were synthesized for the encapsulation of a crystallographically defined ternary V(IV)-curcumin-bipyridine (VCur) complex with proven bioactivity, as potential anticancer agents. The liposomal vesicles were produced via the thin film hydration method employing N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP) and egg phosphatidylcholine lipids and were magnetized through the addition of citric acid surface-modified monodispersed magnetite colloidal magnetic nanoparticles. The obtained nanoformulations were evaluated for their structural and textural properties and shown to have exceptional stability and enhanced solubility in physiological media, demonstrated by the entrapment efficiency and loading capacity results and the in vitro release studies of their cargo. Furthermore, the generated liposomal formulations preserved the superparamagnetic behavior of the employed magnetic core maintaining the physicochemical and morphological requirements for targeted drug delivery applications. The novel nanomaterials were further biologically evaluated for their DNA interaction potential and were found to act as intercalators. The findings suggest that the positively charged magnetic liposomal nanoformulations can generate increased concentration of their cargo at the DNA site, offering a further dimension in the importance of cationic liposomes as nanocarriers of hydrophobic anticancer metal ion complexes for the development of new multifunctional pharmaceutical nanomaterials with enhanced bioavailability and targeted antitumor activity.

Effective targeted antitumor activity of the antimicrobial agent taurolidine against relapsed/refractory neuroblastoma: Cytotoxicity, target modulation and tumor xenograft studies.

e21502 Background: Neuroblastoma (NB) is the most common extracranial solid tumor and one of the most complex and difficult to treat diseases in pediatrics. Currently, even with highly aggressive treatment protocols, the prognosis for patients with high-risk and relapsed NB remains poor. Hence, there is a clear need to identify new agents and novel therapeutic strategies for the treatment of these children. Taurolidine (TRD) is derived from the aminosulfoacid taurine and has known anti-microbial and anti-inflammatory properties. TRD has demonstrated anti-neoplastic activity against a range of aggressive human tumors. We present mechanistic evidence and supportive preclinical data from in vitro and animal models of refractory NB for the development of an early phase clinical trial incorporating TRD. Methods: For in vitro activity studies, a panel of cell lines derived from patients with relapsed NB (n = 6) and normal control cells were treated with increasing concentrations of TRD and cell viability was measured by alamar blue assay. Phase-contrast light microscopy, western blotting, time-lapse video microscopy and analysis of global gene expression by RNA-Seq were used to evaluate target modulation and induction of cell death pathways. Bioluminescence imaging of mice bearing NB xenografts treated with TRD was used to investigate the efficacy of TRD in vivo. Results: Cell survival data showed that TRD is cytotoxic against NB cell lines in vitro (mean IC50 value 100 µM, range 65-135 µM). Phase-contrast and time-lapse video microscopy confirmed the antitumor effects of TRD. Western blot analyses identified that TRD induced target modulation and an effective apoptotic cascade, resulting in PARP cleavage. Gene expression analyses and signaling pathway activation scores indicated alterations in the Notch, MAPK and IL-10 signaling pathways. Xenograft studies further validated the in vivo activity of TRD with decreased tumor burden in treated mice and a measurable improvement in survival. Conclusions: Our study provides key pre-clinical data on the activity and mechanism of action of TRD against NB. The findings support the rationale for further evaluation of TRD for the treatment of relapsed/refractory NB patients in an early phase clinical trial.

Abstract B115: MORAb-202: a folate receptor alpha (FRA)-targeting antibody-drug conjugate, exhibiting targeted antitumor activity and bystander elimination of cancer-associated fibroblasts

Abstract Triple-negative breast cancer (TNBC) is a highly metastatic disease with very poor long-term prognosis. Because of the lack of well-defined molecular targets, traditional chemotherapy is currently the only therapeutic regimen for TNBC. Within this agent class, eribulin has been approved for metastatic breast cancer and the prespecified subgroup analysis in phase 3 clinical trial (study301) revealed that eribulin demonstrated the greatest therapeutic benefit on overall survival of TNBC patients among all other breast cancer subtypes. Folate receptor alpha (FRA) is a GPI-linked membrane glycoprotein that has limited expression in normal tissue, but is highly overexpressed on a large number of cancers of epithelial origin, including non-small cell lung cancer (NSCLC), ovarian cancer, and TNBC. These features make FRA an attractive target for the treatment of TNBC using targeted therapeutic approaches. Farletuzumab, a humanized monoclonal antibody targeting FRA, has been investigated in multiple clinical trials enrolling patients overexpressing FRA. MORAb-202, a novel antibody-drug conjugate (ADC) consisting of farletuzumab conjugated to eribulin, targets FRA overexpressed by a variety of tumor types including TNBC. Upon binding and internalization, it is hypothesized that the liberated eribulin elicits both a direct cytotoxic effect on FRA-positive tumor cells and a bystander effect on FRA-negative tumor cells and, importantly, tumor-associated stromal cells within the tumor microenvironment. To address this hypothesis, we have evaluated the in vivo efficacy of MORAb-202 against a FRA-positive TNBC patient-derived xenograft (PDX) model. Single administration of 5 mg/kg MORAb-202 mediated tumor regression statistically compared to vehicle control (P&amp;lt;0.0001). Additionally, one animal treated with MORAb-202 showed complete and durable tumor regression. In order to examine the effects of MORAb-202 treatment on the tumor microenvironment, we harvested tumors both prior to treatment and five days post-treatment. Immunofluorescent staining was used to assess the target-specific engagement of MORAb-202 to FRA-positive regions of the tumor. Strong membrane staining of MORAb-202 was observed, while no staining was seen in the adjacent stromal regions. MORAb-202 also had demonstrable effects on the tumor microenvironment via reduction of the number of FRA-negative cancer-associated fibroblasts (CAF) present. To further evaluate whether these effects on CAF are part of the mode of action (MOA) of MORAb-202, we tested the antitumor efficacy and effects on the tumor microenvironment in a surrogate FRA-positive in vivo model. Tumors were isolated on days 3, 5, 7, and 9 post-treatment. The reduction in CAF, visualized by alpha-smooth muscle actin staining, was directly proportional to overall tumor reduction. In both studies, single-dose MORAb-202 showed dramatically greater efficacy than molar equivalent dosing of eribulin alone. Histologic analyses revealed unique modes of action of MORAb-202 over antibody or payload alone. These results suggest a potential benefit of MORAb-202 in the treatment of TNBC. Citation Format: Katherine A. Rybinski, Christopher Maddage, Xin Cheng, Earl Albone, Luigi Grasso, Keiji Furuuchi, Toshimitsu Uenaka. MORAb-202: a folate receptor alpha (FRA)-targeting antibody-drug conjugate, exhibiting targeted antitumor activity and bystander elimination of cancer-associated fibroblasts [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B115.

ADCT-301, a Pyrrolobenzodiazepine (PBD) Dimer-Containing Antibody-Drug Conjugate (ADC) Targeting CD25-Expressing Hematological Malignancies.

Despite the many advances in the treatment of hematologic malignancies over the past decade, outcomes in refractory lymphomas remain poor. One potential strategy in this patient population is the specific targeting of IL2R-α (CD25), which is overexpressed on many lymphoma and leukemic cells, using antibody-drug conjugates (ADC). ADCT-301 is an ADC composed of human IgG1 HuMax-TAC against CD25, stochastically conjugated through a dipeptide cleavable linker to a pyrrolobenzodiazepine (PBD) dimer warhead with a drug-antibody ratio (DAR) of 2.3. ADCT-301 binds human CD25 with picomolar affinity. ADCT-301 has highly potent and selective cytotoxicity against a panel of CD25-expressing human lymphoma cell lines. Once internalized, the released warhead binds in the DNA minor groove and exerts its potent cytotoxic action via the formation of DNA interstrand cross-links. A strong correlation between loss of viability and DNA cross-link formation is demonstrated. DNA damage persists, resulting in phosphorylation of histone H2AX, cell-cycle arrest in G2-M, and apoptosis. Bystander killing of CD25-negative cells by ADCT-301 is also observed. In vivo, a single dose of ADCT-301 results in dose-dependent and targeted antitumor activity against both subcutaneous and disseminated CD25-positive lymphoma models. In xenografts of Karpas 299, which expressed both CD25 and CD30, marked superiority over brentuximab vedotin (Adcetris) is observed. Dose-dependent increases in DNA cross-linking, γ-H2AX, and PBD payload staining were observed in tumors in vivo indicating a role as relevant pharmacodynamic assays. Together, these data support the clinical testing of this novel ADC in patients with CD25-expressing tumors. Mol Cancer Ther; 15(11); 2709-21. ©2016 AACR.

IMGN853, a Folate Receptor-α (FRα)–Targeting Antibody–Drug Conjugate, Exhibits Potent Targeted Antitumor Activity against FRα-Expressing Tumors

A majority of ovarian and non-small cell lung adenocarcinoma cancers overexpress folate receptor α (FRα). Here, we report the development of an anti-FRα antibody-drug conjugate (ADC), consisting of a FRα-binding antibody attached to a highly potent maytansinoid that induces cell-cycle arrest and cell death by targeting microtubules. From screening a large panel of anti-FRα monoclonal antibodies, we selected the humanized antibody M9346A as the best antibody for targeted delivery of a maytansinoid payload into FRα-positive cells. We compared M9346A conjugates with various linker/maytansinoid combinations, and found that a conjugate, now denoted as IMGN853, with the N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB) linker and N(2')-deacetyl-N(2')-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4) exhibited the most potent antitumor activity in several FRα-expressing xenograft tumor models. The level of expression of FRα on the surface of cells was a major determinant in the sensitivity of tumor cells to the cytotoxic effect of the conjugate. Efficacy studies of IMGN853 in xenografts of ovarian cancer and non-small cell lung cancer cell lines and of a patient tumor-derived xenograft model demonstrated that the ADC was highly active against tumors that expressed FRα at levels similar to those found on a large fraction of ovarian and non-small cell lung cancer patient tumors, as assessed by immunohistochemistry. IMGN853 displayed cytotoxic activity against FRα-negative cells situated near FRα-positive cells (bystander cytotoxic activity), indicating its ability to eradicate tumors with heterogeneous expression of FRα. Together, these findings support the clinical development of IMGN853 as a novel targeted therapy for patients with FRα-expressing tumors.

Fabrication of modular multifunctional delivery for antitumor drugs based on host–guest recognition

Herein, learning from the idea of the modular concept widely used in ship building, as a design approach that assembles some subdivided smaller modules to a specific ship, a new modular multifunctional drug delivery (MMDD) with excellent biocompatibility was directly prepared by a flexible host–guest interaction between pH-sensitive benzimidazole-graft-dextran (Dex-BM) and pre-synthesized multifunctional cyclodextrins. In this drug system, pH-sensitive Dex-BM acted as the main case and pre-synthesized multifunctional cyclodextrins were the changeable modules. To verify the feasibility of MMDD in cancer chemotherapy, doxorubicin (DOX) was used as a model drug. In vitro drug release experiments indicated that the drug released around 80% from DOX-loaded MMDD at pH 5.3, while approximately 40% of DOX released under the condition of pH 7.4. Moreover, the targeting antitumor activity of DOX-loaded MMDD was investigated in HeLa and HepG2 cells using MTT assays, confocal laser scanning microscopy and flow cytometer, which indicated that the targeted DOX-loaded MMDD provided an efficient drug delivery platform for inhibition of different cancer cells. Meantime, the incorporation of different functional modules into one system was also investigated, simultaneously exhibiting targeting and imaging property. These features suggest that this modular multifunctional drug delivery system can efficiently enhance the inhibition of cellular proliferation in vitro, and according to the needs in clinical treatment, some targeting and imaging molecules can be chosen.

Two-stage extraction of antitumor, antioxidant and antiacetylcholinesterase compounds from Ganoderma lucidum fruiting body

Ganoderma lucidum has been used in oriental medicine for its contribution to vitality and longevity. None of them report about targeted antitumor activity against adenocarcinoma cells, or antioxidant and antiacetylcholinesterase activity for potential application in treatment of Alzheimer's and other neurodegenerative diseases. To date, there are a few studies available concerning supercritical carbon dioxide extraction of biologically active compounds from G. lucidum fruiting body. In our study, two stage extractions of biologically active compounds from G. lucidum were performed. First, supercritical carbon dioxide was used as extraction solvent. Next, the same material was used for hot water isolation of biologically active polysaccharides. Cytotoxicity, antioxidant and antiacetylcholinesterase activity were tested for all obtained extracts. Additionally, the effect of extraction process conditions on the biological activity of extracts was assessed.

Clinical experience of a targeted TCR-IL2 fusion protein in combination with cisplatin (CDDP) in patients (pts) with metastatic melanoma.

e13088 Background: ALT-801 is recombinant human interleukin-2 (IL-2) genetically fused to a single-chain T-cell receptor specific to a peptide antigen of human p53 presented in the context of HLA-A2 positivity. In melanoma xenograft mouse models, this fusion protein alone demonstrated potent, targeted antitumor activity and synergistic efficacy was observed in combination with CDDP. A prior phase I study in pts with metastatic malignancies, including metastatic melanoma, showed that ALT-801 monotherapy could be safely administered and was associated with immunologic changes of potential antitumor relevance (Fishman 2011 CCR 17:7765). Here we report the results of a phase Ib study of ALT-801+ CDDP in pts with metastatic melanoma. Methods: The primary objectives of this multicenter study are to: 1) define an MTD and evaluate the safety of ALT-801+CDDP and 2) assess the objective response rate according to RECIST criteria in treated patients. Eligible pts (histologically confirmed advanced/metastatic melanoma, p53 peptide/HLA-A2+ tumor, chemotherapy naive, PS (ECOG) 0-1, adequate renal and cardiac functions) received 2 consecutive 4-wk courses of CDDP (70 mg/m2 iv, Day 1) and ALT-801 (iv, Days 3, 5, 15, 17 &amp; 19). Pts with SD or better after 2 courses received 2 additional courses of treatment. Up to 5 cohorts (3+3 dose escalation) of ALT-801 (0.04 - 0.12 mg/kg) were planned with a 2-stage design of expanded pt enrollment at the MTD. Results: 22 patients (11M, 11F, 30-74 yrs) were evaluable at ALT-801 dose levels from 0.04 to 0.10 mg/kg. The MTD has not been reached at 0.10 mg/kg ALT-801. Toxicity was manageable and transient. RECIST-confirmed tumor response and disease stabilization was reported for some patients. Overall survival rates (6 mon, 87%; 12 mon, 58%) observed to date suggest durable clinical benefit. Conclusions: ALT-801+CDDP can be safely administered in an out-patient setting to pts with metastatic melanoma. Preliminary evaluation suggests durable clinical activity of the ALT-801+CDDP regimen in melanoma patients. A trial using ALT-801 at the 0.1 mg/kg dose level in combination with a BRAF kinase inhibitor is being planned for patients with metastatic melanoma (CA097550).

Phase I evaluation of the safety of conatumumab (AMG 655) in combination with AMG 479 in patients (pts) with advanced, refractory solid tumors.

3102 Background: Preclinical studies show that evasion of death receptor (DR)-mediated apoptosis may depend on intact growth factor signaling. DR agonists have not previously been combined with IGF-1R inhibitors in published clinical studies. The combination may yield synergistic targeted antitumor activity without added toxicity. This study combined 2 investigational, fully human monoclonal antibodies: conatumumab, a human DR5 agonist; and AMG 479, an IGF-1R antagonist. Methods: Eligible pts had locally advanced or metastatic chemotherapy refractory solid tumors, ECOG PS of 0 or 1, and adequate glycemic control if diabetic. Pts enrolled into 3 sequential dose cohorts and received conatumumab 1, 3, or 15 mg/kg IV and AMG 479 18 mg/kg IV on day 1 of each Q3W cycle. Dose escalation was based on <33% incidence of dose-limiting toxicities (DLT) during the first cycle. Study endpoints included: dose determination, adverse event (AE) incidence, DLT, anti-drug antibodies, and pharmacokinetics (PK) of both drugs. Results: Nine pts were enrolled (3 per cohort) and received treatment for a median (range) of 2 (1-8) cycles; 5 pts were female, 7 had ECOG PS=1, and median (range) age was 58 (24-66) years. Primary tumor types were: CRC (3 pts), soft tissue sarcoma [STS] (4 pts), pancreatic carcinoma (1 pt), and breast carcinoma (1 pt). Seven pts discontinued due to disease progression, 1 due to death. No DLT was observed during phase 1 dose escalation. Treatment-emergent serious AEs occurred in 3 pts: pneumonia in 1 pt and dyspnea and pericardial effusion in 2 pts; none were deemed treatment related. Four pts experienced a minor infusion reaction; all patients received all planned doses. Combining conatumumab and AMG 479 showed little impact on the PK of either drug. Three pts had stable disease for >120 days (2 STS, 1 pancreatic carcinoma) with progressive disease in 5 pts; the duration of stable disease was 42-217 days). One pt remained on therapy for >240 days. The recommended dose for phase 2 is AMG 479 18 mg/kg + conatumumab 15 mg/kg. Conclusions: Conatumumab combined with AMG 479 was well-tolerated and no drug-drug interactions were observed. Phase II development of this combination is ongoing. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Amgen Amgen Amgen Amgen Amgen Amgen

Phase I clinical experience of a targeted TCR-IL2 fusion protein in patients with metastatic malignancies

3040 Background: ALT-801 is recombinant human interleukin-2 (IL-2) genetically fused to a single-chain T-cell receptor specific to a peptide antigen of human p53 presented in the context of HLA-A2 positivity. In tumor xenografts in animal models, this fusion protein demonstrated potent and targeted antitumor activity. Methods: A phase I/IIa multicenter clinical study was initiated to assess safety and clinical response of ALT-801 in patients with various metastatic malignancies. Patients screened positively for HLA-A2 and tumors expressed p53 peptide/HLA-A2 complex. In this dose escalation trial, patients received ALT-801 as a daily intravenous infusion for 4 days followed by a 10-day rest period and 4 additional daily doses. Results: Data from three dosing cohorts (0.015, 0.04, and 0.08 mg/kg/dose) indicate that ALT-801 is well-tolerated up to 0.04 mg/kg/dose. The half-life of ALT-801 is 2.5–4 hours per pharmacokinetic analysis, depending on the dose. Even at the 0.015 mg/kg level, there were sufficient concentrations of ALT-801 in the patients’ serum to fully activate cell lytic activity in vitro. Treatment with ALT-801 also led to immune activation in patients as demonstrated by elevated serum IFN-γ levels as well as an increase in IFN-γ-producing immune cells in patients’ blood. Interestingly, serum TNF-α, a major inducer of hypotension in patients receiving high-dose IL-2 treatment, was not induced in patients receiving ALT-801. Evidence of antitumor activity (i.e., stable disease, tumor shrinkage) was observed in some patients after one or two courses of ALT-801. Conclusions: At a dose of 0.04 mg/kg, ALT-801 is a well-tolerated agent that generated a vigorous immune response and demonstrated clinical efficacy in a group of patients with various metastatic malignancies. An expansion cohort of patients is in progress at 0.04 mg/kg. [Table: see text]

Combining Established and Novel Immunomodulators: Synergistic Inhibition of Major T Cell Effector Functions by Dexamethasone and Dasatinib

Introduction. An increasing body of evidence suggests that in addition to their molecular targeted antitumor activity tyrosine kinase inhibitors (TKIs) exert immunomodulatory effects on T cells. Others (Blake et al 2008; Schade et al 2008) and we (Weichsel et al 2008) have recently shown that dasatinib (a multitargeting TKI) may lead to a complete inhibition of TCR dependent T cell effector functions via the SRC kinases LCK and FYN. Because TKIs act as competitive enzyme inhibitors with short half life, they permit better side effect control in the clinic. We investigated the impact of combining clinically relevant doses of dasatinib (1–100nM) with the established immunosuppressant dexamethasone (1–1000nM) on T cells.Materials and Methods. Purified human CD3+ T cells from healthy blood donors were studied directly ex vivo. Functional outcomes assessed included cytokine production (IL-2), activation (CD69 upregulation), proliferation (CFSE dilution) and apoptosis/necrosis induction. EBV or CMV specific proliferation of antigen specific CD8+ T cells were evaluated applying tetramer technology. To distinguish between rapid non-genomic and genomic effects all assessments compared the impact of pre-treating T-cells with dexamethasone for 1 vs. 24h.Results and Discussion. Complete inhibition of proliferation and activation occurred with dasatinib alone at levels of 50nM and above, whereas application of dexamethasone did not lead to a complete inhibition even at doses up to 1000nM. Dose-dependent inhibition of with the monoclonal CD3 antibody OKT3 induced T cell activation and proliferation was observed with the combination of dasatinib and dexamethasone. Strongest synergistic inhibitory effects of the drug combination were observed for OKT3 induced cytotoxic CD8+ T cell proliferation (mean±SEM given: OKT3 induced 63±5% proliferating T cells after 4 days incubation, OKT3 + 10nM dexamethasone 44±5%, OKT3 + 10nM dasatinib 39±10%, OKT3 + combination 16±4%; n=5; p<0.05). A significant inhibition of OKT3 induced up-regulation of the activation marker CD69 was demonstrated with the combination but not for dexamethasone alone (n=5). Our previously published data on dasatinib alone also showed inhibition of OKT3 induced up-regulation of CD69 expression, and this was potentiated in combination with dexamethasone. The pre-treatment time did not influence the dexamethasone effect except for increased reduction of IL-2 production after 24h vs. 1h pre-incubation. Overall, helper CD4+ T cells were more sensitive to the inhibitory effects of the drug combination regarding activation and proliferation than cytotoxic CD8+ T cells. Of note, synergistic effects occurred primarily in the different memory CD4 and CD8 T cell subsets but not in the naïve CD4 and CD8 T cells (e.g. for CD8+CD45RO+CD27+ memory T cells mean±SEM given for percentage of proliferating cells after 4 days: OKT3 92±1%, OKT3 + 100nM dexamethasone 77±2%, OKT3 + 10nM dasatinib 69±15%, and OKT3 + combination 27±11%; n=5; p<0.05). IL-2 production in purified T cells was significantly reduced (p<0.05) in a dose dependent nature for both dasatinib and dexamethasone compared to the OKT3 stimulated condition, either alone or in combination (n=5). Similarly, activation induced cell death (AICD) was significant reduced when the two drugs were combined, whereas no synergistic effects were observed regarding necrosis inhibition (n=5; p<0.05). In contrast, initial results suggested that dexamethasone did not inhibit clinically relevant EBV or CMV antigen specific CD8+ memory T cell proliferation when used alone and did not show synergistic effects with dasatinib (n=3). This may be due to a reduced sensitivity of the specific viral memory subsets towards dexamethasone.Outlook and Conclusion. As viral reactivation especially with CMV is a major cause for morbidity after allogeneic stem cell transplantation, our results warrant further studies in vitro and in vivo. With an indication that each drug, when combined could be used at reduced dose, this research may then pave the way for synergistic uses of TKIs and glucocorticosteroids in treatment of graft versus host disease or autoimmune diseases potentially without increased risk of infections.

Targeting activity of a TCR/IL-2 fusion protein against established tumors.

We have previously reported that a single-chain T cell receptor/IL-2 fusion protein (scTCR-IL2) exhibits potent targeted antitumor activity in nude mice bearing human tumor xenografts that display cognate peptide/HLA complexes. In this study, we further explore the mechanism of action of this molecule. We compared the biological activities of c264scTCR-IL2, a scTCR-IL2 protein recognizing the aa264-272 peptide of human p53, with that of MART-1scTCR-IL2, which recognizes the MART-1 melanoma antigen (aa27-35). In vitro studies showed that c264scTCR-IL2 and MART-1scTCR-IL2 were equivalent in their ability to bind cell-surface IL-2 receptors and stimulate NK cell responses. In mice, MART-1scTCR-IL2 was found to have a twofold longer serum half-life than c264scTCR-IL2. However, despite its shorter serum half-life, c264scTCR-IL2 showed significantly better antitumor activity than MART-1scTCR-IL2 against p53(+)/HLA-A2(+) tumor xenografts. The more potent antitumor activity of c264scTCR-IL2 correlated with an enhanced capacity to promote NK cell infiltration into tumors. Similar differences in antigen-dependent tumor infiltration were observed with activated splenocytes pre-treated in vitro with c264scTCR-IL2 or MART-1scTCR-IL2 and then transferred into p53(+)/HLA-A2(+) tumor bearing recipients. The data support a model where c264scTCR-IL2 activates immune cells to express IL-2 receptors. Following stable interactions with cell-surface IL-2 receptors, c264scTCR-IL2 fusion molecule enhances the trafficking of immune cells to tumors displaying target peptide/HLA complexes where the immune cells mediate antitumor effects. Thus, this type of fusion molecule could be used directly as a targeted immunotherapeutic or in adoptive cell transfer approaches to activate and improve the anti-cancer activities of immune cells by providing them with pre-selected antigen recognition capability.