Mathematical Modeling Unveils Optimization Strategies for Targeted Radionuclide Therapy of Blood Cancers.

Targeted radionuclide therapy is based on injections of cancer-specific molecules conjugated with radioactive nuclides. Despite the specificity of this treatment, it is not devoid of side-effects limiting its use and is especially harmful for rapidly proliferating organs well perfused by blood, like bone marrow. Optimization of radioconjugates administration accounting for toxicity constraints can increase treatment efficacy. Based on our experiments on disseminated multiple myeloma mouse model treated by 225 Ac-DOTA-daratumumab, we developed a mathematical model which investigation highlighted the following principles for optimization of targeted radionuclide therapy. 1) Nuclide to antibody ratio importance. The density of radioconjugates on cancer cells determines the density of radiation energy deposited in them. Low labeling ratio as well as accumulation of unlabeled antibodies and antibodies attached to decay products in the bloodstream can mitigate cancer radiation damage due to excessive occupation of specific receptors by antibodies devoid of radioactive nuclides. 2) Cancer binding capacity-based dosing. The rate of binding of drug to cancer cells depends on the total number of their specific receptors, which therefore can be estimated from the pharmacokinetic curve of diagnostic radioconjugates. Injection of doses significantly exceeding cancer binding capacity should be avoided since radioconjugates remaining in the bloodstream have negligible efficacy to toxicity ratio. 3) Particle range-guided multi-dosing. The use of short-range particle emitters and high-affinity antibodies allows for robust treatment optimization via initial saturation of cancer binding capacity, enabling redistribution of further injected radioconjugates and deposited dose towards still viable cells that continue expressing specific receptors. Significance: Mathematical modeling yields general principles for optimization of targeted radionuclide therapy in mouse models of multiple myeloma that can be extrapolated on another cancer models and on clinical setting.

Yttrium-90 Anti-CD25-BEAM-Conditioning for Autologous Hematopoietic Cell Transplantation in Peripheral T Cell Lymphoma.

Peripheral T cell lymphomas (PTCL) have a poor prognosis with current treatments. High-dose chemotherapy followed by autologous hematopoietic cell transplant (AHCT) is used as a consolidation strategy after achieving clinical remission with first-line therapy, as well as in chemosensitive relapse if allogeneic transplant is not an option. CD25 is a targetable protein often highly expressed in PTCL. In this phase 1 clinical trial, we tested the addition of beta-emitting 90Y-labeled chimeric anti-CD25 basiliximab (aTac) to BEAM (carmustine, etoposide, cytarabine, melphalan) as conditioning for AHCT in patients with PTCL. Twenty-three AHCT-eligible patients were enrolled, and 20 received therapeutic 90Y-aTac-BEAM AHCT. Radiation doses of 0.4, 0.5 and 0.6 mCi/kg were tested. With no observed dose-limiting toxicities, 0.6 mCi/kg was deemed the recommended phase 2 dose. The most prevalent adverse effect, grade 2 mucositis, was experienced by 80% of patients. As of this report, 6 (30%) of the treated patients had died, 5 due to progressive disease and 1 due to multiple organ failure [median time of death 17 mo (range: 9-21 mo)] post-AHCT. Median follow-up was 24 mo (range: 9-26 mo) overall and 24 mo (range: 13-26 mo) for surviving patients. For patients who received therapeutic 90Y-aTac-BEAM AHCT, the 2-year progression-free and overall survival were 59% (95% CI: 34-77%) and 68% (95% CI: 42-84%), respectively. 90Y-aTac-BEAM appears to be safe as an AHCT conditioning regimen for PTCL, with no increased toxicity over the toxicities historically seen with BEAM alone in this patient population. This trial was registered at www.clinicaltrials.gov as # NCT02342782.

Designing combination therapies for cancer treatment: application of a mathematical framework combining CAR T-cell immunotherapy and targeted radionuclide therapy.

Introduction: Cancer combination treatments involving immunotherapies with targeted radiation therapy are at the forefront of treating cancers. However, dosing and scheduling of these therapies pose a challenge. Mathematical models provide a unique way of optimizing these therapies. Methods: Using a preclinical model of multiple myeloma as an example, we demonstrate the capability of a mathematical model to combine these therapies to achieve maximum response, defined as delay in tumor growth. Data from mice studies with targeted radionuclide therapy (TRT) and chimeric antigen receptor (CAR)-T cell monotherapies and combinations with different intervals between them was used to calibrate mathematical model parameters. The dependence of progression-free survival (PFS), overall survival (OS), and the time to minimum tumor burden on dosing and scheduling was evaluated. Different dosing and scheduling schemes were evaluated to maximize the PFS and optimize timings of TRT and CAR-T cell therapies. Results: Therapy intervals that were too close or too far apart are shown to be detrimental to the therapeutic efficacy, as TRT too close to CAR-T cell therapy results in radiation related CAR-T cell killing while the therapies being too far apart result in tumor regrowth, negatively impacting tumor control and survival. We show that splitting a dose of TRT or CAR-T cells when administered in combination is advantageous only if the first therapy delivered can produce a significant benefit as a monotherapy. Discussion: Mathematical models are crucial tools for optimizing the delivery of cancer combination therapy regimens with application along the lines of achieving cure, maximizing survival or minimizing toxicity.

Targeting Wnt signaling for improved glioma immunotherapy.

Introduction: Despite aggressive standard-of-care therapy, including surgery, radiation, and chemotherapy, glioblastoma recurrence is almost inevitable and uniformly lethal. Activation of glioma-intrinsic Wnt/ß-catenin signaling is associated with a poor prognosis and the proliferation of glioma stem-like cells, leading to malignant transformation and tumor progression. Impressive results in a subset of cancers have been obtained using immunotherapies including anti-CTLA4, anti-PD-1, and anti-PD-L1 or chimeric antigen receptor (CAR) T cell therapies. However, the heterogeneity of tumors, low mutational burden, single antigen targeting, and associated antigen escape contribute to non-responsiveness and potential tumor recurrence despite these therapeutic efforts. In the current study, we determined the effects of the small molecule, highly specific Wnt/CBP (CREB Binding Protein)/ß-catenin antagonist ICG-001, on glioma tumor cells and the tumor microenvironment (TME)-including its effect on immune cell infiltration, blood vessel decompression, and metabolic changes. Methods: Using multiple glioma patient-derived xenografts cell lines and murine tumors (GL261, K-Luc), we demonstrated in vitro cytostatic effects and a switch from proliferation to differentiation after treatment with ICG-001. Results: In these glioma cell lines, we further demonstrated that ICG-001 downregulated the CBP/ß-catenin target gene Survivin/BIRC5-a hallmark of Wnt/CBP/ß-catenin inhibition. We found that in a syngeneic mouse model of glioma (K-luc), ICG-001 treatment enhanced tumor infiltration by CD3+ and CD8+ cells with increased expression of the vascular endothelial marker CD31 (PECAM-1). We also observed differential gene expression and induced immune cell infiltration in tumors pretreated with ICG-001 and then treated with CAR T cells as compared with single treatment groups or when ICG-001 treatment was administered after CAR T cell therapy. Discussion: We conclude that specific Wnt/CBP/ß-catenin antagonism results in pleotropic changes in the glioma TME, including glioma stem cell differentiation, modulation of the stroma, and immune cell activation and recruitment, thereby suggesting a possible role for enhancing immunotherapy in glioma patients.

Neuroprotective potential of intranasally delivered L-myc immortalized human neural stem cells in female rats after a controlled cortical impact injury.

Efficacious stem cell-based therapies for traumatic brain injury (TBI) depend on successful delivery, migration, and engraftment of stem cells to induce neuroprotection. L-myc expressing human neural stem cells (LMNSC008) demonstrate an inherent tropism to injury sites after intranasal (IN) administration. We hypothesize that IN delivered LMNSC008 cells migrate to primary and secondary injury sites and modulate biomarkers associated with neuroprotection and tissue regeneration. To test this hypothesis, immunocompetent adult female rats received either controlled cortical impact injury or sham surgery. LMNSC008 cells or a vehicle were administered IN on postoperative days 7, 9, 11, 13, 15, and 17. The distribution and migration of eGFP-expressing LMNSC008 cells were quantified over 1 mm-thick optically cleared (CLARITY) coronal brain sections from TBI and SHAM controls. NSC migration was observed along white matter tracts projecting toward the hippocampus and regions of TBI. ELISA and Nanostring assays revealed a shift in tissue gene expression in LMNSC008 treated rats relative to controls. LMNSC008 treatment reduced expression of genes and pathways involved in inflammatory response, microglial function, and various cytokines and receptors. Our proof-of-concept studies, although preliminary, support the rationale of using intranasal delivery of LMNSC008 cells for functional studies in preclinical models of TBI and provide support for potential translatability in TBI patients.

Neuroprotective potential of intranasally delivered L-myc immortalized human neural stem cells in female rats after a controlled cortical impact injury.

Efficacious stem cell-based therapies for traumatic brain injury (TBI) depend on successful delivery, migration, and engraftment of stem cells to induce neuroprotection. L-myc expressing human neural stem cells (LMNSC008) demonstrate an inherent tropism to injury sites after intranasal (IN) administration. We hypothesize that IN delivered LMNSC008 cells migrate to primary and secondary injury sites and modulate biomarkers associated with neuroprotection and tissue regeneration. To test this, immunocompetent adult female rats received a controlled cortical impact injury (CCI) or sham surgery. LMNSC008 cells or a vehicle (VEH) were administered IN on postoperative days 7, 9, 11, 13, 15, and 17. The distribution and migration of eGFP-expressing LMNSC008 cells were quantified over 1 mm-thick optically cleared (CLARITY) coronal brain sections from TBI and SHAM controls. NSC migration was observed along white matter tracts projecting toward the hippocampus and regions of TBI. ELISA and Nanostring assays revealed a shift in tissue gene expression in LMNSC008 treated rats relative to controls. LMNSC008 treatment reduced expression of genes and pathways involved in inflammatory response, microglial function, and various cytokines and receptors. The data demonstrate a robust proof-of-concept for LMNSC008 therapy for TBI and provides a strong rationale for IN delivery for translation in TBI patients.

Data driven model discovery and interpretation for CAR T-cell killing using sparse identification and latent variables.

In the development of cell-based cancer therapies, quantitative mathematical models of cellular interactions are instrumental in understanding treatment efficacy. Efforts to validate and interpret mathematical models of cancer cell growth and death hinge first on proposing a precise mathematical model, then analyzing experimental data in the context of the chosen model. In this work, we present the first application of the sparse identification of non-linear dynamics (SINDy) algorithm to a real biological system in order discover cell-cell interaction dynamics in in vitro experimental data, using chimeric antigen receptor (CAR) T-cells and patient-derived glioblastoma cells. By combining the techniques of latent variable analysis and SINDy, we infer key aspects of the interaction dynamics of CAR T-cell populations and cancer. Importantly, we show how the model terms can be interpreted biologically in relation to different CAR T-cell functional responses, single or double CAR T-cell-cancer cell binding models, and density-dependent growth dynamics in either of the CAR T-cell or cancer cell populations. We show how this data-driven model-discovery based approach provides unique insight into CAR T-cell dynamics when compared to an established model-first approach. These results demonstrate the potential for SINDy to improve the implementation and efficacy of CAR T-cell therapy in the clinic through an improved understanding of CAR T-cell dynamics.

Sequential CAR T cell and targeted alpha immunotherapy in disseminated multiple myeloma.

Multiple myeloma (MM) is still an incurable disorder despite improved antibody and cellular therapies against different MM antigens. Single targeted antigens have so far been ineffective against MM with most patients relapsing after initial response. Hence, sequential immunotherapies directed at different targets are expected to perform better than monotherapy alone. Here, we optimized and established in preclinical studies the therapeutic rationale of using targeted alpha therapy (TAT) directed against CD38 antigen (225Ac-DOTA-daratumumab) with CAR T cell therapy directed at CS1 antigen in a systemic MM model. The sequential therapies compared CAR T therapy followed by TAT to TAT followed by CAR T therapy. CAR T cell monotherapy increased median survival from 49 days (d) in untreated controls to 71d with a modest improvement to 89d for 3.7 kBq of TAT given 14d later. When CAR T was followed by 7.4 kBq of TAT 29d later, sequential therapy increased median survival from 47d in untreated controls to 106d, compared to 68d for CAR T monotherapy. When CAR T therapy was followed by untargeted alpha immunotherapy using 7.4 kBq of 225Ac-DOTA-trastuzumab (anti-HER2) antibody 29d later, there was only a slight improvement in response over CAR T monotherapy demonstrating the role of tumor targeting. TAT (7.4 kBq) followed by CAR T therapy was also effective when CAR T therapy was delayed for 21d vs 14d or 28d post TAT, highlighting the importance of timing sequential therapies. Sequential targeted therapies using CS1 CAR T or 225Ac-DOTA-CD38 TAT in either order shows promise over monotherapies alone.

First-In-Human Pilot PET Immunoimaging Study of 64Cu-Anti-Carcinoembryonic Antigen Monoclonal Antibody (hT84.66-M5A) in Patients with Carcinoembryonic Antigen-Producing Cancers.

Background: PET imaging using radiolabeled immunoconstructs shows promise in cancer detection and in assessing tumor response to therapies. The authors report the first-in-human pilot study evaluating M5A, a humanized anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb), radiolabeled with 64Cu in patients with CEA-expressing malignancies. The purpose of this pilot study was to identify the preferred patient population for further evaluation of this agent in an expanded trial. Methods: Patients with CEA-expressing primary or metastatic cancer received 64Cu-DOTA-hT84.66-M5A with imaging performed at 1 and 2 days postinfusion. 64Cu-DOTA-hT84.66-M5A PET scan findings were correlated with CT, MRI, and/or FDG PET scans and with histopathologic findings from planned surgery or biopsy performed postscan. Results: Twenty patients received 64Cu-DOTA-hT84.66-M5A. Twelve patients demonstrated positive images, which were confirmed in 10 patients as tumor by standard-of-care (SOC) imaging, biopsy, or surgical findings. Four of the 8 patients with negative imaging were confirmed as true negative, with the remaining 4 patients having disease demonstrated by SOC imaging or surgery. All 5 patients with locally advanced rectal cancer underwent planned biopsy or surgery after 64Cu-DOTA-hT84.66-M5A imaging (4 patients imaged 6-8 weeks after completing neoadjuvant chemotherapy and radiation therapy) and demonstrated a high concordance between biopsy findings and 64Cu-DOTA-hT84.66-M5A PET scan results. Three patients demonstrated positive uptake at the primary site later confirmed by biopsy and at surgery as residual disease. Two patients with negative scans each demonstrated complete pathologic response. In 5 patients with medullary thyroid cancer, 64Cu-DOTA-hT84.66-M5A identified disease not seen on initial CT scans in 3 patients, later confirmed to be disease by subsequent surgery or MRI. Conclusions: 64Cu-DOTA-hT84.66-M5A demonstrates promise in tumor detection, particularly in patients with locally advanced rectal cancer and medullary thyroid cancer. A successor trial in locally advanced rectal cancer has been initiated to further evaluate this agent's ability to define tumor extent before and assess disease response after neoadjuvant chemotherapy and radiotherapy. clinical trial.gov (NCT02293954).

Mathematical modeling of therapeutic neural stem cell migration in mouse brain with and without brain tumors.

Neural stem cells (NSCs) offer a potential solution to treating brain tumors. This is because NSCs can circumvent the blood-brain barrier and migrate to areas of damage in the central nervous system, including tumors, stroke, and wound injuries. However, for successful clinical application of NSC treatment, a sufficient number of viable cells must reach the diseased or damaged area(s) in the brain, and evidence suggests that it may be affected by the paths the NSCs take through the brain, as well as the locations of tumors. To study the NSC migration in brain, we develop a mathematical model of therapeutic NSC migration towards brain tumor, that provides a low cost platform to investigate NSC treatment efficacy. Our model is an extension of the model developed in Rockne et al. (PLoS ONE 13, e0199967, 2018) that considers NSC migration in non-tumor bearing naive mouse brain. Here we modify the model in Rockne et al. in three ways: (i) we consider three-dimensional mouse brain geometry, (ii) we add chemotaxis to model the tumor-tropic nature of NSCs into tumor sites, and (iii) we model stochasticity of migration speed and chemosensitivity. The proposed model is used to study migration patterns of NSCs to sites of tumors for different injection strategies, in particular, intranasal and intracerebral delivery. We observe that intracerebral injection results in more NSCs arriving at the tumor site(s), but the relative fraction of NSCs depends on the location of injection relative to the target site(s). On the other hand, intranasal injection results in fewer NSCs at the tumor site, but yields a more even distribution of NSCs within and around the target tumor site(s).

Anti-CD25 radioimmunotherapy with BEAM autologous hematopoietic cell transplantation conditioning in Hodgkin lymphoma.

High-risk relapsed or refractory (R/R) classical Hodgkin lymphoma (HL) is associated with poor outcomes after conventional salvage therapy and autologous hematopoietic cell transplantation (AHCT). Post-AHCT consolidation with brentuximab vedotin (BV) improves progression-free survival (PFS), but with increasing use of BV early in the treatment course, the utility of consolidation is unclear. CD25 is often expressed on Reed-Sternberg cells and in the tumor microenvironment in HL, and we hypothesized that the addition of 90Y-antiCD25 (aTac) to carmustine, etoposide, cytarabine, melphalan (BEAM) AHCT would be safe and result in a transplantation platform that is agnostic to prior HL-directed therapy. Twenty-five patients with high-risk R/R HL were enrolled in this phase 1 dose-escalation trial of aTac-BEAM. Following an imaging dose of 111In-antiCD25, 2 patients had altered biodistribution, and a third developed an unrelated catheter-associated bacteremia; therefore, 22 patients ultimately received therapeutic 90Y-aTac-BEAM AHCT. No dose-limiting toxicities were observed, and 0.6 mCi/kg was deemed the recommended phase 2 dose, the dose at which the heart wall would not receive >2500 cGy. Toxicities and time to engraftment were similar to those observed with standard AHCT, though 95% of patients developed stomatitis (all grade 1-2 per Bearman toxicity scale). Seven relapses (32%) were observed, most commonly in patients with ≥3 risk factors. The estimated 5-year PFS and overall survival probabilities among 22 evaluable patients were 68% and 95%, respectively, and non-relapse mortality was 0%. aTac-BEAM AHCT was tolerable in patients with high-risk R/R HL, and we are further evaluating the efficacy of this approach in a phase 2 trial. This trial was registered at www.clinicaltrials.gov as #NCT01476839.

A Mathematical Modeling Approach for Targeted Radionuclide and Chimeric Antigen Receptor T Cell Combination Therapy.

Targeted radionuclide therapy (TRT) has recently seen a surge in popularity with the use of radionuclides conjugated to small molecules and antibodies. Similarly, immunotherapy also has shown promising results, an example being chimeric antigen receptor T cell (CAR-T) therapy in hematologic malignancies. Moreover, TRT and CAR-T therapies possess unique features that require special consideration when determining how to dose as well as the timing and sequence of combination treatments including the distribution of the TRT dose in the body, the decay rate of the radionuclide, and the proliferation and persistence of the CAR-T cells. These characteristics complicate the additive or synergistic effects of combination therapies and warrant a mathematical treatment that includes these dynamics in relation to the proliferation and clearance rates of the target tumor cells. Here, we combine two previously published mathematical models to explore the effects of dose, timing, and sequencing of TRT and CAR-T cell-based therapies in a multiple myeloma setting. We find that, for a fixed TRT and CAR-T cell dose, the tumor proliferation rate is the most important parameter in determining the best timing of TRT and CAR-T therapies.

Intranasally Administered L-Myc-Immortalized Human Neural Stem Cells Migrate to Primary and Distal Sites of Damage after Cortical Impact and Enhance Spatial Learning.

As the success of stem cell-based therapies is contingent on efficient cell delivery to damaged areas, neural stem cells (NSCs) have promising therapeutic potential because they inherently migrate to sites of central nervous system (CNS) damage. To explore the possibility of NSC-based therapy after traumatic brain injury (TBI), isoflurane-anesthetized adult male rats received a controlled cortical impact (CCI) of moderate severity (2.8 mm deformation at 4 m/s) or sham injury (i.e., no cortical impact). Beginning 1-week post-injury, the rats were immunosuppressed and 1 × 106 human NSCs (LM-NS008.GFP.fLuc) or vehicle (VEH) (2% human serum albumen) were administered intranasally (IN) on post-operative days 7, 9, 11, 13, 15, and 17. To evaluate the spatial distributions of the LM-NSC008 cells, half of the rats were euthanized on day 25, one day after completion of the cognitive task, and the other half were euthanized on day 46. 1 mm thick brain sections were optically cleared (CLARITY), and volumes were imaged by confocal microscopy. In addition, LM-NSC008 cell migration to the TBI site by immunohistochemistry for human-specific Nestin was observed at day 39. Acquisition of spatial learning was assessed in a well-established Morris water maze task on six successive days beginning on post-injury day 18. IN administration of LM-NSC008 cells after TBI (TBI + NSC) significantly facilitated spatial learning relative to TBI + VEH rats (p < 0.05) and had no effect on sham + NSC rats. Overall, these data indicate that IN-administered LM-NSC008 cells migrate to sites of TBI damage and that their presence correlates with cognitive improvement. Future studies will expand on these preliminary findings by evaluating other LM-NSC008 cell dosing paradigms and evaluating mechanisms by which LM-NSC008 cells contribute to cognitive recovery.

Comparison of CD38-Targeted α- Versus ß-Radionuclide Therapy of Disseminated Multiple Myeloma in an Animal Model.

Targeted therapies for multiple myeloma (MM) include the anti-CD38 antibody daratumumab, which, in addition to its inherent cytotoxicity, can be radiolabeled with tracers for imaging and with ß- and α-emitter radionuclides for radioimmunotherapy. Methods: We have compared the potential therapeutic efficacy of ß- versus α-emitter radioimmunotherapy using radiolabeled DOTA-daratumumab in a preclinical model of disseminated multiple myeloma. Multiple dose levels were investigated to find the dose with the highest efficacy and lowest toxicity. Results: In a dose­response study with the ß-emitter 177Lu-DOTA-daratumumab, the lowest tested dose, 1.85 MBq, extended survival from 37 to 47 d but did not delay tumor growth. Doses of 3.7 and 7.4 MBq extended survival to 55 and 58 d, respectively, causing a small equivalent delay in tumor growth, followed by regrowth. The higher dose, 11.1 MBq, eradicated the tumor but had no effect on survival compared with untreated controls, because of whole-body toxicity. In contrast, the α-emitter 225Ac-DOTA-daratumumab had a dose-dependent effect, in which 0.925, 1.85, and 3.7 kBq increased survival, compared with untreated controls (35 d), to 47, 52, and 73 d, respectively, with a significant delay in tumor growth for all 3 doses. Higher doses of 11.1 and 22.2 kBq resulted in equivalent survival to 82 d but with significant whole-body toxicity. Parallel studies with untargeted 225Ac-DOTA-trastuzumab conferred no improvement over untreated controls and resulted in whole-body toxicity. Conclusion: We conclude, and mathematic modeling confirms, that maximal biologic doses were achieved by targeted α-therapy and demonstrated 225Ac to be superior to 177Lu in delaying tumor growth and decreasing whole-body toxicity.

Towards integration of 64Cu-DOTA-trastuzumab PET-CT and MRI with mathematical modeling to predict response to neoadjuvant therapy in HER2 + breast cancer.

While targeted therapies exist for human epidermal growth factor receptor 2 positive (HER2 +) breast cancer, HER2 + patients do not always respond to therapy. We present the results of utilizing a biophysical mathematical model to predict tumor response for two HER2 + breast cancer patients treated with the same therapeutic regimen but who achieved different treatment outcomes. Quantitative data from magnetic resonance imaging (MRI) and 64Cu-DOTA-trastuzumab positron emission tomography (PET) are used to estimate tumor density, perfusion, and distribution of HER2-targeted antibodies for each individual patient. MRI and PET data are collected prior to therapy, and follow-up MRI scans are acquired at a midpoint in therapy. Given these data types, we align the data sets to a common image space to enable model calibration. Once the model is parameterized with these data, we forecast treatment response with and without HER2-targeted therapy. By incorporating targeted therapy into the model, the resulting predictions are able to distinguish between the two different patient responses, increasing the difference in tumor volume change between the two patients by > 40%. This work provides a proof-of-concept strategy for processing and integrating PET and MRI modalities into a predictive, clinical-mathematical framework to provide patient-specific predictions of HER2 + treatment response.

Identifying CD38+ cells in patients with multiple myeloma: first-in-human imaging using copper-64-labeled daratumumab.

18F-Fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) is one of the most widely used imaging techniques to detect multiple myeloma (MM). Intracellular FDG uptake depicts in vivo metabolic activity, which can be seen in both malignant and nonmalignant cells, resulting in limited sensitivity and specificity. Our group showed preclinically that tracing MM dissemination using a CD38-directed human antibody, daratumumab, that is radioconjugated with 64Cu via the chelator DOTA (64Cu-daratumumab), led to improved sensitivity and specificity over that of FDG. Here, we report the results of a phase 1 trial designed to (1) assess the safety and feasibility of 64Cu-daratumumab PET/CT and (2) preliminarily evaluate and characterize the ability of 64Cu-daratumumab to accurately detect or exclude MM lesions. A total of 12 daratumumab-naive patients were imaged. Prior to the injection of 15 mCi/5 mg of 64Cu-daratumumab, patients were treated with 0 (n = 3), 10 (n = 3), 45 (n = 3), or 95 mg (n = 3) of unlabeled daratumumab to assess its effect on image quality. No significant adverse events were observed from either unlabeled daratumumab or 64Cu-daratumumab. Of the dose levels tested, 45 mg unlabeled daratumumab was the most optimal in terms of removing background signal without saturating target sites. 64Cu-daratumumab PET/CT provided safe whole-body imaging of MM. A trial comparing the sensitivity and specificity of 64Cu-daratumumab PET/CT with that of FDG PET/CT is planned. This trial was registered at www.clinicaltrials.gov as #NCT03311828.

Mathematical deconvolution of CAR T-cell proliferation and exhaustion from real-time killing assay data.

Chimeric antigen receptor (CAR) T-cell therapy has shown promise in the treatment of haematological cancers and is currently being investigated for solid tumours, including high-grade glioma brain tumours. There is a desperate need to quantitatively study the factors that contribute to the efficacy of CAR T-cell therapy in solid tumours. In this work, we use a mathematical model of predator-prey dynamics to explore the kinetics of CAR T-cell killing in glioma: the Chimeric Antigen Receptor T-cell treatment Response in GliOma (CARRGO) model. The model includes rates of cancer cell proliferation, CAR T-cell killing, proliferation, exhaustion, and persistence. We use patient-derived and engineered cancer cell lines with an in vitro real-time cell analyser to parametrize the CARRGO model. We observe that CAR T-cell dose correlates inversely with the killing rate and correlates directly with the net rate of proliferation and exhaustion. This suggests that at a lower dose of CAR T-cells, individual T-cells kill more cancer cells but become more exhausted when compared with higher doses. Furthermore, the exhaustion rate was observed to increase significantly with tumour growth rate and was dependent on level of antigen expression. The CARRGO model highlights nonlinear dynamics involved in CAR T-cell therapy and provides novel insights into the kinetics of CAR T-cell killing. The model suggests that CAR T-cell treatment may be tailored to individual tumour characteristics including tumour growth rate and antigen level to maximize therapeutic benefit.

Quantitative Evaluation of Intraventricular Delivery of Therapeutic Neural Stem Cells to Orthotopic Glioma.

Neural stem cells (NSCs) are inherently tumor-tropic, which allows them to migrate through normal tissue and selectively localize to invasive tumor sites in the brain. We have engineered a clonal, immortalized allogeneic NSC line (HB1.F3.CD21; CD-NSCs) that maintains its stem-like properties, a normal karyotype and is HLA Class II negative. It is genetically and functionally stable over time and multiple passages, and has demonstrated safety in phase I glioma trials. These properties enable the production of an "off-the-shelf" therapy that can be readily available for patient treatment. There are multiple factors contributing to stem cell tumor-tropism, and much remains to be elucidated. The route of NSC delivery and the distribution of NSCs at tumor sites are key factors in the development of effective cell-based therapies. Stem cells can be engineered to deliver and/or produce many different therapeutic agents, including prodrug activating enzymes (which locally convert systemically administered prodrugs to active chemotherapeutic agents); oncolytic viruses; tumor-targeted antibodies; therapeutic nanoparticles; and extracellular vesicles that contain therapeutic oligonucleotides. By targeting these therapeutics selectively to tumor foci, we aim to minimize toxicity to normal tissues and maximize therapeutic benefits. In this manuscript, we demonstrate that NSCs administered via intracerebral/ventricular (IVEN) routes can migrate efficiently toward single or multiple tumor foci. IVEN delivery will enable repeat administrations for patients through an Ommaya reservoir, potentially resulting in improved therapeutic outcomes. In our preclinical studies using various glioma lines, we have quantified NSC migration and distribution in mouse brains and have found robust migration of our clinically relevant HB1.F3.CD21 NSC line toward invasive tumor foci, irrespective of their origin. These results establish proof-of-concept and demonstrate the potential of developing a multitude of therapeutic options using modified NSCs.

Long-term stability and computational analysis of migration patterns of L-MYC immortalized neural stem cells in the brain.

BACKGROUND: Preclinical studies indicate that neural stem cells (NSCs) can limit or reverse central nervous system (CNS) damage through delivery of therapeutic agents for cell regeneration. Clinical translation of cell-based therapies raises concerns about long-term stability, differentiation and fate, and absence of tumorigenicity of these cells, as well as manufacturing time required to produce therapeutic cells in quantities sufficient for clinical use. Allogeneic NSC lines are in growing demand due to challenges inherent in using autologous stem cells, including production costs that limit availability to patients. METHODS/PRINCIPAL FINDINGS: We demonstrate the long-term stability of L-MYC immortalized human NSCs (LM-NSC008) cells in vivo, including engraftment, migration, and absence of tumorigenicity in mouse brains for up to nine months. We also examined the distributions of engrafted LM-NSC008 cells within brain, and present computational techniques to analyze NSC migration characteristics in relation to intrinsic brain structures. CONCLUSIONS/SIGNIFICANCE: This computational analysis of NSC distributions following implantation provides proof-of-concept for the development of computational models that can be used clinically to predict NSC migration paths in patients. Previously, models of preferential migration of malignant tumor cells along white matter tracts have been used to predict their final distributions. We suggest that quantitative measures of tissue orientation and white matter tracts determined from MR images can be used in a diffusion tensor imaging tractography-like approach to describe the most likely migration routes and final distributions of NSCs administered in a clinical setting. Such a model could be very useful in choosing the optimal anatomical locations for NSC administration to patients to achieve maximum therapeutic effects.

[18F]Fluciclovine PET discrimination between high- and low-grade gliomas.

BACKGROUND: The ability to accurately and non-invasively distinguish high-grade glioma from low-grade glioma remains a challenge despite advances in molecular and magnetic resonance imaging. We investigated the ability of fluciclovine (18F) PET as a means to identify and distinguish these lesions in patients with known gliomas and to correlate uptake with Ki-67. RESULTS: Sixteen patients with a total of 18 newly diagnosed low-grade gliomas (n = 6) and high grade gliomas (n = 12) underwent fluciclovine PET imaging after histopathologic assessment. Fluciclovine PET analysis comprised tumor SUVmax and SUVmean, as well as metabolic tumor thresholds (1.3*, 1.6*, 1.9*) to normal brain background (TBmax, and TBmean). Comparison was additionally made to the proliferative status of the tumor as indicated by Ki-67 values. Fluciclovine uptake greater than normal brain parenchyma was found in all lesions studied. Time activity curves demonstrated statistically apparent flattening of the curves for both high-grade gliomas and low-grade gliomas starting 30 min after injection, suggesting an influx/efflux equilibrium. The best semiquantitative metric in discriminating HGG from LGG was obtained utilizing a metabolic 1 tumor threshold of 1.3* contralateral normal brain parenchyma uptake to create a tumor: background (TBmean1.3) cutoff of 2.15 with an overall sensitivity of 97.5% and specificity of 95.5%. Additionally, using a SUVmax > 4.3 cutoff gave a sensitivity of 90.9% and specificity of 97.5%. Tumor SUVmean and tumor SUVmax as a ratio to mean normal contralateral brain were both found to be less relevant predictors of tumor grade. Both SUVmax (R = 0.71, p = 0.0227) and TBmean (TBmean1.3: R = 0.81, p = 0.00081) had a high correlation with the tumor proliferative index Ki-67. CONCLUSIONS: Fluciclovine PET produces high-contrast images between both low-grade and high grade gliomas and normal brain by visual and semiquantitative analysis. Fluciclovine PET appears to discriminate between low-grade glioma and high-grade glioma, but must be validated with a larger sample size.