Promote Tumor Cell Killing Research Articles

Impact of drug dispersion on tumor-effector dynamics during combined chemo-immunotherapy with sensitivity analysis

Solid cancer remains a serious threat to global health despite decades of progress. Traditional chemotherapy treatment has been used to curb the disease despite its inability to reach cancer cells at high enough quantities leading to adverse toxicity on healthy cells, producing severe side effects, and exacerbating patient suffering. Active targeted nano-drugs (ATNDs) acting as transporter systems have become a viable solution to this shortcoming, offering the possibility of more precise cancer targeting. Therefore, in the present study, we develop a numerical model of capturing ATNDs, incorporating movements within and outside the tumor spheroid and the magnetic forces guiding the nanoparticles. By combining a model of ATNDs transport with a tumor-effector dynamic model based on porous media phenomena, we demonstrate how the models may be combined to model drug-loaded nanoparticle transport in an external magnetic field. We analyze the controlled drug delivery against traditional chemotherapy based on the combined effect of immunotherapy and chemotherapy with the dispersion of nano-drugs to lead a solute cloud toward the tumor. We found that the amalgamation of immune and chemotherapy delivered by ATNDs enhances cancer therapy efficacy compared with conventional chemotherapy. Increased drug convection toward the tumor region presented by ascending values of the Peclet number inhibits the growth of tumor cells and prolongs progression-free survival. Increases in source term and vessel permeability also increase the likelihood of tumor suppression, while raising the hematocrit and magnetic number results in reduced tumor cell killing. Sensitivity analysis of the dynamic model parameters has been discussed. This work demonstrates that ATND-delivery systems can improve therapeutic agent delivery to the tumor tissue, and promote tumor cell killing.

Abstract 5297: CLN-619, a clinical stage MICA/B-specific hIgG1 monoclonal antibody, engages multiple immune effector cells to promote anti-tumor activity

Abstract CLN-619 is a humanized IgG1 monoclonal antibody that targets MICA and MICB (MICA/B) and is currently in phase 1 clinical development in cancer patients (NCT05117476). MICA/B serve as activating signals on target cells for recognition by the NKG2D receptor, which is expressed on NK cells and a subset of T cell populations. MICA/B expression is induced in response to stressed conditions, thereby enabling NKG2D-mediated elimination of target cells. On NK cells, NKG2D is one of many receptors in the complex network of activating and inhibitory receptors, whereby NKG2D pathway activation results in cytokine production, enhancement of ADCC and target cell death. On CD8 T cells, the NKG2D axis plays a costimulatory role in lowering the threshold for lysis upon TCR engagement, and may also drive direct CD8-mediated killing following prior TCR activation. MICA/B is expressed broadly on a range of solid and hematological malignancies. However, tumor cells evade NKG2D-mediated elimination by shedding MICA/B ligand from the cell surface via proteases present in the tumor microenvironment (TME). CLN-619 functions by binding to MICA/B and preventing shedding, thereby increasing MICA/B cell surface expression to restore the NKG2D-MICA/B axis to promote tumor cell killing by NK cells and T cells. Additionally, CLN-619 can mediate ADCC and ADCP by NK cells and macrophages. In preclinical xenograft models, doses as low as 0.03 mg/kg inhibited tumor outgrowth. We investigated the effects of CLN-619 on a variety of primary immune cells including NK cells, T cells and macrophages. In the evaluation of NK cells, we measured CLN-619 modulation of cytokine production and cytotoxicity in the presence of MICA/B expressing target cells. In addition, we demonstrated the contribution of Fc-mediated functions of CLN-619. In the context of T cells, we explored the requirement of TCR co-stimulation or prior antigen exposure in the activation of the NKG2D pathway by CLN-619. In the context of macrophages, we measured the ability of CLN-619 to mediate ADCP against MICA/B-expressing target cells. These data highlight the potential of CLN-619 to engage multiple immune cell types within the TME, which may lead to greater and broader efficacy compared to IO therapies targeting a single immune cell type. Citation Format: Kerry A. Whalen, Catherine C. Henry, Kavya Rakhra, Kristan Meetze, Jennifer S. Michaelson, Patrick A. Baeuerle. CLN-619, a clinical stage MICA/B-specific hIgG1 monoclonal antibody, engages multiple immune effector cells to promote anti-tumor activity [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 5297.

Synergistic reinforcement of immunogenic cell death and transformation of tumor-associated macrophages via an M1-type macrophage membrane-camouflaged ferrous-supply-regeneration nanoplatform

The immune system's role in tumor growth and spread has led to the importance of activating immune function in tumor therapy. We present a strategy using an M1-type macrophage membrane-camouflaged ferrous-supply-regeneration nanoplatform (M1mDDTF) to synergistically reinforce immunogenic cell death (ICD) and transform tumor-associated macrophages (TAMs) against tumors. The M1mDDTF nanoparticles consist of doxorubicin-loaded dendritic mesoporous silica nanoparticles chelated with FeIII-tannic acid (FeIIITA) and coated with M1-type macrophage membranes. In the acidic tumor microenvironment, FeIIITA releases Fe2+ and generates ·OH, aided by near infrared irradiation for enhanced doxorubicin release. Furthermore, the M1mDDTF nanoplatform not only directly kills tumor cells but stimulates ICD, which can increase the proportion of CD86+ CD80+ cells and promote dendritic cell maturation. Particularly, the M1mDDTF nanoplatform can also promote the gradual polarization of TAMs into the M1-type and promote tumor cell killing. This study demonstrates the safety and multifunctionality of M1mDDTF nanoparticles, highlighting their potential for clinical tumor treatment. Statement of significanceMalignant tumors are a global concern and a major cause of death. Nanoparticles' passive targeting is ineffective and hindered by reticuloendothelial system clearance. Therefore, enhancing nanoparticle accumulation in tumors while minimizing toxicity is a challenge. Coating nanoparticles with cell membranes enhances biocompatibility, immune evasion, and specific targeting. This approach has led to the development of numerous cell membrane-mimicking nanomaterials with remarkable properties and functions. This study developed an M1-type macrophage membrane-camouflaged ferrous-supply-regeneration nanoplatform, boosting immunogenic cell death and transforming tumor-associated macrophages. Tannic acid in the tumor microenvironment reduced Fe3+ to Fe2+, generating ·OH. M1mDDTF nanosystem induced M1-type macrophage polarization, inhibiting tumor growth and triggering immune cell death. Safe and versatile, these M1mDDTF nanoparticles hold promise for clinical tumor treatment.

Abstract 3514: Bispecific gamma delta T cell engagers containing butyrophilin 2A1/3A1 heterodimeric fusion protein efficiently activate Vg9Vd2 T cells and promote tumor cell killing

Abstract A primary mechanism of cancer immunotherapy resistance involves downregulation of specific antigens or major histocompatibility complex (MHC) based antigen presentation, which renders tumor cells invisible to αβ T cells, but not γδ T cells. Recently, a two-step model of γδ T cell activation has emerged, wherein one butyrophilin (BTN, i.e., BTN2A1) directly binds the γδ TCR but is only activated if certain molecular patterns (e.g., phosphoantigens) facilitate recruitment of a second BTN (i.e., BTN3A1) into a complex to form a BTN2A1/3A1 heterodimer. The BTN2A1/3A1 complex specifically activates the Vγ9Vδ2 T cells found in the peripheral blood in response to phosphoantigen build up in tumor cells. The unique mechanism of action and specificity of γδ TCR/BTN interactions suggests that therapeutic proteins comprising specific BTN heterodimers could be used to target specific γδ T cell populations to tumor cells which lack expression of high affinity neoantigens or MHC class I molecules. In this study, we generated bispecific γδ T cell engagers (GADLEN) containing heterodimeric BTN2A1 and BTN3A1 extracellular domains (ECD) fused via inert Fc linkers to scFv domains targeting a tumor-antigen (CD19 or CD33) to test their ability to modulate Vγ9Vδ2 T cells and to promote tumor cell killing in co-culture assays. GADLEN induced proliferation, degranulation and cytokine production including IFNγ and TNFα in Vγ9Vδ2 T cells but requires co-stimulation of a natural cytotoxicity receptor (via anti-NKG2D) or T-cell co-stimulatory receptor (via anti-CD28), highlighting the parallels of signal 1 and signal 2 requirements of TCR activation in αβ- and γδ- T cells. In Vγ9Vδ2 T cells and tumor co-culture assays, the addition of GADLEN alone enhanced killing of CD19+ lymphoma cells such as Daudi and Raji, expressing known ligands for CD28 (e.g., CD80 and CD86). Similarly, GADLEN enhanced Vγ9Vδ2 T cell-mediated killing of K562 cells engineered to express CD19, which express MICA/B (ligands for NKG2D) on the cell surface. In addition, the CD33-targeted GADLEN alone was able to specifically kill acute myeloid leukemia cells expressing CD33 and costimulatory ligands (e.g., CD80 and CD86) in a mechanism similar to the CD19-targeted GADLEN construct. These results highlight the ability of GADLENs in promoting targeted killing of tumor cells by providing the “active” heterodimeric BTN2A1 and BTN3A1 that is critical to enhance cytotoxic killing by Vγ9Vδ2 T cells. Furthermore, this study provides mechanistic insights into BTN-mediated activation of Vγ9Vδ2 T cells and offers proof of concept for using antigen-targeted butyrophilin heterodimeric fusion proteins for the treatment of cancer. Citation Format: Anne Lai, Arpita Patel, Faraha Brewer, Kinsley Evans, Kellsey Johannes, Louis Gonzalez, Kyung Jin Yoo, George Fromm, Keith Wilson, Taylor H. Schreiber, Suresh de Silva. Bispecific gamma delta T cell engagers containing butyrophilin 2A1/3A1 heterodimeric fusion protein efficiently activate Vg9Vd2 T cells and promote tumor cell killing [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 3514.

Glycosylation-Dependent Induction of Programmed Cell Death in Murine Adenocarcinoma Cells.

Altered surface glycosylation is a major hallmark of tumor cells associated with aggressive phenotype and poor prognosis. By recognizing specific carbohydrate motifs, lectins can be applied to distinguish tumor from healthy cells based on the expression of glycosylation-dependent markers. Through their ability to bind to specific carbohydrates, lectins induce cell agglutination and cross-link surface glycoproteins, thereby mediating mitogenic and death-inducing effects in various cell types. The carbohydrate-selective cytotoxic effect of lectins also enables their possible application in therapies targeting cancer cells. To clarify the intracellular pathways mediating cell death induced by a group of plant and fungal lectins, we investigated mouse adenocarcinoma MC-38 cells harboring inactive genes involved in apoptosis, necroptosis and pyroptosis. Treatment of MC-38 cells with wheat germ agglutinin, Maackia amurensis lectin I, and Aleuria aurantia lectin induced multiple cell death pathways through reactions that relied on the autophagy machinery without depending on caspase activation. Furthermore, inhibition of de novo protein synthesis by cycloheximide strongly decreased the cytotoxic response, indicating that the lectins investigated induced cell death via effector molecules that are not expressed under normal circumstances and supporting the non-apoptotic nature of cell death. The broad cytotoxic response to lectins can be beneficial for the development of combination therapies targeting tumor cells. Given that tumors acquire resistance to various cytotoxic treatments because of mutations in cell death pathways, compounds inducing broad cytotoxic responses, such as lectins, represent potent sensitizers to promote tumor cell killing.

Abstract LB066: Assessing the safety liability of t-cell bispecific (TCB) antibodies using Ogans-on-Chips technology

Abstract T-cell bispecific antibodies (TCBs) are a promising class of immunotherapeutic agents that promote tumor cell killing by physical crosslinking of effector T-cells to target expressing cells. While TCBs are effective in targeting less-immunogenic tumors, they are subject to safety liabilities in normal tissues, which may express low levels of the target. Preclinical assessment of safety risks is crucial but species differences between human and rodent immune responses necessitate the development of advanced human cell-based models for TCB safety profiling. While conventional killing assays are experimentally tractable, they lack physiological organization and cytoarchitecture and often fail to accurately predict efficacy and off-tumor cytotoxicity. Here, we show the robustness of our Intestine-Chip to capture variability in immune cell activation at physiologically relevant TCB concentrations when immune cells are directly applied to the apical epithelium. With increased confidence in the predictive capabilities of the model, we then confirmed expected regional dependence to gastrointestinal (GI) toxicity of the TCB by showing elevated immune cell activation in the large- versus small-intestine. Further, we have demonstrated vascular recruitment and transmigration of circulating immune cells to the intestinal epithelium to more accurately capture the in vivo mechanisms of TCB-mediated toxicity. Together, these data show that our models are suitable for safety profiling of novel engineered immunotherapies and provide clinically relevant results. Citation Format: S. Jordan Kerns, Ville Kujala, Marianne Kanellias, John Sauld, William Tien-Street, Heather S. Grant, Katia A. Karalis, Geraldine A. Hamilton, Lorna Ewart, Adrian B. Roth, Annie Moisan, Nikolche Gjorevski. Assessing the safety liability of t-cell bispecific (TCB) antibodies using Ogans-on-Chips technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB066.

Modeling of tumor response to macrophage and T lymphocyte interactions in the liver metastatic microenvironment.

The dynamic interactions between macrophages and T-lymphocytes in the tumor microenvironment exert both antagonistic and synergistic functions affecting tumor growth. Extensive experimental effort has been expended to investigate immunotherapeutic strategies targeting macrophage polarization as well as T-cell activation with the goal to promote tumor cell killing and cancer elimination. However, these interactions remain poorly understood, and cancer immunotherapeutic strategies are often disappointing. The complex system encompassing innate and adaptive immune cell activity in response to tumor growth could benefit from a systems perspective built upon mathematical modeling. This study develops a modeling system to help evaluate the effects of macrophage and T-lymphocyte interactions on tumor growth. The system enables simulating the combined cytotoxic and tumor-promoting interactions of these two immune cell populations in a vascularized organ microenvironment, such as in liver metastases. A hypothetical immunotherapeutic strategy is simulated to increase the number of tumor-suppressive (M1-phenotype) vs. tumor-promoting (M2-phenotype) macrophages to gauge their effects on CD8+ T-cells and CD4+ T-helper cells, which in turn affect the macrophage functions. The results highlight the dynamic interactions between macrophages and T-lymphocytes in the tumor microenvironment and show that with the chosen set of parameter values, the overall cytotoxic effect from macrophages and T-lymphocytes obtained by driving the M1:M2 ratio higher could saturate and fail to achieve tumor regression. Further expansion of this modeling platform to include additional tumor-immune cell interactions, coupled with parameters representing particular tumor characteristics, could enable systematic evaluation of immunotherapeutic strategies tailored to patient-tumor specific conditions, including metastatic disease.

Modeling immune cell behavior across scales in cancer.

Detailed, mechanistic models of immune cell behavior across multiple scales in the context of cancer provide clinically relevant insights needed to understand existing immunotherapies and develop more optimal treatment strategies. We highlight mechanistic models of immune cells and their ability to become activated and promote tumor cell killing. These models capture various aspects of immune cells: (a) single‐cell behavior by predicting the dynamics of intracellular signaling networks in individual immune cells, (b) multicellular interactions between tumor and immune cells, and (c) multiscale dynamics across space and different levels of biological organization. Computational modeling is shown to provide detailed quantitative insight into immune cell behavior and immunotherapeutic strategies. However, there are gaps in the literature, and we suggest areas where additional modeling efforts should be focused to more prominently impact our understanding of the complexities of the immune system in the context of cancer.This article is categorized under:Biological Mechanisms > Cell SignalingModels of Systems Properties and Processes > Mechanistic ModelsModels of Systems Properties and Processes > Cellular Models

Data-driven analysis of a mechanistic model of CAR T cell signaling predicts effects of cell-to-cell heterogeneity

Due to the variability of protein expression, cells of the same population can exhibit different responses to stimuli. It is important to understand this heterogeneity at the individual level, as population averages mask these underlying differences. Using computational modeling, we can interrogate a system much more precisely than by using experiments alone, in order to learn how the expression of each protein affects a biological system. Here, we examine a mechanistic model of CAR T cell signaling, which connects receptor-antigen binding to MAPK activation, to determine intracellular modulations that can increase cellular response. CAR T cell cancer therapy involves removing a patient's T cells, modifying them to express engineered receptors that can bind to tumor-associated antigens to promote tumor cell killing, and then injecting the cells back into the patient. This population of cells, like all cell populations, would have heterogeneous protein expression, which could affect the efficacy of treatment. Thus, it is important to examine the effects of cell-to-cell heterogeneity. We first generated a dataset of simulated cell responses via Monte Carlo simulations of the mechanistic model, where the initial protein concentrations were randomly sampled. We analyzed the dataset using partial least-squares modeling to determine the relationships between protein expression and ERK phosphorylation, the output of the mechanistic model. Using this data-driven analysis, we found that only the expressions of proteins relating directly to the receptor and the MAPK cascade, the beginning and end of the network, respectively, are relevant to the cells’ response. We also found, surprisingly, that increasing the amount of receptor present can actually inhibit the cell's ability to respond due to increasing the strength of negative feedback from phosphatases. Overall, we have combined data-driven and mechanistic modeling to generate detailed insight into CAR T cell signaling.

Multi-level Proteomics Identifies CT45 as a Chemosensitivity Mediator and Immunotherapy Target in Ovarian Cancer

Most high-grade serous ovarian cancer (HGSOC) patients develop resistance to platinum-based chemotherapy and recur, but 15% remain disease free over a decade. To discover drivers of long-term survival, we quantitatively analyzed the proteomes of platinum-resistant and -sensitive HGSOC patients from minute amounts of formalin-fixed, paraffin-embedded tumors. This revealed cancer/testis antigen 45 (CT45) as an independent prognostic factor associated with a doubling of disease-free survival in advanced-stage HGSOC. Phospho- and interaction proteomics tied CT45 to DNA damage pathways through direct interaction with the PP4 phosphatase complex. Invitro, CT45 regulated PP4 activity, and its high expression led to increased DNA damage and platinum sensitivity. CT45-derived HLA class I peptides, identified by immunopeptidomics, activate patient-derived cytotoxic Tcells and promote tumor cell killing. This study highlights the power of clinical cancer proteomics to identify targets for chemo- and immunotherapy and illuminate their biological roles.

Abstract 5459: Autophagy and senescence as possible mechanisms leading to proliferative recovery and escape from treatment-induced tumor dormancy

Abstract Despite the ability of chemotherapy to eliminate the majority of tumor cells, some are able to escape cell death and proliferate. In this study we evaluated mechanisms of autophagy and senescence, that in theory allow cells to escape apoptosis and might be responsible for cancer recurrence. Autophagy and senescence, either alone or in concert, may result in temporary growth arrest followed by proliferative recovery. With regard to autophagy, its function may be either cytoprotective, where autophagy inhibition results in sensitization to therapy or nonprotective, where autophagy inhibition does not alter sensitivity to chemotherapy and/or ionizing radiation. To determine the mode of autophagy in murine metastatic carcinoma 4T1 cell lines, autophagy was inhibited pharmacologically using chloroquine, and by genetic silencing of the Atg5 autophagy associated protein. Cells were treated with 1 μM Adriamycin, stained with Acridine Orange to assess autophagy by fluorescent microscopy (autophagosomes appear orange-red) and autophagy quantified by Flow Cytometry. Induction and blockade of autophagy by chloroquine was confirmed by Western blotting for the appearance of LC3B and degradation of p62. Senescence was monitored by β-galactosidase staining and quantified by Flow Cytometry based on C12FDG staining; senescence was confirmed based on the induction of p21 and p16. Adriamycin (doxorubicin) exposure resulted in breast tumor cell death as well as prolonged growth arrest; some of the arrested cells eventually recovered and formed colonies. After exposure to doxorubicin, 4T-1 cells were sorted based on intensity of β-galactosidase staining and increased size (senescent cells experience changes in size and morphology). β-galactosidase positive cells were plated and monitored over time during which some of the cells recovered the capacity to proliferate. In separate studies with mouse mammary carcinoma cells, radiation also promoted cell death and prolonged growth arrest from which some cells were able to recover proliferative capacity. Our data suggests that although chemotherapy and radiation induce prolonged growth arrest and senescence, these features are not permanent and cells are able to escape and re-emerge from the senescent state to generate proliferating daughter cells. As pharmacologic inhibition of autophagy did not result in increased sensitivity to doxorubicin in 4T1 cells (or to radiation in the mouse mammary tumor cells) or interfere with proliferative recovery, we postulate that inhibition of senescence associated pathways may block proliferative recovery and/or promote tumor cell killing in response to chemotherapy and/or radiation. Studies in progress are focused on modulation of c-myc and miR34 levels and the IL1/6/8 signaling axis as potential strategies for interference with senescence and suppression of residual (dormant) tumor growth and cancer recurrence. Citation Format: Liliya Tyutyunyk, Theresa Thekkudan, Tareq Saleh, David A. Gewirtz. Autophagy and senescence as possible mechanisms leading to proliferative recovery and escape from treatment-induced tumor dormancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5459. doi:10.1158/1538-7445.AM2017-5459

Survivin inhibitor YM155 induces mitochondrial dysfunction, autophagy, DNA damage and apoptosis in Bcl-xL silenced glioma cell lines.

Because the anti-apoptotic protein Bcl-xL is overexpressed in glioma, one might expect that inhibiting or silencing this gene would promote tumor cell killing. However, our studies have shown that this approach has limited independent activity, but may tip the balance in favor of apoptosis induction in response to other therapeutic interventions. To address this issue, we performed a pharmacological screen using a panel of signaling inhibitors and chemotherapeutic agents in Bcl-xL silenced cells. Although limited apoptosis induction was observed with a series of inhibitors for receptor tyrosine kinases, PKC inhibitors, Src family members, JAK/STAT, histone deacetylase, the PI3K/Akt/mTOR pathway, MAP kinase, CDK, heat shock proteins, proteasomal processing, and various conventional chemotherapeutic agents, we observed a dramatic potentiation of apoptosis in Bcl-xL silenced cells with the survivin inhibitor, YM155. Treatment with YM155 increased the release of cytochrome c, smac/DIABLO and apoptosis inducing-factor, and promoted loss of mitochondrial membrane potential, activation of Bax, recruitment of LC3-II to the autophagosomes and apoptosis in Bcl-xL silenced cells. We also found an additional mechanism for the augmentation of apoptosis due to abrogation of DNA double-strand break repair mediated by Rad51 repression and enhanced accumulation of γH2AX. In summary, our observations may provide a new insight into the link between Bcl-xL and survivin inhibition for the development of novel therapies for glioma. © 2016 Wiley Periodicals, Inc.

The Four Faces of Autophagy: Implications for Cancer Therapy

It is generally thought that autophagy has two primary and opposing functions in tumor cells in response to stress induced by chemotherapy or radiation. One is the cytoprotective function that can in theory be inhibited for therapeutic advantage by sensitizing the cells to these treatment modalities. The other is the cytotoxic function that is generally not observed with conventional treatment modalities, but that may function to promote tumor cell killing either alone or in association with apoptosis. In this commentary/review, we advance the premise that autophagy is actually populated by at least two additional players. One we have termed the nonprotective form of autophagy, where the cell is apparently carrying out autophagy-mediated degradative functions, but where autophagy inhibition does not lead to perceptible alterations in drug or radiation sensitivity. The other is what we now term the cytostatic form of autophagy in that its activation results in prolonged growth inhibition as well as reduced clonogenic survival (loss of reproductive capacity) but in the absence of actual loss of cell viability through apoptosis or necrosis; however, as is the case with cytototoxic autophagy, inhibition of cytostatic autophagy protects the tumor cell from the agent (drugs or radiation) that promotes the autophagic response. In view of current clinical efforts to exploit autophagy inhibition as a therapeutic strategy for sensitization of malignancies to chemotherapy and radiation, it is critical to recognize that if chemotherapy and/or radiation actually promote autophagy in patient tumors, the autophagy is not of necessity cytoprotective in function.

5-Azacytidine enhances the anti-leukemic activity of lintuzumab (SGN-33) in preclinical models of acute myeloid leukemia

Despite therapeutic advances, the poor prognoses for acute myeloid leukemia (AML) and intermediate and high-risk myelodysplastic syndromes (MDS) point to the need for better treatment options. AML and MDS cells express the myeloid marker CD33, making it amenable to CD33-targeted therapy. Lintuzumab (SGN-33), a humanized monoclonal anti-CD33 antibody undergoing clinical evaluation, induced meaningful responses in a Phase 1 clinical trial and demonstrated anti-leukemic activity in preclinical models. Recently, it was reported that 5-azacytidine (Vidaza™) prolonged the overall survival of a group of high risk MDS and AML patients. To determine whether the combination of lintuzumab and 5-azacytidine would be beneficial, a mouse xenograft model of disseminated AML was used to evaluate the combination. There was a significant reduction in tumor burden and an increase in overall survival in mice treated with lintuzumab and 5-azacytidine. The effects were greater than that obtained with either agent alone. As the in vivo anti-leukemic activity of lintuzumab was dependent upon the presence of mouse effector cells including macrophages and neutrophils, in vitro effector function assays were used to assess the impact of 5-azacytidine on lintuzumab activity. The results show that 5-azacytidine significantly enhanced the ability of lintuzumab to promote tumor cell killing through antibody-dependent cellular cytotoxicity (ADCC) and phagocytic (ADCP) activities. These results suggest that lintuzumab and 5-azacytidine act in concert to promote tumor cell killing. Additionally, these findings provide the rationale to evaluate this combination in the clinic.

Tumor Cell Surface Display of Immunoglobulin Heavy Chain Fc by Gene Transfer as a Means to Mimic Antibody Therapy

We hypothesized that inducing display of the immunoglobulin Fc (IgFc) molecule on the tumor cell surface by gene transfer would promote tumor cell killing by the same mechanisms as antibody-based approaches but would alleviate some of the problems inherent in the use of antibodies for cancer therapy. We expressed the cDNA of the Fc portion of the murine IgG2a heavy chain on the surface of tumor cells such that its C terminus projected away from the tumor cell surface, mimicking a natural antibody-tagging event. In vitro, Fc receptor-positive natural killer (NK) cells specifically recognized and lysed B16 melanoma cells expressing surface IgFc. Macrophages bound to B16-Fc cells significantly more than to parental B16 cells and surface IgFc expression promoted formation of the terminal complement pore complex leading to cell lysis and death. Expression of IgFc dramatically delayed the ability of B16 cells to form tumors in vivo, attributable largely to the effects of NK cells. Furthermore, fluorescence-activated cell-sorting analysis showed that cells from outgrowth B16 IgFc tumors had lost all IgFc expression. When additional immunostimulatory signals were provided at the time of IgFc-mediated tumor cell killing through expression of heat shock protein 70 (hsp70), significant antitumor immunity was generated. Intratumoral delivery of an adenoviral vector expressing IgFc was effective at treating locally accessible tumors but did not impact metastatic disease. However, delivery of adenoviral vectors expressing both IgFc and hsp70 cured both local and metastatic tumors established for 6 days before viral treatment. These data suggest that it is possible to use gene transfer to mimic the beneficial properties of antibody therapy while alleviating some of the associated problems.

704. Tumor Cell Surface Display of the Immunoglobulin Heavy Chain Fc by Gene Transfer as a Means to Mimic Antibody Therapy

We hypothesized that inducing display of the IgFc molecule on the tumor cell surface by gene transfer would promote tumor cell killing by the same mechanisms as antibody based approaches, but would alleviate some of the problems inherent in the use of antibodies for cancer therapy. We expressed the cDNA of the Fc portion of the murine IgG2a heavy chain on the surface of tumor cells thereby mimicking a natural antibody tagging event. In vitro, Fc receptor positive natural killer cells specifically recognized and lysed B16 melanoma cells expressing surface IgFc. Macrophages bound to B16-Fc, cells significantly more than to parental B16 and surface IgFc expression promoted formation of the terminal complement pore complex. Expression of the IgFc dramatically delayed the ability of B16 cells to form tumors in vivo, dependent on macrophages and NK cells.

The functional properties of Fcγ RI, II and III on myeloid cells: A comparative study of killing of erythrocytes and tumor cells mediated through the different Fc receptors

Three distinct Fc receptors for IgG, Fcγ RI, Fcγ RII and Fcγ RIII are known to be associated with human myeloid cells. Using mAb specific for these receptors, and the hydridoma cells lines that produce these mAb, we have examined the ability of each of these receptors on different myeloid cells and cell lines to mediate killing of tumor and red cell targets. Hybridoma cells (HC) expressing anti-Fcγ RI, Fcγ RII or Fcγ RIII upon their surface were used as model self-directed tumor targets. Chicken erythrocytes (CE) were used as another type of target cell and in this case effector cell cytotoxicity was mediated by heteroantibodies (HA) composed of Fab fragments of anti-Fcγ R mAb covalently linked to Fab fragments of rabbit anti-CE antibodies. Monocytes, lymphocytes, polymorphonuclear cells (PMNs) and the myeloid cell lines U937, HL-60 and THP-l were used as effector cells either in their native state or after activation with rIFN-γ. Direct comparison of cytotoxicity by the same effector cell population against both tumor and erythroid targets has permitted definitive evaluation of the ability of the different Fcγ R to promote cytolysis under two different conditions. Monocytes were able to utilize Fcγ RI, Fcγ RII and Fcγ RIII in killing both CE and HC targets, and incubation with rIFN-γ augmented their ability to kill CE, particularly through Fcγ RI. Fcγ RII and Fcγ RIII mediated killing of CE by untreated neutrophils. rIFN-γ induced PMNs to express Fcγ RI and to mediate killing of CE through this receptor. Moreover, HC targets were not lyzed by untreated neutrophils, but rIFN-γ activated neutrophils killed HC bearing surface anti-Fcγ RI and anti-Fcγ RII, but not anti-Fcγ RIII. Myeloid cell lines HL-60 and U937 were unable to perform cytotoxicity without prior culture with rIFN-γ, following which they killed CE through Fcγ RI and Fcγ RII, but were still incapable of HC lysis. THP-l, another myeloid cell line, was cytotoxic to CE through Fcγ RI and Fcγ RII without activation. Following rIFN-γ treatment, cytotoxicity through these two Fcγ R increased and was also mediated by Fcγ RIII but these cells were still unable to kill HC. However, after culture under conditions which promoted a macrophage-like phenotype, THP-l cells acquired the ability to kill HC through Fcγ RI and Fcγ RII. When differentiated THP-l cells were also stimulated with rIFN-γ, killing of HC was augmented and was mediated by Fcγ RI, Fcγ RII and Fcγ RIII. Thus, untreated monocytes performed cytotoxicity of CE and HC through all three Fcγ R, whereas untreated neutrophils performed killing of CE only through Fcγ RII and Fcγ RIII and killed HC only after treatment with IFN-γ and only through Fcγ RI and Fcγ RII. Fcγ RIII on PMNs was unable to promote tumor cell killing even after IFN-γ activation of these cells. These studies indicate that the different Fcγ R on the various effector cell populations have distinct abilities to mediate cytotoxicity of tumor and red cell targets.

Hybrid antibodies in cancer diagnosis and therapy.

Monoclonal Antibodies (Mabs) represent a promising tool for cancer diagnosis and therapy. Administration of MAbs alone or conjugated to cytotoxic agents has been attempted but has significant limitations. Another potentially effective approach is the use of bispecific or bifunctional antibodies where the capacity to recognize the tumor cell and the toxic agent or lymphocyte activation molecule are united in one MAb. The hybrid molecule can be produced by chemical linkage between the two parental antibodies, or alternatively by a biological approach that consists in the fusion of the two selected hybridomas. In the resulting quadroma cell the hybridoma immunoglobulin chains recombine randomly to form the bifunctional MAb. In different in vitro and in vivo models, bifunctional MAbs against tumor and CD3 at nanomolar concentration has been shown to promote tumor cell killing by cytotoxic T cells. Specific localization of chemotherapeutic drugs in xenografted tumors has been demonstrated in mice pretreated with hybrid MAbs. The advantages of the hybrid MAb approach are that it should reduce the MAb biodistribution problem and that it involves no chemical manipulation between the functional agent and the MAb molecules.