Small Molecule CXCR4 Antagonist Research Articles

Multivalent CXCR4-targeting nanobody formats differently affect affinity, receptor clustering, and antagonism

The chemokine receptor CXCR4 is involved in the development and migration of stem and immune cells but is also implicated in tumor progression and metastasis for a variety of cancers. Antagonizing ligand (CXCL12)-induced CXCR4 signaling is, therefore, of therapeutic interest. Currently, there are two small-molecule CXCR4 antagonists on the market for the mobilization of hematopoietic stem cells. Other molecules with improved potencies and safety profiles are being developed for different indications, including cancer. Moreover, multiple antagonistic nanobodies targeting CXCR4 displayed similar or better potencies as compared to the CXCR4-targeting molecule AMD3100 (Plerixafor), which was further enhanced through avid binding of bivalent derivatives. In this study, we aimed to compare the affinities of various multivalent nanobody formats which might be differently impacted by avidity. By fusion to a flexible GS-linker, Fc-region of human IgG1, different C4bp/CLR multimerization domains, or via site-directed conjugation to a trivalent linker scaffold, we generated different types of multivalent nanobodies with varying valencies ranging from bivalent to decavalent. Of these, C-terminal fusion, especially to human Fc, was most advantageous with a 2-log-fold and 3-log-fold increased potency in inhibiting CXCL12-mediated Gαi- or β-arrestin recruitment, respectively. Overall, we describe strategies for generating multivalent and high-potency CXCR4 antagonistic nanobodies able to induce receptor clustering and conclude that fusion to an Fc-tail results in the highest avidity effect irrespective of the hinge linker.

Trans-IV restriction: a new configuration for metal bis-cyclam complexes as potent CXCR4 inhibitors.

The chemokine receptor CXCR4 is implicated in multiple diseases including inflammatory disorders, cancer growth and metastasis, and HIV/AIDS. CXCR4 targeting has been evaluated in treating cancer metastasis and therapy resistance. Cyclam derivatives, most notably AMD3100 (Plerixafor™), are a common motif in small molecule CXCR4 antagonists. However, AMD3100 has not been shown to be effective in cancer treatment as an individual agent. Configurational restriction and transition metal complex formation increases receptor binding affinity and residence time. In the present study, we have synthesized novel trans-IV locked cyclam-based CXCR4 inhibitors, a previously unexploited configuration, and demonstrated their higher affinity for CXCR4 binding and CXCL12-mediated signaling inhibition compared to AMD3100. These results pave the way for even more potent CXCR4 inhibitors that may provide significant efficacy in cancer therapy.

Trial in Progress: An Open-Label, Multi-Center Phase 2 Study to Assess the Safety and Efficacy of Burixafor (GPC-100) and Propranolol with and without G-CSF for the Mobilization of Stem Cells in Patients with Multiple Myeloma Undergoing Autologous Stem Cell Transplant

Background and Significance: Poor stem cell mobilization with failure to produce the target cell dose of ≥2x10 6 CD34 cells/kg required for autologous stem cell transplant (ASCT) occurs in 15-50% of patients with lymphoma or multiple myeloma (MM). Newer therapies may also have a negative impact on mobilization, supporting a need for newer treatment options. Burixafor (GPC-100) is a potent and selective small molecule antagonist of CXCR4. Previous clinical trials with burixafor alone or in combination with G-CSF have demonstrated safe and effective mobilization of stem cells. Preclinical murine studies demonstrated that burixafor in combination with propranolol is a potent mobilizer of white blood cells and mouse stem cells, and mobilization was further augmented with the addition of G-CSF. The goal of the current clinical study is to evaluate the efficacy of burixafor and propranolol in the presence and absence of G-CSF. The triple combination is predicted to be best in class and can target patients at risk for poor mobilization while burixafor plus propranolol will allow for an option when G-CSF is not tolerated, such as in sickle cell disease or patients with autoimmune disease. Study Design and Methods: This study (NCT05561751) is an open-label, randomized, multi-site (U.S. only) Phase 2 trial. Approximately 40 participants will be enrolled across 8-10 sites and randomized in a 1:1 ratio to one of two treatment arms: Burixafor in combination with propranolol or burixafor in combination with propranolol and G-CSF. The study will employ a Bayesian Optimal Phase 2 (BOP2) design for participant enrollment to characterize the safety and clinical activity of burixafor. Participants must be at least 18 years of age, be diagnosed with MM and be eligible for ASCT. Key exclusion criteria include: no more than one year of therapy administered prior to stem cell mobilization, previous stem cell transplant, history of another malignancy, and history of poorly controlled or uncontrolled cardiovascular or pulmonary disease. All participants will self-administer 30 mg propranolol orally twice daily from Days 1 to 8. For participants in the treatment arm receiving G-CSF, they will receive SC injections of 10g/kg/day G-CSF in the afternoon on Days 3 to 7. On days 7 and 8, participants will receive 3.14 mg/kg dose of burixafor via IV and undergo leukapheresis for stem cell collection two hours post drug administration. Participants may undergo additional optional days of the treatment regimen and stem cell collection, per Investigator's discretion to meet institutional standards. The primary objective is to determine the proportion of patients that will achieve at least ≥2 x 10 6 CD34 + cells/kg in 2 leukapheresis sessions following the treatment regimens. Key secondary objectives include: assessment of safety and tolerability of GPC-100 and propranolol with and without G-CSF, evaluation of the pharmacodynamics by determining levels of circulating CD34 + cell counts and levels of the chemokine CXCL12 in peripheral blood, and assessment of mean time to ANC and platelet count recovery after ASCT. Exploratory objectives are also included to discover key biomarkers for patient selection, to evaluate cancer/stem/immune cell profiles, and to evaluate treatment impact on patient quality of life.

GPC-100, a novel CXCR4 antagonist, improves in vivo hematopoietic cell mobilization when combined with propranolol.

Autologous Stem Cell Transplant (ASCT) is increasingly used to treat hematological malignancies. A key requisite for ASCT is mobilization of hematopoietic stem cells into peripheral blood, where they are collected by apheresis and stored for later transplantation. However, success is often hindered by poor mobilization due to factors including prior treatments. The combination of G-CSF and GPC-100, a small molecule antagonist of CXCR4, showed potential in a multiple myeloma clinical trial for sufficient and rapid collection of CD34+ stem cells, compared to the historical results from the standards of care, G-CSF alone or G-CSF with plerixafor, also a CXCR4 antagonist. In the present study, we show that GPC-100 has high affinity towards the chemokine receptor CXCR4, and it potently inhibits β-arrestin recruitment, calcium flux and cell migration mediated by its ligand CXCL12. Proximity Ligation Assay revealed that in native cell systems with endogenous receptor expression, CXCR4 co-localizes with the beta-2 adrenergic receptor (β2AR). Co-treatment with CXCL12 and the β2AR agonist epinephrine synergistically increases β-arrestin recruitment to CXCR4 and calcium flux. This increase is blocked by the co-treatment with GPC-100 and propranolol, a non-selective beta-adrenergic blocker, indicating a functional synergy. In mice, GPC-100 mobilized more white blood cells into peripheral blood compared to plerixafor. GPC-100 induced mobilization was further amplified by propranolol pretreatment and was comparable to mobilization by G-CSF. Addition of propranolol to the G-CSF and GPC-100 combination resulted in greater stem cell mobilization than the G-CSF and plerixafor combination. Together, our studies suggest that the combination of GPC-100 and propranolol is a novel strategy for stem cell mobilization and support the current clinical trial in multiple myeloma registered as NCT05561751 at www.clinicaltrials.gov.

Abstract 2649: Orally bioavailable small molecule CXCR4 antagonists with enhanced efficacy in mouse models of genitourinary cancers

Abstract Purpose: Pro-angiogenic and immune cells expressing chemokine receptor CXCR4 traffic along concentration gradients of the chemokine ligand CXCL12, which disseminates from stromal niches. The CXCR4/CXCL12 axis is hijacked by various cancer types characterized by dramatic CXCR4 and/or CXCL12 upregulation. This chemokine network misregulation causes hyperactivation of CXCR4-mediated processes, leading to (1) excessive stimulation of protective tumor-stromal interactions, (2) metastatic expansion of CXCR4+ cancer cells to CXCL12-rich sites, and (3) intratumoral infiltration of CXCR4+ immunosuppressive cells. Accordingly, CXCR4 antagonists have significant therapeutic potential against cancer. While clinical CXCR4 inhibitors are severely limited by suboptimal pharmacokinetic (PK) properties and off-target activities, several of our tetrahydroisoquinoline-based CXCR4 antagonists exhibit superior selectivity and PK profiles to these clinical comparators (e.g., X4P-001). Methods: To test the hypothesis that improved PK profiles would translate to enhanced efficacy, our lead CXCR4 antagonist EMU-116 was compared to X4P-001 head-to-head in three mouse models of genitourinary cancers. First, human 786.0 renal cell carcinoma (RCC) xenograft-bearing female nude mice were treated p.o., q.d. with vehicle, axitinib (30 mg/kg), X4P-001 (100 mg/kg), EMU-116 (3, 10, or 30 mg/kg), X4P + axitinib, or EMU-116 + axitinib. Subcutaneous tumor volume was monitored using calipers. Second, nude male mice bearing intratibial, luciferase-expressing human PC-3 prostate cancer xenografts were treated with vehicle (p.o., q.d.), docetaxel (10 mg/kg i.p. weekly), X4P-001 (10 or 30 mg/kg p.o., q.d.), EMU-116 (10 or 30 mg/kg p.o., q.d.), X4P + axitinib, or EMU + axitinib. Intratibial tumor volume was monitored via luminescence imaging. Third, syngeneic RENCA RCC tumor-bearing female Balb/c mice were treated p.o., q.d. with vehicle, X4P-001 (30 mg/kg), or EMU-116 (30 mg/kg). Subcutaneous tumor volume was monitored using calipers, and immune cell subsets in bone marrow, blood, tumor, and tumor-draining lymph nodes were quantified via flow cytometry. Results: In the RCC xenograft model in combination with axitinib, EMU-116 (3 or 10 mg/kg) was equally effective at decreasing tumor burden compared to X4P-001 (100 mg/kg), whereas EMU-116 (30 mg/kg) was more effective. In the bone metastatic prostate cancer xenograft model, EMU-116 was as or more effective than X4P-001 when paired with docetaxel. In the syngeneic RCC model, EMU-116 mobilized T cells more effectively than X4P-001. Conclusion: EMU-116, at the same or lower dose, was more efficacious than X4P-001 in these models. These results highlight EMU-116 as a clinical candidate with significant therapeutic potential to synergize with chemotherapeutics, targeted therapies, and immuno-oncology agents. Citation Format: Eric J. Miller, Carrie Q. Sun, Petra Gregorova, Edgars Jecs, Yesim Altas Tahirovic, Robert J. Wilson, Huy H. Nguyen, Savita K. Sharma, Perry Bartsch, Zachary Sticher, Levi Moellering, Priscilla Davidson, Ryan Jajosky, Michael D'Erasmo, Manohar Saindane, Zafer Sahin, Nicholas S. Akins, Alexander A. Kolykhalov, Lawrence Wilson, Rebecca S. Arnold, John A. Petros, Haydn Kissick, Lingjie Xu, Yi Jiang, Dennis C. Liotta. Orally bioavailable small molecule CXCR4 antagonists with enhanced efficacy in mouse models of genitourinary cancers [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 2649.

Study of Renal Accumulation of Targeted Polycations in Acute Kidney Injury.

Acute kidney injury (AKI) is a global healthcare burden characterized by rapid loss of renal function and high morbidity and mortality. Chemokine receptor CXCR4 participates in the renal infiltration of immune cells following injury and in local inflammatory enhancement. Injured renal tubule cells overexpress CXCR4, which could be used as a target for improved drug delivery in AKI. Plerixafor is a small-molecule CXCR4 antagonist that has shown beneficial effects against AKI and has been previously developed into a polymeric analog (polymeric plerixafor, PP). With the goal of gaining a better understanding of how overall charge and hydrophilicity affect renal accumulation of PP, we have synthesized PP copolymers containing hydroxyl, carboxyl, primary amine, and alkyl moieties using Michael-type addition copolymerization. All synthesized copolymers showed excellent CXCR4-binding and inhibiting ability in vitro and improved cellular uptake in hypoxia-reoxygenation stimulated mouse tubule cells. Analysis of serum protein binding revealed that polymers with hydroxyl group modification showed the least amount of protein binding. Biodistribution of the polymers was tested in a unilateral ischemia reperfusion-induced AKI mouse model. The results showed significant differences in accumulation in the injured kidneys depending on the net charge and hydrophilicity of the polymers. The findings of this study will guide the development of polymeric drug carriers for targeted delivery to injured kidneys for better AKI therapy.

A fragment integrational approach to GPCR inhibition: Identification of a high affinity small molecule CXCR4 antagonist

Targeting the protein-protein interactions involving CXCR4, a member of chemokine receptor family and G-protein-coupled receptor superfamily, has become an attractive therapeutic strategy for HIV-1 infection, hematopoietic stem cell mobilization, and cancer metastasis. As such, new small molecule CXCR4 antagonists are needed to offer therapeutic alternatives with enhanced clinical outcomes. Here, employing a fragment integrational approach we designed and synthesized a new and potent small molecule CXCR4 antagonist (named as HF51116), as well as a fluorescent (FITC)-labeled HF51116 (FITC-HF51116). HF51116 exhibited very high CXCR4 binding affinity with IC50 of 12 nM in competitive binding with a CXCR4 specific antibody 12G5, which is comparable to the wild type chemokines or synthetic peptides of much larger molecular sizes. Direct binding measurement using FITC-HF51116 further revealed the compound's high CXCR4 affinity. HF51116 strongly antagonized SDF-1α-induced cell migration, calcium mobilization, and CXCR4 internalization. Furthermore, HF51116 inhibited HIV-1 infection via CXCR4, demonstrating its antiviral therapeutic potential. The mechanism of HF51116-CXCR4 interaction was analyzed by site-directed mutagenesis and molecular modeling which suggested that the compound recognizes the minor and major subpockets of CXCR4. Its binding to CXCR4 was found to block G protein-dependent downstream signal pathways as detected by luciferase reporter assays. With its potent bioactivities and asymmetric structure amenable to chemical diversification, HF51116 may serve as a prototype for developing a new class of CXCR4-targeted therapeutics and proof of the concept of similar strategies for studying other GPCRs.

Preliminary Clinical Response Data from a Phase 1b Study of Mavorixafor in Combination with Ibrutinib in Patients with Waldenström's Macroglobulinemia with MYD88 and CXCR4 Mutations

Preliminary Clinical Response Data from a Phase 1b Study of Mavorixafor in Combination with Ibrutinib in Patients with Waldenström's Macroglobulinemia with MYD88 and CXCR4 Mutations

An optimized derivative of an endogenous CXCR4 antagonist prevents atopic dermatitis and airway inflammation

Aberrant CXCR4/CXCL12 signaling is involved in many pathophysiological processes such as cancer and inflammatory diseases. A natural fragment of serum albumin, named EPI-X4, has previously been identified as endogenous peptide antagonist and inverse agonist of CXCR4 and is a promising compound for the development of improved analogues for the therapy of CXCR4-associated diseases. To generate optimized EPI-X4 derivatives we here performed molecular docking analysis to identify key interaction motifs of EPI-X4/CXCR4. Subsequent rational drug design allowed to increase the anti-CXCR4 activity of EPI-X4. The EPI-X4 derivative JM#21 bound CXCR4 and suppressed CXCR4-tropic HIV-1 infection more efficiently than the clinically approved small molecule CXCR4 antagonist AMD3100. EPI-X4 JM#21 did not exert toxic effects in zebrafish embryos and suppressed allergen-induced infiltration of eosinophils and other immune cells into the airways of animals in an asthma mouse model. Moreover, topical administration of the optimized EPI-X4 derivative efficiently prevented inflammation of the skin in a mouse model of atopic dermatitis. Thus, rationally designed EPI-X4 JM#21 is a novel potent antagonist of CXCR4 and the first CXCR4 inhibitor with therapeutic efficacy in atopic dermatitis. Further clinical development of this new class of CXCR4 antagonists for the therapy of atopic dermatitis, asthma and other CXCR4-associated diseases is highly warranted.

Design, synthesis, and biological characterization of a new class of symmetrical polyamine-based small molecule CXCR4 antagonists

CXCR4, a well-studied coreceptor of human immunodeficiency virus type 1 (HIV-1) entry, recognizes its cognate ligand SDF-1α (also named CXCL12) which plays many important roles, including regulating immune cells, controlling hematopoietic stem cells, and directing cancer cells migration. These pleiotropic roles make CXCR4 an attractive target to mitigate human disorders. Here a new class of symmetrical polyamines was designed and synthesized as potential small molecule CXCR4 antagonists. Among them, a representative compound 21 (namely HF50731) showed strong CXCR4 binding affinity (mean IC50 = 19.8 nM) in the CXCR4 competitive binding assay. Furthermore, compound 21 significantly inhibited SDF-1α-induced calcium mobilization and cell migration, and blocked HIV-1 infection via antagonizing CXCR4 coreceptor function. The structure-activity relationship analysis, site-directed mutagenesis, and molecular docking were conducted to further elucidate the binding mode of compound 21, suggesting that compound 21 could primarily occupy the minor subpocket of CXCR4 and partially bind in the major subpocket by interacting with residues W94, D97, D171, and E288. Our studies provide not only new insights for the fragment-based design of small molecule CXCR4 antagonists for clinical applications, but also a new and effective molecular probe for CXCR4-targeting biological studies.

Efficacy evaluation and mechanism study on inhibition of breast cancer cell growth by multimodal targeted nanobubbles carrying AMD070 and ICG

To construct targeted nanobubbles carrying both small-molecule CXCR4 antagonist AMD070 and light-absorbing material indocyanine green (ICG), and to study their in vitro multimodal imaging, as well as their mechanism and efficacy of inhibition of breast cancer cell growth. Nanobubbles carrying AMD070 and ICG (ICG-TNBs) were constructed by carbodiimide reaction and mechanical oscillation. The physical characteristics and in vitro multimodal imaging were determined. The binding potential of ICG-TNBs to human breast cancer cells were observed by laser confocal microscopy. CCK-8 and flow cytometry were used to analyze the role of ICG-TNBs + US in inhibiting proliferation and inducing apoptosis of tumor cells. Flow cytometry and Western blotting are used to analyse the ROS generation and molecular mechanisms. ICG-TNBs had a particle size of 497.0 ± 29.2 nm and a Zeta potential of −8.05 ± 0.73 mV. In vitro multimodal imaging showed that the image signal intensity of ICG-TNBs increased with concentration. Targeted binding assay confirmed that ICG-TNBs could specifically bind to MCF-7 cells (CXCR4 positive), but not to MDA-MB-468 cells (CXCR4 negative). CCK-8 assay and flow cytometry analysis showed that ICG-TNBs + US could significantly inhibit the growth of MCF-7 breast cancer cells and promote their apoptosis. Flow cytometry and Western blotting showed that ICG-TNBs + US could significantly raise generation of ROS, reduce the expression of CXCR4, inhibit phosphorylation of Akt, and increase the expression of Caspase3 and Cleaved-caspase3. This indicated that ICG-TNBs could effectively inhibit and block the SDF-1/CXCR4 pathway, thus leading to the apoptosis of MCF-7 cells. ICG-TNBs can specifically bind to CXCR4 positive breast cancer cells, furthermore inhibit growth and promote apoptosis of breast cancer cells combined with ultrasonic irradiation by blocking the SDF-1/CXCR4 pathway. This study introduces a novel concept, method and mechanism for integration of targeted diagnosis and treatment of breast cancer.

The CXCL12-CXCR4/CXCR7 axis as a mechanism of immune resistance in gastrointestinal malignancies.

Single agent checkpoint inhibitor therapy has not been effective for most gastrointestinal solid tumors, but combination therapy with drugs targeting additional immunosuppressive pathways is being attempted. One such pathway, the CXCL12-CXCR4/CXCR7 chemokine axis, has attracted attention due to its effects on tumor cell survival and metastasis as well as immune cell migration. CXCL12 is a small protein that functions in normal hematopoietic stem cell homing in addition to repair of damaged tissue. Binding of CXCL12 to CXCR4 leads to activation of G protein signaling kinases such as P13K/mTOR and MEK/ERK while binding to CXCR7 leads to β-arrestin mediated signaling. While some gastric and colorectal carcinoma cells have been shown to make CXCL12, the primary source in pancreatic cancer and peritoneal metastases is cancer-associated fibroblasts. Binding of CXCL12 to CXCR4 and CXCR7 on tumor cells leads to anti-apoptotic signaling through Bcl-2 and survivin upregulation, as well as promotion of the epithelial-to-mesechymal transition through the Rho-ROCK pathway and alterations in cell adhesion molecules. High levels of CXCL12 seen in the bone marrow, liver, and spleen could partially explain why these are popular sites of metastases for many tumors. CXCL12 is a chemoattractant for lymphocytes at lower levels, but becomes chemorepellant at higher levels; it is unclear exactly what gradient exists in the tumor microenvironment and how this influences tumor-infiltrating lymphocytes. AMD3100 (Plerixafor or Mozobil) is a small molecule CXCR4 antagonist and is the most frequently used drug targeting the CXCL12-CXCR4/CXCR7 axis in clinical trials for gastrointestinal solid tumors currently. Other small molecules and monoclonal antibodies against CXCR4 are being trialed. Further understanding of the CXCL12- CXCR4/CXCR7 chemokine axis in the tumor microenvironment will allow more effective targeting of this pathway in combination immunotherapy.

<p>Preparation Of Nanobubbles Modified With A Small-Molecule CXCR4 Antagonist For Targeted Drug Delivery To Tumors And Enhanced Ultrasound Molecular Imaging</p>

PurposeTo construct nanobubbles (PTX-AMD070 NBs) for targeted delivery of paclitaxel (PTX) and AMD070, examine their performance in ultrasound molecular imaging of breast cancer and cervical cancer and their therapeutic effect combined with ultrasound targeted nanobubble destruction (UTND).Materials and methodsPTX-AMD070 NBs were prepared via an amide reaction, and the particle size, zeta potential, encapsulation rate and drug loading efficiency were examined. Laser confocal microscopy and flow cytometry were used to analyze the targeted binding ability of PTX-AMD070 NBs to CXCR4+ MCF-7 cells and C33a cells. The effect of PTX-AMD070 NBs combined with UTND on cell proliferation inhibition and apoptosis induction was detected by CCK-8 assays and flow cytometry. The contrast-enhanced imaging features of PTX-AMD070 NBs and paclitaxel-loaded nanobubbles were compared in xenograft tumors. The penetration ability of PTX-AMD070 NBs in xenograft tissues was evaluated by immunofluorescence. The therapeutic effect of PTX-AMD070 NBs combined with UTND on xenograft tumors was assessed.ResultsPTX-AMD070 NBs showed a particle size of 494.3±61.2 nm, a zeta potential of −22.4±1.75 mV, an encapsulation rate with PTX of 53.73±7.87%, and a drug loading efficiency with PTX of 4.48±0.66%. PTX-AMD070 NBs displayed significantly higher targeted binding to MCF-7 cells and C33a cells than that of PTX NBs (P<0.05), and combined with UTND manifested a more pronounced effect in inhibiting cell proliferation and promoting apoptosis than other treatments. PTX-AMD070 NBs aggregated specifically in xenograft tumors in vivo, and significantly improved the image quality. Compared with other treatment groups, PTX-AMD070 NBs combined with UTND exhibited the smallest tumor volume and weight, and the highest degree of apoptosis and necrosis.ConclusionPTX-AMD070 NBs improved the ultrasound imaging effect in CXCR4+ xenograft tumors and facilitated targeted therapy combined with UTND. Therefore, this study provides an effective method for the integration of ultrasound molecular imaging and targeted therapy of malignant tumors.

Design and evaluation of a CXCR4 targeting peptide 4DV3 as an HIV entry inhibitor and a ligand for targeted drug delivery

The feasibility of utilizing the cell surface chemokine receptor CXCR4 for human immunodeficiency virus (HIV) entry inhibition and as an intracellular portal for targeted drug delivery was evaluated. Novel DV3 ligands (1DV3, 2DV3, and 4DV3) were designed, synthesized and conjugated to various probes (fluorescein isothiocyanate (FITC) or biotin) and cargos with sizes ranging from 10 to 50 nm (polyethylene glycol (PEG), streptavidin, and a polymeric nanoparticle). 4DV3 conjugated probes inhibited HIV-1 entry into the CXCR4-expressing reporter cell line TZM-bl (IC50 at 553 nM) whereas 1DV3 and 2DV3 did not. 4DV3 also inhibited binding of anti-CXCR4 antibody 44,708 to TZM-bl cells with nanomolar potency, while the small-molecule CXCR4 antagonist AMD3100 did not. Molecular modeling suggested simultaneous binding of a single 4DV3 molecule to four CXCR4 molecules. Differences in CXCR4-binding sites could explain the discrete inhibitory effects observed for 4DV3, the 44,708 antibody and AMD3100. In the Sup-T1 cell chemotaxis assay, the 4DV3 ligand functioned as a CXCR4 allosteric enhancer. In addition, 4DV3 ligand-conjugated cargos with sizes ranging from 10 to 50 nm were taken up into CXCR4-expressing Sup-T1 and TZM-bl cells, demonstrating that CXCR4 could serve as a drug delivery portal for nanocarriers. The uptake of 4DV3 functionalized nanocarriers combined with the allosteric interaction with CXCR4 suggests enhanced endocytosis occurs when 4DV3 is the targeting ligand. The current results indicate that 4DV3 might serve as a prototype for a new type of dual function ligand, one that acts as a HIV-1 entry inhibitor and a CXCR4 drug delivery targeting ligand.

Development of a novel 99mTc‐labeled small molecular antagonist for CXCR4 positive tumor imaging

The chemokine receptor 4 (CXCR4) has been an attractive molecular target for tumor imaging, because it is overexpressed in many tumor types and involved in tumor progression and metastasis. The purpose of this study is to examine the CXCR4 targeting properties of 99m Tc-labeled AMD3465, a small molecule antagonist of CXCR4. 99m Tc-AMD3465 was prepared in high yield (>95%) and stable in mice serum at least for 4hours. In vitro cell binding experiments were performed with Chinese hamster ovary (CHO), MCF-7 (breast cancer), and CHO-CXCR4 (CHO stably transfected to express CXCR4) cell lines. Small animal single photon emission computed tomography/computed tomography imaging studies in nude mice bearing MCF-7 and CHO xenografts showed that the uptakes of the radiotracer in MCF-7 tumors were significantly higher than those in the CXCR4-negative CHO tumors (P<0.05), and the MCF-7 tumors uptake could be blocked with an excess of unlabeled AMD3465 (P<0.05). These results suggested that 99m Tc-AMD3465 could be a potential single photon emission computed tomography radiotracer for CXCR4 imaging.

Small molecule CXCR4 antagonists block the HIV-1 Nef/CXCR4 axis and selectively initiate the apoptotic program in breast cancer cells.

The chemokine receptor CXCR4 plays an integral role in the development of highly metastatic breast cancer and in the pathogenesis of chronic HIV infection. In this study, we compared the effects of CXCR4 antagonists on apoptosis induction in hematopoietic cells and in tumor cells. We incubated cells expressing CXCR4 with a series of CXCR4 antagonists and subsequently exposed the cultures to a pro-apoptotic peptide derived from the HIV-1 Nef protein (NefM1). The NefM1 peptide contains residues 50–60 of Nef and was previously shown to be the sequence necessary for Nef to initiate the apoptotic program through CXCR4 signaling. We found that several of the compounds studied potently blocked Nef-induced apoptosis in Jurkat T-lymphocyte cells. Interestingly, many of the same compounds selectively triggered apoptosis in MDA-MB-231 breast cancer cells, in some cases at sub-nanomolar concentrations. None of the compounds were toxic to lymphocyte, monocyte or macrophage cells, suggesting that aggressive breast cancer carcinomas may be selectively targeted and eliminated using CXCR4-based therapies without additional cytotoxic agents. Our results also demonstrate that not all CXCR4 antagonists are alike and that the observed anti-Nef and pro-apoptotic effects are chemically tunable. Collectively, these findings suggest our CXCR4 antagonists have promising clinical utility for HIV or breast cancer therapies as well as being useful probes to examine the link between CXCR4 and apoptosis.

Discovery of Tetrahydroisoquinoline-Containing CXCR4 Antagonists with Improved in Vitro ADMET Properties.

CXCR4 is a seven-transmembrane receptor expressed by hematopoietic stem cells and progeny, as well as by ≥48 different cancers types. CXCL12, the only chemokine ligand of CXCR4, is secreted within the tumor microenvironment, providing sanctuary for CXCR4+ tumor cells from immune surveillance and chemotherapeutic elimination by (1) stimulating prosurvival signaling and (2) recruiting CXCR4+ immunosuppressive leukocytes. Additionally, distant CXCL12-rich niches attract and support CXCR4+ metastatic growths. Accordingly, CXCR4 antagonists can potentially obstruct CXCR4-mediated prosurvival signaling, recondition the CXCR4+ leukocyte infiltrate from immunosuppressive to immunoreactive, and inhibit CXCR4+ cancer cell metastasis. Current small molecule CXCR4 antagonists suffer from poor oral bioavailability and off-target liabilities. Herein, we report a series of novel tetrahydroisoquinoline-containing CXCR4 antagonists designed to improve intestinal absorption and off-target profiles. Structure-activity relationships regarding CXCR4 potency, intestinal permeability, metabolic stability, and cytochrome P450 inhibition are presented.

Abstract 1633: Immunotherapy for malignant mesothelioma that combines a mesothelia-targeted immune-activating protein and CXCL12/CXCR4 blockade

Abstract Background and Purpose: There is a significant unmet need for new treatment strategies for malignant mesothelioma (MM). Despite relevant advances in many cancer treatment areas, including improvements in diagnosis, staging, and the clinical course of treated patients, MM remains a highly lethal disease. The purpose of this study is to develop a combination immunotherapy for MM, which involves a fusion protein to target and evoke a cellualr immune response to mesothelin (MSLN) and the blockade of CXCL12/CXCR4 pathway to mobilize cytotoxic effector cells into tumors. Experimental Procedures: The efficacy of the MSLN targeted immune activating fusion protein (scFv-MtbHsp70), FDA-approved small molecule CXCR4 antagonist AMD3100 (plerixafor), and the combination were evaluated in two syngeneic and orthotopic murine models of MM in immune competent C57BL/6 mice. Mice received 4 intraperitoneal (i.p.) treatments from 7 days post i.p. injection of luciferase-expressing 40L and AE17 cells. Tumor growth was monitored by in vivo imaging of luciferase activity with an IVIS Spectrum. Survival time was calculated as life span from the day of tumor inoculation. In immunological studies, mice were sacrificed 4 weeks after tumor cell inoculation. Immune cells from spleens and tumors were labeled with antibodies against CD3, CD4, CD8, CD25 and Foxp3 antibodies, and examined by flow cytometry. Splenocytes were stimulated with MSLN and assessed for intracellular IFN-γ production by flow cytometry. Results: In both murine mesothelioma models, the fusion protein scFv-MtbHsp70 alone delayed tumor growth and prolonged mouse survival, which was associated with increased tumor infiltration by CD3+CD8+ T cells. Treatment enhanced the cytotoxic function of tumor-specific CD3+CD8+ T cells by evoking dendritic cell activation as well as antigen presentation and cross presentation. AMD3100 alone reduced the proportion of CD4+CD25+Foxp3+ Treg cells in tumors and decreased PD-1 expression on CD3+CD8+ T cells. The combination of the fusion protein and AMD3100 further significantly slowed tumor growth and enhanced mouse survival while augmenting tumor-specific CD8+ T-cell immune responses and abrogating intratumoral immunosuppression. Conclusion: Our findings demonstrated for the first time the synergistic effect of combination of MSLN-targeted immune-activating fusion protein scFv-MtbHsp70 and AMD3100 in treatment of MM in mice. This is a new therapeutic strategy which may significantly prolong survival of patients with this disease. Citation Format: Huabiao Chen, Binghao Li, Yang Zeng, Patrick Reeves, Qiuyan Liu, Ann Sluder, Jeffrey Gelfand, Timothy Brauns, Mark Poznansky. Immunotherapy for malignant mesothelioma that combines a mesothelia-targeted immune-activating protein and CXCL12/CXCR4 blockade [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 1633. doi:10.1158/1538-7445.AM2017-1633

Continuous blockade of CXCR4 results in dramatic mobilization and expansion of hematopoietic stem and progenitor cells

AbstractInteraction between the chemokine receptor CXCR4 and its chief ligand CXCL12 plays a critical role in the retention and migration of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM) microenvironment. In this study, qualitative and quantitative effects of long-term pharmacologic inhibition of the CXCR4/CXCL12 axis on the HSPC compartment were investigated by using 3 structurally unrelated small molecule CXCR4 antagonists. A >10-fold increase in mobilization efficiency was achieved by administering the antagonists as a subcutaneous continuous infusion for 2 weeks compared to a single bolus injection. A concurrent increase in self-renewing proliferation leading to a twofold to fourfold expansion of the HSPC pool in the BM was observed. The expanded BM showed a distinct repopulating advantage when tested in serial competitive transplantation experiments. Furthermore, major changes within the HSPC niche associated with previously described HSPC expansion strategies were not detected in bones treated with a CXCR4 antagonist infusion. Our data suggest that prolonged but reversible pharmacologic blockade of the CXCR4/CXCL12 axis represents an approach that releases HSPC with efficiency superior to any other known mobilization strategy and may also serve as an effective method to expand the BM HSPC pool.

Expansion and Maintenance of Hematopoietic Stem and Progenitor Cells in Course of Long-Term Inhibition of CXCR4/CXCL12 Signaling

During the past two decades peripheral blood stem cells have become the favored graft source for HSCT with 80 % of allogeneic and almost 100 % of autologous HSCT performed with mobilized blood. The critical role of the interaction between the chemokine receptor CXCR4 and its chief ligand CXCL12 for retention and migration of hematopoietic stem and progenitor cells (HSPC) has been well established. Interference with CXCR4/CXCL12 signalling iscurrentlybeing exploited as a strategy to mobilize HSPC indirectly with the most clinically relevant mobilizing agent to date, G-CSF as well as directly with the bicyclam CXCR4 antagonist Plerixafor (AMD3100).In this study, qualitative and quantitative effects of long-term pharmacologic inhibition of CXCR4/CXCL12 axis within the HSPC compartment were investigated in healthy C57BL/6 mice using the non-peptidic small molecule CXCR4 antagonists Plerixafor and ALT1188 along with the Protein-EpitopeMimeticsInhibitor POL5551.Up to 12-14 fold higher mobilization efficiency was achieved by applying the antagonists via two weeks of continuous infusion (up to 8-10x104 CFU-C and LSK/ml) as compared to bolus treatment (4-6x103 CFU-C and LSK/ml) or 5-day course of G-CSF (3-6x103 CFU-C/ml).Despite dramatic increase in numbers of circulating HSPC, the BM HSPC pool dis not decrease; in fact it expanded up to 2-4-fold compared to steady state reservoir (sham-operated control mice) as measured by immunophenotypical (LSK SLAM) and functional (e.g. serial competitive transplantation) properties of the cells. Thus, in contrast to genetically CXCR4 ablatedHSPC, the reversible long-term blockade of the receptor did not diminish the long-term repopulating capacity of HSPC.Cell cycle analysis showed a 2-3-fold increase in cycling activity of BM HSPC: only 10-20% of LSK and 30-40 % of LSK SLAM cells were found to be quiescent (in G0 phase of the cell cycle) after two weeks of CXCR4 antagonist infusion versus 50-60 % of LSK and 70 % of LSK SLAM found in G0 under homeostatic conditions. This increased proliferation was very similar to the one induced transiently at day 3 G-CSF treatmentand would conceivably explain the sustained mobilization without concomitant depletion of the BM HSPC pool. Profiling of differentially treated BM HSC (LSK SLAM) via microarray analysis did not reveal substantial effects of CXCR4 inhibitor infusion on the expression signature.Ofnote, major cytological changes typically associated with G-CSF induced mobilization, e.g. depletion of bone lining osteoblast lineage cells and macrophages, were not detected in continuous infusion of POL5551 exposed BM suggesting limitedeffects within the BM niche compartment. Moreover analysis of the BM HSPC after different washout periods at the end of continuous infusion treatment revealed a rapid (within 1-3 days after discontinuation of infusion) reestablishment of steady state HSPC numbers in the BM.Our data suggest that prolonged pharmacologic blockade of the CXCR4/CXCL12 axis using multiple small molecule inhibitorsrepresents an approach thatreleasesHSPCwith efficiency superiorto any other knownmobilization strategybut also may serve as an effective method induce cell cycling and thus expand BM HSPCs. [Display omitted] DisclosuresLevesque:GlycoMimetics: Equity Ownership.