Precision-engineered Peptide and Protein Analogs: Establishing a New Discovery Platform for Potent GPCR Modulators.

Numerous members of the human G protein-coupled receptor (GPCR) superfamily are receptors of therapeutic interest. GPCRs are considered to be highly tractable for drug discovery, representing the targets of approximately one-third of currently licensed drugs. These successful drug discovery outcomes cover only a relatively small subset of the superfamily, however, and many other attractive receptors have proven to present significant challenges. Among these difficult GPCRs are those whose natural ligands are peptides and proteins. In this review we explain the obstacles faced by GPCR drug discovery campaigns, with particular focus on those related to peptide and protein GPCRs. We describe a novel and promising approach for these targets based on engineering of their natural ligands and describe an integrated discovery platform that allows potent ligand analogs to be discovered rapidly and efficiently. Finally, we present a case study involving the chemokine receptor CCR5 to show that this approach can be used to generate new drugs for peptide and protein GPCR targets combining best-in-class potency with tunable signaling activity.

SARS-CoV-2 infection as a trigger of humoral response against apolipoprotein A-1.

BACKGROUND: Unravelling autoimmune targets triggered by SARS-CoV-2 infection may provide crucial insights into the physiopathology of the disease and foster the development of potential therapeutic candidate targets and prognostic tools. We aimed at determining (a) the association between anti-SARS-CoV-2 and anti-apoA-1 humoral response and (b) the degree of linear homology between SARS-CoV-2, apoA-1 and Toll-like receptor 2 (TLR2) epitopes. DESIGN: Bioinformatics modelling coupled with mimic peptides engineering and competition experiments were used to assess epitopes sequence homologies. Anti-SARS-CoV-2 and anti-apoA-1 IgG as well as cytokines were assessed by immunoassays on a case-control (n = 101), an intensive care unit (ICU; n = 126) and a general population cohort (n = 663) with available samples in the pre and post-pandemic period. RESULTS: Using bioinformatics modelling, linear sequence homologies between apoA-1, TLR2 and Spike epitopes were identified but without experimental evidence of cross-reactivity. Overall, anti-apoA-1 IgG levels were higher in COVID-19 patients or anti-SARS-CoV-2 seropositive individuals than in healthy donors or anti-SARS-CoV-2 seronegative individuals (P < .0001). Significant and similar associations were noted between anti-apoA-1, anti-SARS-CoV-2 IgG, cytokines and lipid profile. In ICU patients, anti-SARS-CoV-2 and anti-apoA-1 seroconversion rates displayed similar 7-day kinetics, reaching 82% for anti-apoA-1 seropositivity. In the general population, SARS-CoV-2-exposed individuals displayed higher anti-apoA-1 IgG seropositivity rates than nonexposed ones (34% vs 16.8%; P = .004). CONCLUSION: COVID-19 induces a marked humoral response against the major protein of high-density lipoproteins. As a correlate of poorer prognosis in other clinical settings, such autoimmunity signatures may relate to long-term COVID-19 prognosis assessment and warrant further scrutiny in the current COVID-19 pandemic.

Structural basis of the activation of the CC chemokine receptor 5 by a chemokine agonist.

The human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in inflammation and is involved in cancer, HIV, and COVID-19. Despite its importance as a drug target, the molecular activation mechanism of CCR5, i.e., how chemokine agonists transduce the activation signal through the receptor, is yet unknown. Here, we report the cryo-EM structure of wild-type CCR5 in an active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric Gi protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist chemokines. The N terminus of agonist chemokines pushes onto specific structural motifs at the bottom of the orthosteric pocket that activate the canonical GPCR microswitch network. This activation mechanism differs substantially from other CC chemokine receptors that bind chemokines with shorter N termini in a shallow binding mode involving unique sequence signatures and a specialized activation mechanism.

Arrestin Recruitment to C-C Chemokine Receptor 5: Potent C-C Chemokine Ligand 5 Analogs Reveal Differences in Dependence on Receptor Phosphorylation and Isoform-Specific Recruitment Bias.

C-C chemokine receptor 5 (CCR5) is a chemokine receptor belonging to the G protein-coupled receptor (GPCR) superfamily. An established anti-human immunodeficiency virus drug target, CCR5 is attracting significant additional interest in both cancer and neuroinflammation. Several N-terminally engineered analogs of C-C chemokine ligand 5 (CCL5), a natural ligand of CCR5, are highly potent CCR5 inhibitors. The inhibitory mechanisms of certain analogs relate to modulation of receptor desensitization, but the cellular and molecular mechanisms have not been fully elucidated. Here we made use of a collection of CCR5 phosphorylation mutants and arrestin variants to investigate how CCL5 analogs differ from CCL5 in their capacity to elicit both CCR5 phosphorylation and arrestin recruitment, with reference to the current "core" and "tail" interaction model for arrestin-GPCR interaction. We showed that CCL5 recruits both arrestin 2 and arrestin 3 to CCR5 with recruitment, particularly of arrestin 2, strongly dependent on the arrestin tail interaction. 5P12-RANTES does not elicit receptor phosphorylation or arrestin recruitment. In contrast, PSC-RANTES induces CCR5 hyperphosphorylation, driving enhanced arrestin recruitment with lower dependence on the arrestin tail interaction. 5P14-RANTES induces comparable levels of receptor phosphorylation to CCL5, but arrestin recruitment is absolutely dependent on the arrestin tail interaction, and in one of the cellular backgrounds used, recruitment showed isoform bias toward arrestin 3 versus arrestin 2. No evidence for ligand-specific differences in receptor phosphorylation patterns across the four implicated serine residues was observed. Our results improve understanding of the molecular pharmacology of CCR5 and help further elucidate the inhibitory mechanisms of a group of potent inhibitors. SIGNIFICANCE STATEMENT: C-C chemokine receptor 5 (CCR5) is a key drug target for human immunodeficiency virus, cancer, and inflammation. Highly potent chemokine analog inhibitors act via the modulation of receptor desensitization, a process initiated by the recruitment of arrestin proteins. This study shows that potent C-C chemokine ligand 5 analogs differ from each other and from the parent chemokine in the extent and quality of CCR5-arrestin association that they elicit, providing valuable insights into CCR5 pharmacology and cell biology that will facilitate the development of new medicines targeting this important receptor.

IFN-γ is a therapeutic target in paraneoplastic cerebellar degeneration.

Paraneoplastic neurological disorders result from an autoimmune response against neural self-antigens that are ectopically expressed in neoplastic cells. In paraneoplastic disorders associated to autoantibodies against intracellular proteins, such as paraneoplastic cerebellar degeneration (PCD), current data point to a major role of cell-mediated immunity. In an animal model, in which a neo-self-antigen was expressed in both Purkinje neurons and implanted breast tumor cells, immune checkpoint blockade led to complete tumor control at the expense of cerebellum infiltration by T cells and Purkinje neuron loss, thereby mimicking PCD. Here, we identify 2 potential therapeutic targets expressed by cerebellum-infiltrating T cells in this model, namely α4 integrin and IFN-γ. Mice with PCD were treated with anti-α4 integrin antibodies or neutralizing anti-IFN-γ antibodies at the onset of neurological signs. Although blocking α4 integrin had little or no impact on disease development, treatment using the anti-IFN-γ antibody led to almost complete protection from PCD. These findings strongly suggest that the production of IFN-γ by cerebellum-invading T cells plays a major role in Purkinje neuron death. Our successful preclinical use of neutralizing anti-IFN-γ antibody for the treatment of PCD offers a potentially new therapeutic opportunity for cancer patients at the onset of paraneoplastic neurological disorders.

CCR5: Established paradigms and new frontiers for a 'celebrity' chemokine receptor.

Because of the level of attention it received due to its role as the principal HIV coreceptor, CCR5 has been described as a 'celebrity' chemokine receptor. Here we describe the development of CCR5 inhibitory strategies that have been developed for HIV therapy and which are now additionally being considered for use in HIV prevention and cure. The wealth of CCR5-related tools that have been developed during the intensive investigation of CCR5 as an HIV drug target can now be turned towards the study of CCR5 as a model chemokine receptor. We also summarize what is currently known about the cell biology and pharmacology of CCR5, providing an update on new areas of investigation that have emerged in recent research. Finally, we discuss the potential of CCR5 as a drug target for diseases other than HIV, discussing the evidence linking CCR5 and its natural chemokine ligands with inflammatory diseases, particularly neuroinflammation, and certain cancers. These pathologies may provide new uses for the strategies for CCR5 blockade originally developed to combat HIV/AIDS.

Characterisation of preproendothelin-1 derived peptides identifies Endothelin-Like Domain Peptide as a modulator of Endothelin-1.

Endothelin-1 (ET-1) is involved in the pathogenesis of cardiac and renal diseases, and in the progression of tumour growth in cancer, but current diagnosis and treatment remain inadequate. Peptides derived from the 212 amino acid precursor preproendothelin-1 (ppET-1) may have utility as biomarkers, or cause biological effects that are unaffected by endothelin receptor antagonists. Here, we used specific immunoassays and LC-MS/MS to identify NT-proET-1 (ppET-1[18-50]), Endothelin-Like Domain Peptide (ELDP, ppET-1[93-166]) and CT-proET-1 (ppET-1[169-212]) in conditioned media from cultured endothelial cells. Synthesis of these peptides correlated with ET-1, and plasma ELDP and CT-proET-1 were elevated in patients with chronic heart failure. Clearance rates of NT-proET-1, ELDP and CT-proET-1 were determined after i.v. injection in anaesthetised rats. CT-proET-1 had the slowest systemic clearance, hence providing a biological basis for it being a better biomarker of ET-1 synthesis. ELDP contains the evolutionary conserved endothelin-like domain sequence, which potentially confers biological activity. On isolated arteries ELDP lacked direct vasoconstrictor effects. However, it enhanced ET-1 vasoconstriction and prolonged the increase in blood pressure in anaesthetised rats. ELDP may therefore contribute to disease pathogenesis by augmenting ET-1 responses.

Dual display: phage selection driven by co-engagement of two targets by two different antibody fragments.

Antibody phage display technology has supported the emergence of numerous therapeutic antibodies. The development of bispecific antibodies, a promising new frontier in antibody therapy, could be facilitated by new phage display approaches that enable pairs of antibodies to be co-selected based on co-engagement of their respective targets. We describe such an approach, making use of two complementary leucine zipper domains that heterodimerize with high affinity. Phagemids encoding a first antibody fragment (scFv) fused to phage coat protein via the first leucine zipper are rescued in bacteria expressing a second scFv fused to the second leucine zipper as a soluble periplasmic protein, so that it is acquired by phage during assembly. Using a soluble scFv specific for a human CD3-derived peptide, we show that its acquisition by phage displaying an irrelevant antibody is sufficiently robust to drive selection of rare phage (1 in 10(5)) over three rounds of panning. We then set up a model selection experiment using a cell line expressing the chemokine receptor CCR5 fused to the CD3 peptide together with a panel of phage clones capable displaying either an anti-CCR5 scFv or an irrelevant antibody, with or without the capacity to acquire the soluble anti-CD3 scFv. In this experiment we showed that rare phage (1 in 10(5)) capable of displaying the two different scFvs can be specifically enriched over four rounds of panning. This approach has the potential to be applied to the identification of pairs of ligands capable of co-engaging two different user-defined targets, which would facilitate the discovery of novel bispecific antibodies.

Association between anti-apolipoprotein A-1 antibodies and cardiovascular disease in the general population. Results from the CoLaus study.

We aimed to determine the association between autoantibodies against apolipoprotein A-1 (anti-apoA-1 IgG) and prevalent cardiovascular (CV) disease (CVD) as well as markers of CV risk in the general population. Cross-sectional data were obtained from 6649 subjects (age 52.6 ± 10.7 years, 47.4 % male) of the population-based CoLaus study. CVD was defined as myocardial infarction, angina pectoris, percutaneous revascularisation or bypass grafting for ischaemic heart disease stroke or transient ischaemic attack, and was assessed according to standardised medical records. Anti-apoA-1 IgG and biological markers were measured by ELISA and conventional automated techniques, respectively. Prevalence of high anti-apoA-1 IgG levels in the general population was 19.9 %. Presence of anti-apoA-1 IgG was significantly associated with CVD [odds ratio 1.34, 95 % confidence interval (1.05-1.70), p=0.018], independently of established CV risk factors (CVRFs) including age, sex, hypertension, smoking, diabetes, low and high-density lipoprotein cholesterol levels. The n=455 (6.8 %) study participants with a history of CVD (secondary prevention subgroup) presented higher median anti-ApoA-1 IgG values compared with subjects without CVD (p=0.029). Among patients in the secondary prevention subgroup, those with positive anti-apoA-1 IgG levels had lower HDL (p=0.002) and magnesium (p=0.001) levels, but increased uric acid and high-sensitivity C-reactive protein levels (p=0.022, and p<0.001, respectively) compared to patients with negative anti-apoA-1 IgG levels. In conclusion, anti-apoA-1 IgG levels are independently associated with CVD in the general population and also related to CV biomarkers in secondary prevention. These findings indicate that anti-apoA-1 IgG may represent a novel CVRF and need further study in prospective cohorts.

Enhancing Antitumor Immune Responses by Optimized Combinations of Cell-penetrating Peptide-based Vaccines and Adjuvants.

Cell penetrating peptides (CPPs) from the protein ZEBRA are promising candidates to exploit in therapeutic cancer vaccines, since they can transport antigenic cargos into dendritic cells and induce tumor-specific T cells. Employing CPPs for a given cancer indication will require engineering to include relevant tumor-associated epitopes, administration with an appropriate adjuvant, and testing for antitumor immunity. We assessed the importance of structural characteristics, efficiency of in vitro transduction of target cells, and choice of adjuvant in inducing the two key elements in antitumor immunity, CD4 and CD8 T cells, as well as control of tumor growth in vivo. Structural characteristics associated with CPP function varied according to CPP truncations and cargo epitope composition, and correlated with in vitro transduction efficiency. However, subsequent in vivo capacity to induce CD4 and CD8 T cells was not always predicted by in vitro results. We determined that the critical parameter for in vivo efficacy using aggressive mouse tumor models was the choice of adjuvant. Optimal pairing of a particular ZEBRA-CPP sequence and antigenic cargo together with adjuvant induced potent antitumor immunity. Our results highlight the irreplaceable role of in vivo testing of novel vaccine constructs together with adjuvants to select combinations for further development.

A scalable low-cost cGMP process for clinical grade production of the HIV inhibitor 5P12-RANTES in Pichia pastoris.

In the continued absence of an effective anti-HIV vaccine, approximately 2 million new HIV infections occur every year, with over 95% of these in developing countries. Calls have been made for the development of anti-HIV drugs that can be formulated for topical use to prevent HIV transmission during sexual intercourse. Because these drugs are principally destined for use in low-resource regions, achieving production costs that are as low as possible is an absolute requirement. 5P12-RANTES, an analog of the human chemokine protein RANTES/CCL5, is a highly potent HIV entry inhibitor which acts by achieving potent blockade of the principal HIV coreceptor, CCR5. Here we describe the development and optimization of a scalable low-cost production process for 5P12-RANTES based on expression in Pichia pastoris. At pilot (150 L) scale, this cGMP compliant process yielded 30 g of clinical grade 5P12-RANTES. As well as providing sufficient material for the first stage of clinical development, this process represents an important step towards achieving production of 5P12-RANTES at a cost and scale appropriate to meet needs for topical HIV prevention worldwide.

The Human Autoantibody Response to Apolipoprotein A-I Is Focused on the C-Terminal Helix: A New Rationale for Diagnosis and Treatment of Cardiovascular Disease?

BACKGROUND: Cardiovascular disease (CVD) is the leading cause of death worldwide and new approaches for both diagnosis and treatment are required. Autoantibodies directed against apolipoprotein A-I (ApoA-I) represent promising biomarkers for use in risk stratification of CVD and may also play a direct role in pathogenesis. METHODOLOGY: To characterize the anti-ApoA-I autoantibody response, we measured the immunoreactivity to engineered peptides corresponding to the different alpha-helical regions of ApoA-I, using plasma from acute chest pain cohort patients known to be positive for anti-ApoA-I autoantibodies. PRINCIPAL FINDINGS: Our results indicate that the anti-ApoA-I autoantibody response is strongly biased towards the C-terminal alpha-helix of the protein, with an optimized mimetic peptide corresponding to this part of the protein recapitulating the diagnostic accuracy for an acute ischemic coronary etiology (non-ST segment elevation myocardial infarction and unstable angina) obtainable using intact endogenous ApoA-I in immunoassay. Furthermore, the optimized mimetic peptide strongly inhibits the pathology-associated capacity of anti-ApoA-I antibodies to elicit proinflammatory cytokine release from cultured human macrophages. CONCLUSIONS: In addition to providing a rationale for the development of new approaches for the diagnosis and therapy of CVD, our observations may contribute to the elucidation of how anti-ApoA-I autoantibodies are elicited in individuals without autoimmune disease.

Definition of human apolipoprotein A-I epitopes recognized by autoantibodies present in patients with cardiovascular diseases.

Autoantibodies to apolipoprotein A-I (anti-apoA-I IgG) have been shown to be both markers and mediators of cardiovascular disease, promoting atherogenesis and unstable atherosclerotic plaque. Previous studies have shown that high levels of anti-apoA-I IgGs are independently associated with major adverse cardiovascular events in patients with myocardial infarction. Autoantibody responses to apoA-I can be polyclonal and it is likely that more than one epitope may exist. To identify the specific immunoreactive peptides in apoA-I, we have developed a set of methodologies and procedures to isolate, purify, and identify novel apoA-I endogenous epitopes. First, we generated high purity apoA-I from human plasma, using thiophilic interaction chromatography followed by enzymatic digestion specifically at lysine or arginine residues. Immunoreactivity to the different peptides generated was tested by ELISA using serum obtained from patients with acute myocardial infarction and high titers of autoantibodies to native apoA-I. The immunoreactive peptides were further sequenced by mass spectrometry. Our approach successfully identified two novel immunoreactive peptides, recognized by autoantibodies from patients suffering from myocardial infarction, who contain a high titer of anti-apoA-I IgG. The discovery of these epitopes may open innovative prognostic and therapeutic opportunities potentially suitable to improve current cardiovascular risk stratification.

A novel µ-conopeptide, CnIIIC, exerts potent and preferential inhibition of NaV1.2/1.4 channels and blocks neuronal nicotinic acetylcholine receptors.

BACKGROUND AND PURPOSE: The µ-conopeptide family is defined by its ability to block voltage-gated sodium channels (VGSCs), a property that can be used for the development of myorelaxants and analgesics. We characterized the pharmacology of a new µ-conopeptide (µ-CnIIIC) on a range of preparations and molecular targets to assess its potential as a myorelaxant. EXPERIMENTAL APPROACH: µ-CnIIIC was sequenced, synthesized and characterized by its direct block of elicited twitch tension in mouse skeletal muscle and action potentials in mouse sciatic and pike olfactory nerves. µ-CnIIIC was also studied on HEK-293 cells expressing various rodent VGSCs and also on voltage-gated potassium channels and nicotinic acetylcholine receptors (nAChRs) to assess cross-interactions. Nuclear magnetic resonance (NMR) experiments were carried out for structural data. KEY RESULTS: Synthetic µ-CnIIIC decreased twitch tension in mouse hemidiaphragms (IC(50) = 150 nM), and displayed a higher blocking effect in mouse extensor digitorum longus muscles (IC = 46 nM), compared with µ-SIIIA, µ-SmIIIA and µ-PIIIA. µ-CnIIIC blocked Na(V)1.4 (IC(50) = 1.3 nM) and Na(V)1.2 channels in a long-lasting manner. Cardiac Na(V)1.5 and DRG-specific Na(V)1.8 channels were not blocked at 1 µM. µ-CnIIIC also blocked the α3ß2 nAChR subtype (IC(50) = 450 nM) and, to a lesser extent, on the α7 and α4ß2 subtypes. Structure determination of µ-CnIIIC revealed some similarities to α-conotoxins acting on nAChRs. CONCLUSION AND IMPLICATIONS: µ-CnIIIC potently blocked VGSCs in skeletal muscle and nerve, and hence is applicable to myorelaxation. Its atypical pharmacological profile suggests some common structural features between VGSCs and nAChR channels.

Efficient orthogonal bioconjugation of dendrimers for synthesis of bioactive nanoparticles.

Nanoparticles carrying biologically active functional sets (e.g., targeting moiety, payload, tracer) have potential use in a wide range of clinical applications. Though complex, such constructions should, as far as possible, have a defined molecular architecture and be monodisperse. However, the existing methods to achieve this goal are unsuitable for the incorporation of peptides and proteins, and those that provide for orthogonal introduction of two different types of functional element are incompatible with the use of commercially available materials. In this study, we have developed approaches for the production of nanoparticles based on commercially available polyamidoamine (PAMAM) dendrimers. First, we identified an optimized oxime conjugation strategy under which complex dendrimers can be fully decorated not only with model peptides, but also with recombinant proteins (insulin was taken as an example). Second, we developed a strategy based on a two-chain covalent heterodendrimer (a "diblock") based on cystamine core PAMAM dendrimers and used it to generate heterodendrimers, into which a peptide array and a mannose array were orthogonally introduced. Finally, by incorporating a functionalized linker into the diblock architecture we were able to site-specifically introduce a third functional element into the nanoparticle. We exemplified this approach using fluorescein, a mannose array, and a peptide array as the three functionalities. We showed that incorporation of a mannose array into a nanoparticle strongly and specifically enhances uptake by sentinel cells of the immune system, an important property for vaccine delivery applications. These PAMAM dendrimer-based approaches represent a robust and versatile platform for the development of bioactive nanoparticles.

Deciphering the code for retroviral integration target site selection.

Upon cell invasion, retroviruses generate a DNA copy of their RNA genome and integrate retroviral cDNA within host chromosomal DNA. Integration occurs throughout the host cell genome, but target site selection is not random. Each subgroup of retrovirus is distinguished from the others by attraction to particular features on chromosomes. Despite extensive efforts to identify host factors that interact with retrovirion components or chromosome features predictive of integration, little is known about how integration sites are selected. We attempted to identify markers predictive of retroviral integration by exploiting Precision-Recall methods for extracting information from highly skewed datasets to derive robust and discriminating measures of association. ChIPSeq datasets for more than 60 factors were compared with 14 retroviral integration datasets. When compared with MLV, PERV or XMRV integration sites, strong association was observed with STAT1, acetylation of H3 and H4 at several positions, and methylation of H2AZ, H3K4, and K9. By combining peaks from ChIPSeq datasets, a supermarker was identified that localized within 2 kB of 75% of MLV proviruses and detected differences in integration preferences among different cell types. The supermarker predicted the likelihood of integration within specific chromosomal regions in a cell-type specific manner, yielding probabilities for integration into proto-oncogene LMO2 identical to experimentally determined values. The supermarker thus identifies chromosomal features highly favored for retroviral integration, provides clues to the mechanism by which retrovirus integration sites are selected, and offers a tool for predicting cell-type specific proto-oncogene activation by retroviruses.

V-79 Chinese hamster cells irradiated with antiprotons, a study of peripheral damage due to medium and long range components of the annihilation radiation.

PURPOSE: Radiotherapy of cancer carries a perceived risk of inducing secondary cancer and other damage due to dose delivered to normal tissue. While expectedly small, this risk must be carefully analysed for all modalities. Especially in the use of exotic particles like pions and antiprotons, which annihilate and produce a mixed radiation field when interacting with normal matter nuclei, the biological effective dose far out of field needs to be considered in evaluating this approach. We describe first biological measurements to address the concern that medium and long range annihilation products may produce a significant background dose and reverse any benefits of higher biological dose in the target area. MATERIALS AND METHODS: Using the Antiproton Decelerator (AD) at CERN (Conseil Européen pour la Recherche Nucléaire) we irradiated V-79 Chinese Hamster cells embedded in gelatine using an antiproton beam with fluence ranging from 4.5 x 10(8) to 4.5 x 10(9) particles, and evaluated the biological effect on cells located distal to the Bragg peak using clonogenic survival and the COMET assay. RESULTS: Both methods show a substantial biological effect on the cells in the entrance channel and the Bragg Peak area, but any damage is reduced to levels well below the effect in the entrance channel 15 mm distal to the Bragg peak for even the highest particle fluence used. CONCLUSIONS: The annihilation radiation generated by antiprotons stopping in biological targets causes an increase of the penumbra of the beam but the effect rapidly decreases with distance from the target volume. No major increase in the biological effect is found in the far field outside of the primary beam.

An engineered CX3CR1 antagonist endowed with anti-inflammatory activity.

Chemokines are mainly involved in the recruitment of leukocytes into tissues, a key feature of inflammation. Through its unique receptor CX3CR1, the chemokine CX3CL1 participates in diverse inflammatory processes including arterial atherosclerosis and cerebral or renal inflammation. Using a phage display strategy, we engineered a hCX3CL1 analog (named F1) with a modified N terminus. F1 bound specifically to cells expressing hCX3CR1 and had a K(d) value close to that of native CX3CL1. F1 was not a signaling molecule and did not induce chemotaxis, calcium flux, or CX3CR1 internalization. However, it potently inhibited the CX3CL1-induced calcium flux and chemotaxis in CX3CR1-expressing primary cells of human and murine origin with an IC(50) of 5-50 nM. It also efficiently inhibited the cell adhesion mediated by the CX3CL1-CX3CR1 axis. Finally, in a noninfectious murine model of peritonitis, F1 strongly inhibited macrophage accumulation. These data reveal a prototype molecule that is the first bona fide antagonist of hCX3CR1. This molecule could be used as a lead compound for the development of a novel class of anti-inflammatory substances that act by inhibiting CX3CR1.

Highly potent, fully recombinant anti-HIV chemokines: reengineering a low-cost microbicide.

New prevention strategies for use in developing countries are urgently needed to curb the worldwide HIV/AIDS epidemic. The N-terminally modified chemokine PSC-RANTES is a highly potent entry inhibitor against R5-tropic HIV-1 strains, with an inhibitory mechanism involving long-term intracellular sequestration of the HIV coreceptor, CCR5. PSC-RANTES is fully protective when applied topically in a macaque model of vaginal HIV transmission, but it has 2 potential disadvantages related to further development: the requirement for chemical synthesis adds to production costs, and its strong CCR5 agonist activity might induce local inflammation. It would thus be preferable to find a recombinant analogue that retained the high potency of PSC-RANTES but lacked its agonist activity. Using a strategy based on phage display, we set out to discover PSC-RANTES analogs that contain only natural amino acids. We sought molecules that retain the potency and inhibitory mechanism of PSC-RANTES, while trying to reduce CCR5 signaling to as low a level as possible. We identified 3 analogues, all of which exhibit in vitro potency against HIV-1 comparable to that of PSC-RANTES. The first, 6P4-RANTES, resembles PSC-RANTES in that it is a strong agonist that induces prolonged intracellular sequestration of CCR5. The second, 5P12-RANTES, has no detectable G protein-linked signaling activity and does not bring about receptor sequestration. The third, 5P14-RANTES, induces significant levels of CCR5 internalization without detectable G protein-linked signaling activity. These 3 molecules represent promising candidates for further development as topical HIV prevention strategies.

Engineered CCR5 superagonist chemokine as adjuvant in anti-tumor DNA vaccination.

Chemokine receptors are promising targets for enhancing T-cell immunity and anti-cancer therapy. CCL5 is a potential adjuvant for DNA vaccination. We postulated that CCR5 superagonists could be even more effective. A CCR5 superagonist derived from natural CCL5 by directed in vitro evolution, namely 1P7, is used as a DNA vaccine adjuvant and expressed as fused chemokine-Ig (1P7-Ig). We show that OVA+1P7-Ig DNA co-inoculation induced higher frequencies of OVA-specific CD8 lymphocytes than OVA+CCL5-Ig or controls and gave an even better protection against tumor growth in a CCR5-dependant manner. Our results indicate that CCR5-superagonists may provide potent adjuvants for vaccines.