Antigen specificity can be irrelevant to immunocytokine efficacy and biodistribution

Cytokine therapy can activate potent, sustained antitumor responses, but collateral toxicity often limits dosages. Although antibody–cytokine fusions (immunocytokines) have been designed with the intent to localize cytokine activity, systemic dose-limiting side effects are not fully ameliorated by attempted tumor targeting. Using the s.c. B16F10 melanoma model, we found that a nontoxic dose of IL-2 immunocytokine synergized with tumor-specific antibody to significantly enhance therapeutic outcomes compared with immunocytokine monotherapy, concomitant with increased tumor saturation and intratumoral cytokine responses. Examination of cell subset biodistribution showed that the immunocytokine associated mainly with IL-2R–expressing innate immune cells, with more bound immunocytokine present in systemic organs than the tumor microenvironment. More surprisingly, immunocytokine antigen specificity and Fcγ receptor interactions did not seem necessary for therapeutic efficacy or biodistribution patterns because immunocytokines with irrelevant specificity and/or inactive mutant Fc domains behaved similarly to tumor-specific immunocytokine. IL-2–IL-2R interactions, rather than antibody–antigen targeting, dictated immunocytokine localization; however, the lack of tumor targeting did not preclude successful antibody combination therapy. Mathematical modeling revealed immunocytokine size as another driver of antigen targeting efficiency. This work presents a safe, straightforward strategy for augmenting immunocytokine efficacy by supplementary antibody dosing and explores underappreciated factors that can subvert efforts to purposefully alter cytokine biodistribution.National Cancer Institute (U.S.) (Grant CA174795)National Science Foundation (U.S.). Graduate Research Fellowshi

More accurate process understanding from process characterization studies using Monte Carlo simulation, regularized regression, and classification models

Establishment of an appropriate control strategy with defined operating ranges (OR) predicted to meet a target product profile is a critical component of commercializing new biologics under the Quality by Design (QbD) approach. Process characterization (PC) studies are performed to expand process understanding by achieving two main goals: 1) determining which process parameters have significant effects on quality attributes and 2) establishing models describing the relationships between these critical process parameters (CPP) and critical quality attributes (CQA). Risk assessment and design of experiments (DOE) techniques are effectively deployed in the industry to identify parameters to study and build process understanding. However, the true value of the data produced by these studies can be compromised by the inherent flaws with traditional data analysis techniques. In particular, p-value based methods such as stepwise regression are prone to generate false positives and overestimated parameter coefficients. Many of the deficiencies of traditional stepwise regression can be alleviated by applying Monte Carlo cross validation (MCCV) and simulations to stepwise algorithms. These methods can greatly enhance process understanding and assist in the selection of CPPs. Regularized regression methods such as LASSO, ridge, and elastic net are also designed to overcome many of the issues inherent in techniques based on ordinary least squares. However, a superior strategy is to build multiple models using a variety of techniques and use the insights gained from each to establish the relationships between CPPs and CQAs. Use of complementary methods during data analysis allows more informed decisions to be made during model construction. Please click Additional Files below to see the full abstract

Temporally Programmed CD8α+ DC Activation Enhances Combination Cancer Immunotherapy

Numerous synergistic cancer immunotherapy combinations have been identified, but the effects of relative dose timing are rarely considered. In established syngeneic mouse tumor models, we found that staggering interferon-α (IFNα) administration after, rather than before or simultaneously with, serum-persistent interleukin-2 (IL-2) and tumor-specific antibody significantly increased long-term survival. Successful combination therapy required IFNα-induced activation of cross-presenting CD8α[superscript +] dendritic cells (DCs) following the release of antigenic tumor debris by the IL-2- and antibody-mediated immune response. Due to decreased phagocytic ability post-maturation, DCs activated too early captured less antigen and could not effectively prime CD8[superscript +] T cells. Temporally programming DC activation to occur after tumoricidal activity enhanced tumor control by multiple distinct combination immunotherapies, highlighting dose schedule as an underappreciated factor that can profoundly affect the success of multi-component immunotherapies.National Cancer Institute (U.S.) (Grants NCI CA174795 and CA101830)Ludwig Center for Molecular Oncology at MIT (Graduate Fellowship)Thomas and Stacey Siebel Foundation (Scholarship)National Science Foundation (U.S.). Graduate Research Fellowship ProgramHertz Foundation (Fellowship

A graphene-based physiometer array for the analysis of single biological cells

A significant advantage of a graphene biosensor is that it inherently represents a continuum of independent and aligned sensor-units. We demonstrate a nanoscale version of a micro-physiometer – a device that measures cellular metabolic activity from the local acidification rate. Graphene functions as a matrix of independent pH sensors enabling subcellular detection of proton excretion. Raman spectroscopy shows that aqueous protons p-dope graphene – in agreement with established doping trajectories, and that graphene displays two distinct pKa values (2.9 and 14.2), corresponding to dopants physi- and chemisorbing to graphene respectively. The graphene physiometer allows micron spatial resolution and can differentiate immunoglobulin (IgG)-producing human embryonic kidney (HEK) cells from non-IgG-producing control cells. Population-based analyses allow mapping of phenotypic diversity, variances in metabolic activity, and cellular adhesion. Finally we show this platform can be extended to the detection of other analytes, e.g. dopamine. This work motivates the application of graphene as a unique biosensor for (sub)cellular interrogation.National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051)U.S. Army Research LaboratoryUnited States. Army Research Office. Institute for Soldier Nanotechnologies (Contract W911NF-13-D-0001)National Institute for Biomedical Imaging and Bioengineering (U.S.) (Grant P41EB015871-27)Skolkovo Institute of Science and Technolog

T cell mediated combination immunotherapy

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, February 2016.Cataloged from PDF version of thesis. "September 2015."Includes bibliographical references (pages 128-131).Immunotherapy is a broad treatment strategy that harnesses the immune system to fight off a particular condition or disease. Cancer immunotherapy is the specific application of agents designed to interact or stimulate the immune system to fight off tumors. Treatments as diverse as passive antibody therapy, cytokine support, and comprehensive adoptive T cell transfer make up the broad field of immunotherapeutics. Due to the naturally complex interactions inherent in the immune system, there are many options for therapeutic intervention, however, this same complexity makes it extremely difficult to optimize treatment strategies. Because of this, research into developing new immunotherapies, optimizing existing immunotherapies, and designing new combinations of immunotherapies is still critical in the fight against cancer. Although there have been ongoing successes of individual immunotherapies in the clinic, the complexity and interdependence of the immune system suggests that any single therapeutic intervention will be insufficient to reject established malignancies. Increased interest in applying combinations of immunotherapies in the clinic requires more thorough preclinical work to guide the designs of these studies. The work presented in this thesis focuses on developing combinations of immunotherapies to treat preclinical models of cancer, as well as studying the underlying mechanism of tumor control. T cells are potent mediators of cytotoxicity and when properly used in adoptive cell transfer (ACT) protocols, can be highly effective in the treatment of cancer. ACT consists of three steps: 1) harvesting and purifying T cells from the patient, 2) enriching or modifying the T cells to become tumor specific, and 3) reinfusing the T cells along with supporting therapies. Therapies given alongside ACT are often adjuvants designed to enhance T cell response. However, focusing therapies only on enhancing the activity of the transferred T cells may miss out on synergistic effects when other parts of the immune system are simultaneously engaged. To study the effect of adjuvant therapy on ACT, a preclinical murine model was analyzed. Large, established B16F10 tumors were controlled when pmel-1 T cells were given with a course of supportive MSA-IL2 cytokine therapy, however, no cures were observed. When a course of TA99 antibody therapy was added alongside ACT, a high rate of cures was observed. Flow cytometry of both circulating and tumor infiltrating pmel-1 cells showed massive expansion and activation. Additionally, tumor infiltration of neutrophils, NK cells, and DCs were greatly enhanced by adjuvant therapy. DCs in the tumor draining lymph nodes were largely unchanged by the therapies. Engagement of the humoral immune response was also observed in both treatment cases. Surprisingly, antibody therapy did not substantially alter any of the mechanistic observations made in this study, despite its critical role in achieving cures of tumors. While ACT is a highly effective therapy, its clinical applicability is hindered by the complexity of performing T cell transplants and manipulations. A more optimal solution would involve purely injectable treatments that could elicit the same level of tumor specific T cell response in conjunction with potent recruitment of the adaptive immune system against tumors. To achieve this, working in collaboration with the Irvine Lab, combinations of immunotherapy using up to four different components were tested to identify critical factors in the successful rejection of established tumors in preclinical models. The four components of tumor targeting antibody, cytokine support, checkpoint blockade, and cancer vaccine acted synergistically to reject tumors from B16F10, TC-1, and DD-Her2/neu cell lines. The cancer vaccine elicited large numbers of tumor-specific T cells, and acted as a replacement for ACT. By analyzing subset combinations of this full treatment, the roles of each therapeutic component were identified. CD8 T cells and cross-presenting DCs were critical to curing subcutaneous tumors. Cytokine therapy was indispensable for effective tumor control, promoted immune cell infiltration into the tumor, and led to an increase in DCs. In combination with the other therapies, vaccination against a tumor antigen elicited a strong immunological memory response that was able to reject subsequent tumor rechallenge, as well as promote antigen spreading to new epitopes. Successful combinations were demonstrated to be dependent on the recruitment of both the adaptive and innate branches of the immune system. Finally, the efficacy of this combination of treatments was demonstrated by controlling the growth of induced tumors in a BRaf/Pten model. Combination immunotherapy promises a future where synergistic treatments are specifically tailored to individual cancers leading to highly effective responses. However, determining the optimal combination of therapies, the complexity of dosing strategies, and the availability of targeted treatments are all barriers that must be overcome. The analysis presented here will make a significant contribution to the body of knowledge on immunotherapy as it has shown the importance of combining orthogonal immunotherapies in order to get durable cures to established tumors. These results will hopefully encourage combinations of orthogonally acting therapies based on T cells to achieve stronger clinical responses. By determining the necessary requirements for a strong, synergistic response to tumorous growths, more effective combination immunotherapy protocols may be designed in the future.by Cary F. Opel.Ph. D

Artificial Anti-Tumor Opsonizing Proteins with Fibronectin Scaffolds Engineered for Specificity to each of the Murine FcγR Types

© 2018 Elsevier Ltd We have engineered a panel of novel Fn3 scaffold-based proteins that bind with high specificity and affinity to each of the individual mouse Fcγ receptors (mFcγR). These binders were expressed as fusions to anti-tumor antigen single-chain antibodies and mouse serum albumin, creating opsonizing agents that invoke only a single mFcγR response rather than the broader activity of natural Fc isotypes, as well as all previously reported Fc mutants. This panel isolated the capability of each of the four mFcγRs to contribute to macrophage phagocytosis of opsonized tumor cells and in vivo tumor growth control with these monospecific opsonizing fusion proteins. All activating receptors (mFcγRI, mFcγRIII, and mFcγRIV) were capable of driving specific tumor cell phagocytosis to an equivalent extent, while mFcγRII, the inhibitory receptor, did not drive phagocytosis. Monospecific opsonizing fusion proteins that bound mFcγRI alone controlled tumor growth to an extent similar to the most active IgG2a murine isotype. As expected, binding to the inhibitory mFcγRII did not delay tumor growth, but unexpectedly, mFcγRIII also failed to control tumor growth. mFcγRIV exhibited detectable but lesser tumor-growth control leading to less overall survival compared to mFcγRI. Interestingly, in vivo macrophage depletion demonstrates their importance in tumor control with mFcγRIV engagement, but not with mFcγRI. This panel of monospecific mFcγR-binding proteins provides a toolkit for isolating the functional effects of each mFcγR in the context of an intact immune system

Synergistic Innate and Adaptive Immune Response to Combination Immunotherapy with Anti-Tumor Antigen Antibodies and Extended Serum Half-Life IL-2

Cancer immunotherapies under development have generally focused on either stimulating T cell immunity or driving antibody-directed effector functions of the innate immune system such as antibody-dependent cell-mediated cytotoxicity (ADCC). We find that a combination of an anti-tumor antigen antibody and an untargeted IL-2 fusion protein with delayed systemic clearance induces significant tumor control in aggressive isogenic tumor models via a concerted innate and adaptive response involving neutrophils, NK cells, macrophages, and CD8+ T cells. This combination therapy induces an intratumoral “cytokine storm” and extensive lymphocyte infiltration. Adoptive transfer of anti-tumor T cells together with this combination therapy leads to robust cures of established tumors and development of immunological memory.National Cancer Institute (U.S.) (Grant NCI CA174795)National Institute of General Medical Sciences (U.S.). Biotechnology Training ProgramNational Science Foundation (U.S.). Graduate Research Fellowship Progra