Exit pathways of therapeutic antibodies from the brain and retention strategies.

Treating brain diseases requires therapeutics to pass the blood-brain barrier (BBB) which is nearly impermeable for large biologics such as antibodies. Several methods now facilitate crossing or circumventing the BBB for antibody therapeutics. Some of these exploit receptor-mediated transcytosis, others use direct delivery bypassing the BBB. However, successful delivery into the brain does not preclude exit back to the systemic circulation. Various mechanisms are implicated in the active and passive export of antibodies from the central nervous system. Here we review findings on active export via transcytosis of therapeutic antibodies - in particular, the role of the neonatal Fc receptor (FcRn) - and discuss a possible contribution of passive efflux pathways such as lymphatic and perivascular drainage. We point out open questions and how to address these experimentally. In addition, we suggest how emerging findings could aid the design of the next generation of therapeutic antibodies for neurologic diseases.

HDR-based CRISPR/Cas9-mediated Knockout of PD-L1 in C57BL/6 Mice.

The immune-inhibitory molecule programmed cell death ligand 1 (PD-L1) has been shown to play a role in pathologies such as autoimmunity, infections, and cancer. The expression of PD-L1 not only on cancer cells but also on non-transformed host cells is known to be associated with cancer progression. Generation of PD-L1 deficiency in the murine system enables us to specifically study the role of PD-L1 in physiological processes and diseases. One of the most versatile and easy to use site-specific gene editing tools is the CRISPR/Cas9 system, which is based on an RNA-guided nuclease system. Similar to its predecessors, the Zinc finger nucleases or transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 catalyzes double-strand DNA breaks, which can result in frameshift mutations due to random nucleotide insertions or deletions via non-homologous end joining (NHEJ). Furthermore, although less frequently, CRISPR/Cas9 can lead to insertion of defined sequences due to homology-directed repair (HDR) in the presence of a suitable template. Here, we describe a protocol for the knockout of PD-L1 in the murine C57BL/6 background using CRISPR/Cas9. Targeting of exon 3 coupled with the insertion of a HindIII restriction site leads to a premature stop codon and a loss-of-function phenotype. We describe the targeting strategy as well as founder screening, genotyping, and phenotyping. In comparison to NHEJ-based strategy, the presented approach results in a defined stop codon with comparable efficiency and timelines as NHEJ, generates convenient founder screening and genotyping options, and can be swiftly adapted to other targets.

Attenuation of peripheral serotonin inhibits tumor growth and enhances immune checkpoint blockade therapy in murine tumor models.

Platelet-derived peripheral serotonin has pleiotropic effects on coagulation, metabolism, tissue regeneration, and cancer growth; however, the effect of serotonin on the tumor microenvironment remains understudied. Peripheral serotonin­deficient (Tph1−/−) mice displayed reduced growth of subcutaneous and orthotopically injected syngeneic murine pancreatic and colorectal cancers with enhanced accumulation of functional CD8+ T cells compared to control C57BL/6 mice, resulting in extended overall survival. Subcutaneous and orthotopic syngeneic tumors from Tph1−/− mice expressed less programmed cell death 1 ligand 1 (PD-L1), suggesting serotonin-mediated regulation. Serotonin enhanced expression of PD-L1 on mouse and human cancer cells in vitro via serotonylation, which is the formation of covalent bonds between glutamine residues and serotonin, resulting in activation of small G proteins. Serotonin concentrations in metastases of patients with abdominal tumors negatively correlated to the number of CD8+ tumor-infiltrating T cells. Depletion of serotonin cargo or inhibition of serotonin release from thrombocytes decreased growth of syngeneic pancreatic and colorectal tumors in wild-type mice, increased CD8+ T cell influx, and decreased PD-L1 expression. Pharmacological serotonin depletion with oral fluoxetine or intraperitoneal injection of the TPH1 inhibitor telotristat augmented the effects of programmed cell death protein 1 (PD-1) checkpoint blockade and triggered long-term tumor control in mice subcutaneously inoculated with syngeneic colorectal and pancreatic tumors. Overall, peripheral serotonin weakens effector functions of CD8+ T cells within tumors. Clinically approved serotonin targeting agents alone or in combination with PD-1 blockade provided long-term control of established tumors in murine models, warranting further investigation of the clinical translatability of these findings.

Enzymatic Dissociation Induces Transcriptional and Proteotype Bias in Brain Cell Populations.

Different cell isolation techniques exist for transcriptomic and proteotype profiling of brain cells. Here, we provide a systematic investigation of the influence of different cell isolation protocols on transcriptional and proteotype profiles in mouse brain tissue by taking into account single-cell transcriptomics of brain cells, proteotypes of microglia and astrocytes, and flow cytometric analysis of microglia. We show that standard enzymatic digestion of brain tissue at 37 °C induces profound and consistent alterations in the transcriptome and proteotype of neuronal and glial cells, as compared to an optimized mechanical dissociation protocol at 4 °C. These findings emphasize the risk of introducing technical biases and biological artifacts when implementing enzymatic digestion-based isolation methods for brain cell analyses.

Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery.

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an approach provides a safe delivery method by-passing the blood brain barrier (BBB), thus allowing treatment with therapeutics with poor BBB-permeability or those for which systemic exposure is not desired, e.g., due to toxicity. CED requires optimization of the catheter design, injection protocol, and properties of the infusate. With this protocol we describe how to perform CED of a solution containing up to 20 µg of an antibody into the caudate putamen of mice. It describes preparation of step catheters, testing them in vitro and performing the CED in mice using a ramping injection program. The protocol can be readily adjusted for other infusion volumes and can be used for injecting various tracers or pharmacologically active or inactive substances, including chemotherapeutics, cytokines, viral particles, and liposomes.

CSF1R-dependent myeloid cells are required for NK­mediated control of metastasis.

Myeloid leukocytes are essentially involved in both tumor progression and control. We show that neo-adjuvant treatment of mice with an inhibitor of CSF1 receptor (CSF1R), a drug that is used to deplete tumor-associated macrophages, unexpectedly promoted metastasis. CSF1R blockade indirectly diminished the number of NK cells due to a paucity of myeloid cells that provide the survival factor IL-15 to NK cells. Reduction of the number of NK cells resulted in increased seeding of metastatic tumor cells to the lungs but did not impact on progression of established metastases. Supplementation of mice treated with CSF1R-inhibitor with IL-15 restored numbers of NK cells and diminished metastasis. Our data suggest that CSF1R blockade should be combined with administration of IL-15 to reduce the risk of metastasis.

Stromal Expression of Activated Leukocyte Cell Adhesion Molecule Promotes Lung Tumor Growth and Metastasis.

Activated leukocyte cell adhesion molecule (ALCAM) is expressed on various cell types, including leukocytes, endothelial cells, and certain tumor cells. Although ALCAM expression on tumor cells has been linked to tumor invasion and metastatic spread, the contribution of ALCAM expressed in cells forming the tumor stroma to cancer progression has not been investigated. In this study, ALCAM-deficient (ALCAM-/-) mice were used to evaluate the role of ALCAM in lung tumor growth and metastasis. ALCAM-/- mice displayed an altered blood vascular network in the lung and the diaphragm, indicative of an angiogenetic defect. The absence of ALCAM expression in cells forming the stromal tumor microenvironment profoundly affected lung tumor growth in three different i.v. metastasis models. In the case of Lewis lung carcinoma (LLC), an additional defect in tumor cell homing to the lungs and a resulting reduction in the number of lung tumor nodules were observed. Similarly, when LLC cells were implanted subcutaneously for the study of spontaneous tumor cell metastasis, the rate of LLC metastasis to the lungs was profoundly reduced in ALCAM-/- mice. Taken together, our work demonstrates for the first time the in vivo contribution of ALCAM to angiogenesis and reveals a novel role of stromally expressed ALCAM in supporting tumor growth and metastatic spread.

Rational Combination of Immunotherapies with Clinical Efficacy in Mice with Advanced Cancer.

In the context of cancer, naïve T cells are insufficiently primed and become progressively dysfunctional. Boosting antitumor responses by blocking PD-1 or CTLA-4 results in durable clinical responses only in a limited proportion of cancer patients, suggesting that other pathways must be targeted to improve clinical efficacy. Our preclinical study in TRAMP mice comparing 14 different immune interventions identified anti-CD40 + IL2/anti-IL2 complexes + IL12Fc as a uniquely efficacious treatment that prevents tolerance induction, promotes priming of sustained, protective tumor-specific CD8(+) T cells, and cures late-stage cancer when given together with adoptively transferred tumor-specific T cells. We propose that improving signals 2 (costimulation) and 3 (cytokines) together with fresh tumor-specific, rather than boosting of dysfunctional preexisting memory, T cells represents a potent therapy for advanced cancer.

Targeting of FLT3-ITD kinase contributes to high selectivity of imidazoacridinone C-1311 against FLT3-activated leukemia cells.

Drugs targeting receptor tyrosine kinase FLT3 are of particular interest since activating FLT3-internal tandem duplication (ITD) mutations abundantly occur in fatal acute myeloid leukemias (AMLs). Imidazoacridinone C-1311, a DNA-reactive inhibitor of topoisomerase II, has been previously shown to be a potent and selective inhibitor of recombinant FLT3. Here, we expand those findings by studying its effect on leukemia cells with wild-type FLT3, FLT3-ITD mutant and no FLT3 receptor. While brief C-1311 exposure blocked wild-type and FLT3-ITD activity, profound and sustained inhibition was achieved only for FLT3-ITD mutants. C-1311 inhibited FLT3 downstream pathways (MAPK and AKT) independent of FLT3 status, yet translation to decreased viability was significant in FLT3-ITD cells. RNA interference against FLT3-ITD reduced cytotoxic effect and apoptosis induced by C-1311, indicating selective inhibition of FLT3-ITD crucial for high efficacy of drug against activated leukemia cells. Cellular responses in treated FLT3-ITD mutants included G1 and G2/M phase arrest, moderate inhibition of Bcl-2, caspase-3 activation, PARP cleavage, and depolarization of mitochondria. Consistent with selective decrease in FLT3-ITD activity, C-1311 remarkably reduced antiapoptotic survivin mRNA and protein expression, correlating well with enhanced apoptosis of FLT3-ITD cells. No survivin decrease and respectively lower level of apoptosis was found in wild-type and null-FLT3 cells. Combination of C-1311 with cytarabine or doxorubicin again showed distinct synergistic activity in FLT3-ITD-positive cells. The ability of C-1311 to selectively target constitutively active FLT3, suggests a favorable therapeutic index for AML carrying FLT3-ITD mutations. Thus further preclinical and clinical studies addressing its potency against FLT3-ITD kinase is well justified.

The role of FLT3 kinase as an AML therapy target.

FMS-like tyrosine kinase-3 (FLT3) is a tyrosine kinase receptor involved in the survival and expansion of hematopoietic stem progenitors. A constitutively activated, mutated form of FLT3, is expressed in approximately 30% of de novo acute myeloid leukemia (AML) and about 6% of acute lymphoblastic leukemia (ALL) cases. Since mutant FLT3 has emerged as an attractive therapeutic target, there are several FLT3 inhibitors currently undergoing evaluation in different phases of clinical trials. However, although many aspects of the intracellular signaling mediated by oncogenic FLT3 have been revealed, what is the best strategy to inhibit FLT3 and how FLT3 inhibitors should be developed for AML treatment is poorly defined. Despite promising in vitro studies, where most FLT3 inhibitors show potent efficacy at nanomolar concentrations, clinical responses in AML patients are moderate and temporary. Furthermore, under prolonged therapy, FLT3 mutation-positive leukemic cells rapidly develop resistance to FLT3 inhibitors when used as monotherapy. Considering that there is no uniform mechanism of resistance triggered by FLT3 inhibitors, it will be necessary to develop new agents that target FLT3, and that can be used consecutively or in combination with conventional cytotoxic therapeutics. On the other hand, given that overexpression of FLT3 ligand (FL), occurring after myelosuppressive therapy, reduces the efficacy of FLT3 inhibitors, targeting both FL and FLT3 kinase, might be more effective approach in AML treatment. Here, we summarize up-to-date studies on FLT3 structure, its mutation status and role in malignant signal trafficking. We also review why FLT3 targeted therapies have not revolutionized AML treatment.