Evaluation of the novel therapeutic anti-CCR7 antibody CAP-100 as an add-on therapy in chronic lymphocytic leukemia patients receiving venetoclax.

The Bruton's tyrosine kinase inhibitor ibrutinib and the B-cell lymphoma 2 anti-apoptotic protein inhibitor venetoclax provide high response rates in chronic lymphocytic leukemia (CLL). However, there is a growing number of patients that relapse after treatment or show refractory disease, thus new targets and agents are still needed. We have previously reported the chemokine receptor CCR7 as a relevant deregulated target in CLL and have developed CAP-100, a novel therapeutic anti-CCR7 antibody that is under evaluation for relapse/refractory CLL (NCT04704323). While CCR7 expression has been shown to be down-modulated in CLL patients treated with ibrutinib, whether venetoclax acts in a similar manner remains unaddressed. Here, we aimed to document the impact of venetoclax on CCR7 expression in CLL cells, as well as on the pre-clinical activity of CAP-100. To this end, we addressed CCR7 expression by flow cytometry and the antibody efficacy by means of in vitro chemotactic and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. Our data indicate that venetoclax treatment did not significantly modify CCR7 expression pattern nor CAP-100 mechanisms of action. Together, these findings support CAP-100 as an adjuvant therapy to venetoclax that may introduce additional modes of action in CLL therapy.

Targeting CCR7-PI3Kγ overcomes resistance to tyrosine kinase inhibitors in ALK-rearranged lymphoma.

Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) show potent efficacy in several ALK-driven tumors, but the development of resistance limits their long-term clinical impact. Although resistance mechanisms have been studied extensively in ALK-driven non-small cell lung cancer, they are poorly understood in ALK-driven anaplastic large cell lymphoma (ALCL). Here, we identify a survival pathway supported by the tumor microenvironment that activates phosphatidylinositol 3-kinase γ (PI3K-γ) signaling through the C-C motif chemokine receptor 7 (CCR7). We found increased PI3K signaling in patients and ALCL cell lines resistant to ALK TKIs. PI3Kγ expression was predictive of a lack of response to ALK TKI in patients with ALCL. Expression of CCR7, PI3Kγ, and PI3Kδ were up-regulated during ALK or STAT3 inhibition or degradation and a constitutively active PI3Kγ isoform cooperated with oncogenic ALK to accelerate lymphomagenesis in mice. In a three-dimensional microfluidic chip, endothelial cells that produce the CCR7 ligands CCL19/CCL21 protected ALCL cells from apoptosis induced by crizotinib. The PI3Kγ/δ inhibitor duvelisib potentiated crizotinib activity against ALCL lines and patient-derived xenografts. Furthermore, genetic deletion of CCR7 blocked the central nervous system dissemination and perivascular growth of ALCL in mice treated with crizotinib. Thus, blockade of PI3Kγ or CCR7 signaling together with ALK TKI treatment reduces primary resistance and the survival of persister lymphoma cells in ALCL.

Dasatinib-induced spleen contraction leads to transient lymphocytosis.

The tyrosine kinase inhibitor dasatinib is approved for Philadelphia chromosome-positive leukemia, including chronic myeloid leukemia (CML). Although effective and well tolerated, patients typically exhibit a transient lymphocytosis after dasatinib uptake. To date, the underlying physiological process linking dasatinib to lymphocytosis remains unknown. Here, we used a small rodent model to examine the mechanism of dasatinib-induced lymphocytosis, focusing on lymphocyte trafficking into and out of secondary lymphoid organs. Our data indicate that lymphocyte homing to lymph nodes and spleen remained unaffected by dasatinib treatment. In contrast, dasatinib promoted lymphocyte egress from spleen with kinetics consistent with the observed lymphocytosis. Unexpectedly, dasatinib-induced lymphocyte egress occurred independently of canonical sphingosine-1-phosphate-mediated egress signals; instead, dasatinib treatment led to a decrease in spleen size, concomitant with increased splenic stromal cell contractility, as measured by myosin light chain phosphorylation. Accordingly, dasatinib-induced lymphocytosis was partially reversed by pharmacological inhibition of the contraction-promoting factor Rho-rho associated kinase. Finally, we uncovered a decrease in spleen size in patients with CML who showed lymphocytosis immediately after dasatinib treatment, and this reduction was proportional to the magnitude of lymphocytosis and dasatinib plasma levels. In summary, our work provides evidence that dasatinib-induced lymphocytosis is a consequence of drug-induced contractility of splenic stromal cells.

The Merger of Benzophenone HAT Photocatalysis and Silyl Radical-Induced XAT Enables Both Nickel-Catalyzed Cross-Electrophile Coupling and 1,2-Dicarbofunctionalization of Olefins.

A strategy for both cross-electrophile coupling and 1,2-dicarbofunctionalization of olefins has been developed. Carbon-centered radicals are generated from alkyl bromides by merging benzophenone hydrogen atom transfer (HAT) photocatalysis and silyl radical-induced halogen atom transfer (XAT) and are subsequently intercepted by a nickel catalyst to forge the targeted C(sp3)-C(sp2) and C(sp3)-C(sp3) bonds. The mild protocol is fast and scalable using flow technology, displays broad functional group tolerance, and is amenable to a wide variety of medicinally relevant moieties. Mechanistic investigations reveal that the ketone catalyst, upon photoexcitation, is responsible for the direct activation of the silicon-based XAT reagent (HAT-mediated XAT) that furnishes the targeted alkyl radical and is ultimately involved in the turnover of the nickel catalytic cycle.

Ibrutinib Does Not Impact CCR7-Mediated Homeostatic Migration in T-Cells from Chronic Lymphocytic Leukemia Patients.

Bruton's tyrosine kinase inhibitor ibrutinib has significantly changed treatment landscape in chronic lymphocytic leukemia (CLL). Growing evidence supports ibrutinib to work beyond the effect on tumor cells by means of, for example, restoring functionality of the T-cell compartment and increasing circulating T-cell numbers. Recent evidence suggests T-cell enhanced expansion, rather than increased egress from secondary lymphoid organs (SLO), as a root cause for ibrutinib-induced lymphocytosis. However, whether the latter physiological change is also a consequence of a forced retention in blood remains undisclosed. Since CCR7 is the main chemokine receptor taking over the homing of T-cells from peripheral compartments to lymph nodes and other SLO, we aimed to investigate the impact of ibrutinib on CCR7 functionality in T-cells. To this end, we documented receptor expression in T-cells from a large cohort of ibrutinib-treated CLL patients, and performed different in vivo and in vitro migration models. Overall, our data confirm that CCR7 expression or receptor-mediated migration in CLL T-cells is not affected by ibrutinib. Furthermore, it does not modulate CCR7-driven homing nor nodal interstitial migration. Together, our results support that ibrutinib-induced CLL T-cell accumulation in the blood stream is not derived from an impairment of CCR7-driven recirculation between the SLO and bloodstream, and therefore T-cell expansion is the most plausible cause.

Accelerated and Scalable C(sp3)-H Amination via Decatungstate Photocatalysis Using a Flow Photoreactor Equipped with High-Intensity LEDs.

Carbon-nitrogen bonds are ubiquitous in biologically active compounds, prompting synthetic chemists to design various methodologies for their preparation. Arguably, the ideal synthetic approach is to be able to directly convert omnipresent C-H bonds in organic molecules, enabling even late-stage functionalization of complex organic scaffolds. While this approach has been thoroughly investigated for C(sp2)-H bonds, only few examples have been reported for the direct amination of aliphatic C(sp3)-H bonds. Herein, we report the use of a newly developed flow photoreactor equipped with high intensity chip-on-board LED technology (144 W optical power) to trigger the regioselective and scalable C(sp3)-H amination via decatungstate photocatalysis. This high-intensity reactor platform enables simultaneously fast results gathering and scalability in a single device, thus bridging the gap between academic discovery (mmol scale) and industrial production (>2 kg/day productivity). The photocatalytic transformation is amenable to the conversion of both activated and nonactivated hydrocarbons, leading to protected hydrazine products by reaction with azodicarboxylates. We further validated the robustness of our manifold by designing telescoped flow approaches for the synthesis of pyrazoles, phthalazinones and free amines.

Effect of ibrutinib on CCR7 expression and functionality in chronic lymphocytic leukemia and its implication for the activity of CAP-100, a novel therapeutic anti-CCR7 antibody.

CAP-100 is a novel therapeutic antibody directed against the ligand binding site of human CCR7. This chemokine receptor is overexpressed in chronic lymphocytic leukemia (CLL) and orchestrates the homing of CLL cells into the lymph node. Previous studies, on a very limited number of samples, hypothesized that the Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib might induce loss of surface CCR7 levels in CLL cells. CAP-100 will be evaluated in clinical trials as a therapy for relapse/refractory CLL patients, who have received at least two systemic therapies (NCT04704323). As nowadays many relapse/refractory CLL patients will have received ibrutinib as a prior therapy, we aimed to investigate in a large cohort of CLL patients the impact of this BTKi on CCR7 expression and functionality as well as on the therapeutic activity of CAP-100. Our data confirm that ibrutinib moderately down-regulates the very high expression of CCR7 in CLL cells but has no apparent effect on CCR7-induced chemotaxis. Moreover, CLL cells are perfectly targetable by CAP-100 which led to a complete inhibition of CCR7-mediated migration and induced strong target cell killing through antibody-dependent cell-mediated cytotoxicity, irrespective of previous or contemporary ibrutinib administration. Together, these results validate the therapeutic utility of CAP-100 as a next-line single-agent therapy for CLL patients who failed to ibrutinib and confirm that CAP-100 and ibrutinib have complementary non-overlapping mechanisms of action, potentially allowing for combination therapy.

Self-Optimization of Continuous Flow Electrochemical Synthesis Using Fourier Transform Infrared Spectroscopy and Gas Chromatography.

A continuous-flow electrochemical synthesis platform has been developed to enable self-optimization of reaction conditions of organic electrochemical reactions using attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR) and gas chromatography (GC) as online real-time monitoring techniques. We have overcome the challenges in using ATR FT-IR as the downstream analytical methods imposed when a large amount of hydrogen gas is produced from the counter electrode by designing two types of gas-liquid separators (GLS) for analysis of the product mixture flowing from the electrochemical reactor. In particular, we report an integrated GLS with an ATR FT-IR probe at the reactor outlet to give a facile and low-cost solution to determining the concentrations of products in gas-liquid two-phase flow. This approach provides a reliable method for quantifying low-volatile analytes, which can be problematic to be monitored by GC. Two electrochemical reactions the methoxylation of 1-formylpyrrolidine and the oxidation of 3-bromobenzyl alcohol were investigated to demonstrate that the optimal conditions can be located within the pre-defined multi-dimensional reaction parameter spaces without intervention of the operator by using the stable noisy optimization by branch and FIT (SNOBFIT) algorithm.

CCR7 in Blood Cancers - Review of Its Pathophysiological Roles and the Potential as a Therapeutic Target.

According to the classical paradigm, CCR7 is a homing chemokine receptor that grants normal lymphocytes access to secondary lymphoid tissues such as lymph nodes or spleen. As such, in most lymphoproliferative disorders, CCR7 expression correlates with nodal or spleen involvement. Nonetheless, recent evidence suggests that CCR7 is more than a facilitator of lymphatic spread of tumor cells. Here, we review published data to catalogue CCR7 expression across blood cancers and appraise which classical and novel roles are attributed to this receptor in the pathogenesis of specific hematologic neoplasms. We outline why novel therapeutic strategies targeting CCR7 might provide clinical benefits to patients with CCR7-positive hematopoietic tumors.

Rapid Optimization of Photoredox Reactions for Continuous-Flow Systems Using Microscale Batch Technology.

Photoredox catalysis has emerged as a powerful and versatile platform for the synthesis of complex molecules. While photocatalysis is already broadly used in small-scale batch chemistry across the pharmaceutical sector, recent efforts have focused on performing these transformations in process chemistry due to the inherent challenges of batch photocatalysis on scale. However, translating optimized batch conditions to flow setups is challenging, and a general approach that is rapid, convenient, and inexpensive remains largely elusive. Herein, we report the development of a new approach that uses a microscale high-throughput experimentation (HTE) platform to identify optimal reaction conditions that can be directly translated to flow systems. A key design point is to simulate the flow-vessel pathway within a microscale reaction plate, which enables the rapid identification of optimal flow reaction conditions using only a small number of simultaneous experiments. This approach has been validated against a range of widely used photoredox reactions and, importantly, was found to translate accurately to several commercial flow reactors. We expect that the generality and operational efficiency of this new HTE approach to photocatalysis will allow rapid identification of numerous flow protocols for scale.

Decatungstate-Mediated C(sp3 )-H Heteroarylation via Radical-Polar Crossover in Batch and Flow.

Photocatalytic hydrogen atom transfer is a very powerful strategy for the regioselective C(sp3 )-H functionalization of organic molecules. Herein, we report on the unprecedented combination of decatungstate hydrogen atom transfer photocatalysis with the oxidative radical-polar crossover concept to access the direct net-oxidative C(sp3 )-H heteroarylation. The present methodology demonstrates a high functional group tolerance (40 examples) and is scalable when using continuous-flow reactor technology. The developed protocol is also amenable to the late-stage functionalization of biologically relevant molecules such as stanozolol, (-)-ambroxide, podophyllotoxin, and dideoxyribose.

Targeting cancer homing into the lymph node with a novel anti-CCR7 therapeutic antibody: the paradigm of CLL.

Lymph node (LN) is a key tissue in the pathophysiology of mature blood cancers, especially for chronic lymphocytic leukemia (CLL). Within the multiple de-regulated pathways affecting CLL homeostasis, the CC-chemokine receptor 7 (CCR7) grants homing of CLL cells into the LN where protective environments foster tumor progression. To cover the lack of specific therapies targeting the CCR7-dependence of CLL to enter into the LN, and aiming to displace the disease from LN, we generated CAP-100, an antibody that specifically binds to hCCR7 and neutralizes its ligand-binding site and signaling. In various in vitro and in vivo preclinical models CAP-100 strongly inhibited CCR7-induced migration, extravasation, homing, and survival in CLL samples. Moreover, it triggered potent tumor cell killing, mediated by host immune mechanisms, and was effective in xenograft models of high-risk disease. Additionally, CAP-100 showed a favorable toxicity profile on relevant hematopoietic subsets. Our results validated CAP-100 as a novel therapeutic tool to prevent the access of CLL cells, and other neoplasia with nodal-dependence, into the LN niches, thus hitting a central hub in the pathogenesis of cancer. The first-in-human clinical trial (NCT04704323), which will evaluate this novel therapeutic approach in CLL patients, is pending.

Of Lymph Nodes and CLL Cells: Deciphering the Role of CCR7 in the Pathogenesis of CLL and Understanding Its Potential as Therapeutic Target.

The lymph node (LN) is an essential tissue for achieving effective immune responses but it is also critical in the pathogenesis of chronic lymphocytic leukemia (CLL). Within the multitude of signaling pathways aberrantly regulated in CLL the homeostatic axis composed by the chemokine receptor CCR7 and its ligands is the main driver for directing immune cells to home into the LN. In this literature review, we address the roles of CCR7 in the pathophysiology of CLL, and how this chemokine receptor is of critical importance to develop more rational and effective therapies for this malignancy.

Vaccinate fast but leave no one behind: a call to action for COVID-19 vaccination in Spain.

During the first five months of 2021, Spain's COVID-19 vaccination campaign progressed slowly and failed to reach marginalised populations. Here, we discuss how, despite recent improvements, it remains important to further engage key stakeholders to ensure nobody is left behind.

Synthesis of Enantiopure Unnatural Amino Acids by Metallaphotoredox Catalysis.

We describe herein a two-step process for the conversion of serine to a wide array of optically pure unnatural amino acids. This method utilizes a photocatalytic cross-electrophile coupling between a bromoalkyl intermediate and a diverse set of aryl halides to produce artificial analogues of phenylalanine, tryptophan, and histidine. The reaction is tolerant of a broad range of functionalities and can be leveraged toward the scalable synthesis of valuable pharmaceutical scaffolds via flow technology.

CCR7 as a novel therapeutic target in t-cell PROLYMPHOCYTIC leukemia.

T-cell prolymphocytic leukemia (T-PLL) is a poor prognostic disease with very limited options of efficient therapies. Most patients are refractory to chemotherapies and despite high response rates after alemtuzumab, virtually all patients relapse. Therefore, there is an unmet medical need for novel therapies in T-PLL. As the chemokine receptor CCR7 is a molecule expressed in a wide range of malignancies and relevant in many tumor processes, the present study addressed the biologic role of this receptor in T-PLL. Furthermore, we elucidated the mechanisms of action mediated by an anti-CCR7 monoclonal antibody (mAb) and evaluated whether its anti-tumor activity would warrant development towards clinical applications in T-PLL. Our results demonstrate that CCR7 is a prognostic biomarker for overall survival in T-PLL patients and a functional receptor involved in the migration, invasion, and survival of leukemic cells. Targeting CCR7 with a mAb inhibited ligand-mediated signaling pathways and induced tumor cell killing in primary samples. In addition, directing antibodies against CCR7 was highly effective in T-cell leukemia xenograft models. Together, these findings make CCR7 an attractive molecule for novel mAb-based therapeutic applications in T-PLL, a disease where recent drug screen efforts and studies addressing new compounds have focused on chemotherapy or small molecules. SUPPLEMENTARY INFORMATION: Supplementary information accompanies this paper at 10.1186/s40364-020-00234-z.

Evaluation of therapeutic targeting of CCR7 in acute graft-versus-host disease.

Graft-versus-host disease (GVHD) is the main complication after allogeneic hematopoietic stem cell transplantation. We previously unveiled a correlation between proportions of C-C motif chemokine receptor 7 (CCR7)+ T cells in the apheresis and the risk of developing GVHD. We wanted to evaluate in vivo whether apheresis with low proportion of CCR7+ cells or treatment with an anti-human CCR7 monoclonal antibody (mAb) were suitable strategies to prevent or treat acute GVHD in preclinical xenogeneic models. Therapeutic anti-CCR7 mAb was the most effective strategy in both prophylactic and therapeutic settings where antibody drastically reduced in vivo lymphoid organ infiltration of donor CCR7+ T cells, extended lifespan and solved clinical signs. The antibody neutralized in vitro migration of naïve and central memory T cells toward CCR7 ligands and depleted target CCR7+ subsets through complement activation. Both mechanisms of action spared CCR7- subsets, including effector memory and effector memory CD45RA+ T cells which may mediate graft versus leukemia effect and immunity against infections. Accordingly, the numbers of donor CCR7+ T cells in the apheresis were not associated to cytomegalovirus reactivation or the recurrence of the underlying disease. These findings provide a promising new strategy to prevent and treat acute GVHD, a condition where new specific, safety and effective treatment is needed.

Immediate Effects of Dasatinib on the Migration and Redistribution of Naïve and Memory Lymphocytes Associated With Lymphocytosis in Chronic Myeloid Leukemia Patients.

Introduction: Dasatinib is a dual SRC/ABL tyrosine kinase inhibitor used to treat chronic myeloid leukemia (CML) that is known to have unique immunomodulatory effects. In particular, dasatinib intake typically causes lymphocytosis, which has been linked to better clinical response. Since the underlying mechanisms are unknown and SRC family kinases are involved in many cell motility processes, we hypothesized that the movement and migration of lymphocytes is modulated by dasatinib. Patients, Materials and Methods: Peripheral blood samples from CML patients treated with second-line dasatinib were collected before and 2 h after the first dasatinib intake, and follow-up samples from the same patients 3 and 6 months after the start of therapy. The migratory capacity and phenotype of lymphocytes and differential blood counts before and after drug intake were compared for all study time-points. Results: We report here for the first time that dasatinib intake is associated with inhibition of peripheral blood T-cell migration toward the homeostatic chemokines CCL19 and CCL21, which control the trafficking toward secondary lymphoid organs, mainly the lymph nodes. Accordingly, the proportion of lymphocytes in blood expressing CCR7, the chemokine receptor for both CCL19 and CCL21, decreased after the intake including both naïve CD45RA+ and central memory CD45RO+ T-cells. Similarly, naïve B-cells diminished with dasatinib. Finally, such changes in the migratory patterns did not occur in those patients whose lymphocyte counts remained unchanged after taking the drug. Discussion: We, therefore, conclude that lymphocytosis induced by dasatinib reflects a pronounced redistribution of naïve and memory populations of all lymphocyte subsets including CD4+ and CD8+ T-cells and B-cells.

Lilly Research Award Program (LRAP): A Successful Academia-Industry Partnership Model in the Context of Flow Chemistry for Drug Discovery.

The Lilly Research Award Program (LRAP) provides academic researchers worldwide with a gate to partner with Lilly internal scientists who are working on basic and applied research to collaboratively advance novel impactful projects. The pre-competitive nature of these projects is the most relevant feature as it permits the shared publication of the research outcomes immediately. In this article, this highly successful initiative is reviewed in the context of general academia-industry collaborations and the lessons learned from different shared projects, in the area of innovative continuous flow chemistry, will be discussed.

Photochemical benzylic bromination in continuous flow using BrCCl3 and its application to telescoped p-methoxybenzyl protection.

BrCCl3 represents a rarely used benzylic brominating reagent with complementary reactivity to other reagents. Its reactivity has been revisited in continuous flow, revealing compatibility with electron-rich aromatic substrates. This has brought about the development of a p-methoxybenzyl bromide generator for PMB protection, which was successfully demonstrated on a pharmaceutically relevant intermediate on 11 g scale, giving 91% yield and a PMB-Br space-time-yield of 1.27 kg L-1 h-1.