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Checkpoint inhibitors (CPI) have revolutionized the treatment paradigm for advanced solid tumors; however, there remains an opportunity to improve response rates and outcomes. In preclinical models, 4-1BB costimulation synergizes with CPIs targeting the programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) axis by activating cytotoxic T-cell–mediated antitumor immunity. DuoBody-PD-L1×4-1BB (GEN1046) is an investigational, first-in-class bispecific immunotherapy agent designed to act on both pathways by combining simultaneous and complementary PD-L1 blockade and conditional 4-1BB stimulation in one molecule. GEN1046 induced T-cell proliferation, cytokine production, and antigen-specific T-cell–mediated cytotoxicity superior to clinically approved PD-(L)1 antibodies in human T-cell cultures and exerted potent antitumor activity in transplantable mouse tumor models. In dose escalation of the ongoing first-in-human study in heavily pretreated patients with advanced refractory solid tumors (NCT03917381), GEN1046 demonstrated pharmacodynamic immune effects in peripheral blood consistent with its mechanism of action, manageable safety, and early clinical activity [disease control rate: 65.6% (40/61)], including patients resistant to prior PD-(L)1 immunotherapy.Significance:DuoBody-PD-L1×4-1BB (GEN1046) is a first-in-class bispecific immunotherapy with a manageable safety profile and encouraging preclinical and early clinical activity. With its ability to confer clinical benefit in tumors typically less sensitive to CPIs, GEN1046 may fill a clinical gap in CPI-relapsed or refractory disease or as a combination therapy with CPIs.See related commentary by Li et al., p. 1184.This article is highlighted in the In This Issue feature, p. 1171

Supplementary Figure from Preclinical Characterization and Phase I Trial Results of a Bispecific Antibody Targeting PD-L1 and 4-1BB (GEN1046) in Patients with Advanced Refractory Solid Tumors

Supplemental Tables Supplemental Figures Supplemental Methods

Previous cancer vaccination trials often aimed to activate CD8+ cytotoxic T-cell (CTL) responses with short (8–10mer) peptides and targeted CD4+ helper T cells (TH) with HLA class II–binding longer peptides (12–16 mer) that were derived from tumor antigens. Accordingly, a study of immunomonitoring focused on the detection of CTL responses to the short, and TH responses to the long, peptides. The possible induction of concurrent TH responses to short peptides was widely neglected. In a recent phase I vaccination trial, 53 patients with different solid cancers were vaccinated with EMD640744, a cocktail of five survivin-derived short (9- or 10-mer) peptides in Montanide ISA 51VG. We monitored 49 patients and found strong CD8+ T-cell responses in 63% of the patients. In addition, we unexpectedly found CD4+ TH cell responses against at least two of the five short peptides in 61% (23/38) of the patients analyzed. The two peptides were recognized by HLA-DP4– and HLA-DR–restricted TH1 cells. Some short peptide–reactive (sp)CD4 T cells showed high functional avidity. Here, we show that a short peptide vaccine is able to activate a specific CD4+ T-cell repertoire in many patients, facilitating a strong combined CD4+/CD8+ T-cell response. Cancer Immunol Res; 4(1); 18–25. ©2015 AACR.

Short peptide vaccine induces CD4+ T helper cells in patients with different solid cancers - desription of methods according to MIATA guidelines(MIATA checklist)

(1) Gating strategy, (2) representative examples of the time course of spCD4 responses, (3) individual cytokine profile and magnitude of spCD4 responses of all patients with detected spCD4 T cell response

Higher-order death receptor 5 (DR5) clustering can induce tumor cell death; however, therapeutic compounds targeting DR5 have achieved limited clinical efficacy. We describe HexaBody-DR5/DR5, an equimolar mixture of two DR5-specific IgG1 antibodies with an Fc-domain mutation that augments antibody hexamerization after cell surface target binding. The two antibodies do not compete for binding to DR5 as demonstrated using binding competition studies, and binding to distinct epitopes in the DR5 extracellular domain was confirmed by crystallography. The unique combination of dual epitope targeting and increased IgG hexamerization resulted in potent DR5 agonist activity by inducing efficient DR5 outside-in signaling and caspase-mediated cell death. Preclinical studies in vitro and in vivo demonstrated that maximal DR5 agonist activity could be achieved independent of Fc gamma receptor–mediated antibody crosslinking. Most optimal agonism was observed in the presence of complement complex C1, although without inducing complement-dependent cytotoxicity. It is hypothesized that C1 may stabilize IgG hexamers that are formed after binding of HexaBody-DR5/DR5 to DR5 on the plasma membrane, thereby strengthening DR5 clustering and subsequent outside-in signaling. We observed potent antitumor activity in vitro and in vivo in large panels of patient-derived xenograft models representing various solid cancers. The results of our preclinical studies provided the basis for an ongoing clinical trial exploring the activity of HexaBody-DR5/DR5 (GEN1029) in patients with malignant solid tumors.

BackgroundDuoBody-PD-L1×4-1BB (GEN1046) is a class-defining, bispecific immunotherapy designed to induce an antitumor immune response by simultaneous and complementary PD-L1 blockade and conditional 4-1BB stimulation. Encouraging clinical activity and manageable safety were observed during dose escalation in the ongoing phase 1/2a trial in patients with advanced solid tumors (NCT03917381). We report exploratory pharmacodynamic analyses and potential biomarkers of response in an expansion cohort of patients with PD-(L)1–R/R NSCLC.MethodsPatients with metastatic/unresectable NSCLC who had multiple lines of prior systemic therapy, including a checkpoint inhibitor, received flat-dose DuoBody-PD-L1×4-1BB (100 mg) intravenously every 3 weeks. Immunophenotyping of peripheral blood and measurements of soluble immune mediators were evaluated in serial blood samples in cycles 1–2. Tumor PD-L1 and 4-1BB expression and additional immune markers were evaluated by immunohistochemistry in core needle tumor biopsy specimens collected before treatment and at cycle 2.ResultsAs of May 2021, 40 patients with PD-(L)1–R/R NSCLC were enrolled (median age, 63 years). Treatment with DuoBody-PD-L1×4-1BB elicited pharmacodynamic modulation of immune endpoints within the first 2 cycles. Induction of peripheral IFN-y, CXCL9/10, and expansion of peripheral CD8+ effector memory T cells and activated NK cells were observed starting at cycle 1 (>2-fold from baseline) and maintained or increased through cycle 2. Based on 9 paired tumor biopsy samples, increased PD-L1 and 4-1BB expression and cytotoxic CD8+/GZMB+ cell density were detected following treatment. In a subset of patients with clinical response (n=5 confirmed PRs), a trend of greater induction of IFN-y, CXCL9/10, and activated NK cells was observed vs nonresponders. Disease control rates were higher in patients who had progressed on prior anti–PD-1 therapy within 8 months (64% [16/25]) from the first dose of DuoBody-PD-L1×4-1BB. As expected, among patients with evaluable baseline tumors (n=26), most with any degree of tumor reduction (best change, ConclusionsIn patients with NSCLC who progressed on PD-(L)1 therapy, DuoBody-PD-L1×4-1BB elicited pharmacodynamic effects consistent with its proposed mechanism of action. Relationships between disease control and PD-L1 tumoral expression, as well as time from last prior anti–PD-1 therapy, were observed. These findings support that patient selection and/or anti–PD-1 combination therapy may lead to improved clinical efficacy. Further analyses are ongoing and updated results will be presented.AcknowledgementsThe authors thank Hrefna Kristin Johannsdottir, Lei Pang, and Kate Sasser at Genmab A/S and Friederike Gieseke at BioNTech SE for their valuable contributions. This trial was funded by Genmab A/S and BioNTech SE.Trial RegistrationNCT03917381Ethics ApprovalThis trial is undertaken following full approval of the final protocol, amendments, informed consent form, applicable recruiting materials, and subject compensation programs by the Independent Ethics Committee/Institutional Review Board.ConsentWritten informed consent, in accordance with principles that originated in the Declaration of Helsinki 2013, current ICH guidelines including ICH-GCP E6(R2), applicable regulatory requirements, and sponsor policy, was provided by the patients.

BackgroundDuoBody-PD-L1×4-1BB (GEN1046) is a class-defining bispecific antibody, designed to elicit an anti-tumor immune response by simultaneous and complementary blockade of PD-L1 on tumor cells and conditional stimulation of 4-1BB on T-cells and NK cells. Optimizing target engagement for a bispecific antibody is challenging, as it involves binding with two targets, and predicting trimer levels in tumors based on affinity of individual arms and target expression. Here we describe a semimechanistic, physiologically based pharmacokinetic/pharmacodynamic (PK/PD) model that predicts a dosing regimen for DuoBody-PD-L1×4-1BB, which results in the formation of maximum levels of a therapeutically active 4-1BB-bispecific antibody-PD-L1 trimolecular complex (trimer), and optimal PD-L1 receptor occupancy (RO).MethodsAn integrated semimechanistic PK/PD model that describes the distribution of DuoBody-PD-L1×4-1BB into central and peripheral compartments and partitioning into tumor/lymph nodes was developed. The model used PK/PD data and physiological parameters from the literature for parameterizations of PD-L1 and 4-1BB expression levels and T-cell trafficking. The model incorporates dynamic binding of DuoBody-PD-L1×4-1BB to its targets to predict trimer formation and RO for PD-L1 in tumors. Model parameters were calibrated to match in vitro PD studies, such as analyses of T-cell proliferation and cytokine release, as well as clinical PK data. Sensitivity to model assumptions were assessed by varying PK/PD parameters, and assessing their impact on trimer formation and PD-L1 RO. The model was subsequently used to explore in vivo trimer levels and PD-L1 RO in tumors at various dosing regimens.ResultsThe model was able to adequately describe the PK of DuoBody-PD-L1×4-1BB in the central compartment. Simulations showed a bell-shaped response for average trimer levels in tumors that peaked at 100 mg every 3 weeks (Q3W), with doses >100 mg resulting in reduced trimer formation. Average PD-L1 receptor occupancy at the 100 mg dose was predicted to be approximately 70% over 21 days and increased at higher doses. Based on these model predictions, and available safety, anti-tumor activity, and PD data from the ongoing GCT1046-01 trial (NCT03917381), 100 mg Q3W was chosen as the expansion dose for further evaluation in Part 2 of the study.ConclusionsThis semimechanistic PK/PD model provides a novel approach for dose selection of bispecific antibodies such as DuoBody-PD-L1×4-1BB, by using preclinical and clinical PK/PD data to predict formation of optimal trimer levels and PD-L1 receptor occupancy.AcknowledgementsThe authors thank Friederike Gieseke and Zuzana Jirakova at BioNTech SE; Kalyanasundaram Subramanian at Applied Biomath LLC for their valuable contributions.Trial RegistrationWritten informed consent, in accordance with principles that originated in the Declaration of Helsinki 2013, current ICH guidelines including ICH-GCP E6(R2), applicable regulatory requirements, and sponsor policy, was provided by the patients.

Checkpoint inhibitors (CPI) have revolutionized the treatment paradigm for advanced solid tumors; however, there remains an opportunity to improve response rates and outcomes. In preclinical models, 4-1BB costimulation synergizes with CPIs targeting the programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) axis by activating cytotoxic T-cell–mediated antitumor immunity. DuoBody-PD-L1×4-1BB (GEN1046) is an investigational, first-in-class bispecific immunotherapy agent designed to act on both pathways by combining simultaneous and complementary PD-L1 blockade and conditional 4-1BB stimulation in one molecule. GEN1046 induced T-cell proliferation, cytokine production, and antigen-specific T-cell–mediated cytotoxicity superior to clinically approved PD-(L)1 antibodies in human T-cell cultures and exerted potent antitumor activity in transplantable mouse tumor models. In dose escalation of the ongoing first-in-human study in heavily pretreated patients with advanced refractory solid tumors (NCT03917381), GEN1046 demonstrated pharmacodynamic immune effects in peripheral blood consistent with its mechanism of action, manageable safety, and early clinical activity [disease control rate: 65.6% (40/61)], including patients resistant to prior PD-(L)1 immunotherapy.Significance:DuoBody-PD-L1×4-1BB (GEN1046) is a first-in-class bispecific immunotherapy with a manageable safety profile and encouraging preclinical and early clinical activity. With its ability to confer clinical benefit in tumors typically less sensitive to CPIs, GEN1046 may fill a clinical gap in CPI-relapsed or refractory disease or as a combination therapy with CPIs.See related commentary by Li et al., p. 1184.This article is highlighted in the In This Issue feature, p. 1171

Targeted therapies and immunotherapy have shown promise in patients with non-small cell lung cancer (NSCLC). However, the majority of patients fail or become resistant to treatment, emphasizing the need for novel treatments. In this study, we confirm the prognostic value of levels of AXL, a member of the TAM receptor tyrosine kinase family, in NSCLC and demonstrate potent antitumor activity of the AXL-Targeting antibody-drug conjugate enapotamab vedotin across different NSCLC subtypes in a mouse clinical trial of human NSCLC. Tumor regression or stasis was observed in 17/61 (28%) of the patient-derived xenograft (PDX) models and was associated with AXL mRNA expression levels. Significant single-Agent activity of enapotamab vedotin was validated in vivo in 9 of 10 AXL-expressing NSCLC xenograft models. In a panel of EGFR-mutant NSCLC cell lines rendered resistant to EGFR inhibitors in vitro, we observed de novo or increased AXL protein expression concomitant with enapotamab vedotin mediated cytotoxicity. Enapotamab vedotin also showed antitumor activity in vivo in 3 EGFR-mutant, EGFR inhibitor resistant PDX models, including an osimertinib-resistant NSCLC PDX model. In summary, enapotamab vedotin has promising therapeutic potential in NSCLC. The safety and preliminary efficacy of enapotamab vedotin are currently being evaluated in the clinic across multiple solid tumor types, including NSCLC.

Background: Hyperclustering of death receptor 5 (DR5) and subsequent induction of apoptosis, which normally occurs upon binding of its ligand TRAIL, can be mimicked by agonistic antibodies (Abs). However, clinical efficacy of conventional DR5 Abs has been disappointing. To improve antibody-mediated DR5 clustering on cancer cells, we utilized the HexaBody® antibody technology. This antibody platform is based on the discovery that IgG molecules can organize into hexamers through intermolecular Fc-Fc interactions upon binding to membrane-bound targets. HexaBody molecules are IgG1 Abs with a single point mutation in the Fc domain that enhances hexamerization. HexaBody-DR5/DR5 is a 1:1 mixture of two non-competing anti-DR5 HexaBody molecules that induces potent caspase-dependent apoptosis through hexamer-dependent hyperclustering of DR5 on the cell surface. We previously showed that HexaBody-DR5/DR5 induced superior potency compared to conventional DR5 antibodies in vitro and in vivo. Methods: To obtain preclinical proof-of-concept for targeting specific solid tumors using HexaBody-DR5/DR5, we performed a patient-derived xenograft (PDX) mouse clinical trial (1 mouse per group design) using a large number of colorectal (CRC) (100), gastric (19), urothelial (13), non-small cell lung cancer (NSCLC) (90), and triple-negative breast cancer (TNBC) (20) PDX models. Results: HexaBody-DR5/DR5 showed potent anti-tumor activity (tumor stasis or tumor regression) in a substantial proportion of the CRC (36%), gastric (42%) and urothelial (54%) models. Additional in vivo studies comparing single vs multiple dosing demonstrated that maximal anti-tumor activity was already achieved after a single dose of HexaBody-DR5/DR5. A Phase 1/2 clinical trial to determine the RP2D and assess clinical safety of HexaBody-DR5/DR5 in cancer patients is currently ongoing (NCT03576131), from which a case study will be presented. Conclusions: Potent anti-tumor activity of HexaBody-DR5/DR5 was observed in large and diverse panels of CRC, gastric and urothelial PDX models, providing preclinical rationale for the clinical evaluation of HexaBody-DR5/DR5 in these indications. Citation Format: Marije B Overdijk, Michael Cecchini, Kristin Strumane, Marcel Brandhorst, Andreas Lingnau, Paul W.H.I. Parren, Merete Ellekilde-Pedersen, Ulf Forssmann, Tahamtan Ahmadi, A. Kate Sasser, Janine Schuurman, Esther C.W. Breij, Patricia LoRusso. HexaBody-DR5/DR5 (GEN1029) shows potent preclinical antitumor activity in a variety of patient-derived xenograft (PDX) tumor models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C025. doi:10.1158/1535-7163.TARG-19-C025

PURPOSE Survivin is a member of the inhibitor-of-apoptosis family. Essential for tumor cell survival and overexpressed in most cancers, survivin is a promising target for anti-cancer immunotherapy. Immunogenicity has been demonstrated in multiple cancers. Nonetheless, few clinical trials have demonstrated survivin-vaccine-induced immune responses. EXPERIMENTAL DESIGN This phase I trial was conducted to test whether vaccine EMD640744, a cocktail of five HLA class I-binding survivin peptides in Montanide(®) ISA 51 VG, promotes anti-survivin T-cell responses in patients with solid cancers. The primary objective was to compare immunologic efficacy of EMD640744 at doses of 30, 100, and 300 μg. Secondary objectives included safety, tolerability, and clinical efficacy. RESULTS In total, 49 patients who received ≥2 EMD640744 injections with available baseline- and ≥1 post-vaccination samples [immunologic-diagnostic (ID)-intention-to-treat] were analyzed by ELISpot- and peptide/MHC-multimer staining, revealing vaccine-activated peptide-specific T-cell responses in 31 patients (63 %). This cohort included the per study protocol relevant ID population for the primary objective, i.e., T-cell responses by ELISpot in 17 weeks following first vaccination, as well as subjects who discontinued the study before week 17 but showed responses to the treatment. No dose-dependent effects were observed. In the majority of patients (61 %), anti-survivin responses were detected only after vaccination, providing evidence for de novo induction. Best overall tumor response was stable disease (28 %). EMD640744 was well tolerated; local injection-site reactions constituted the most frequent adverse event. CONCLUSIONS Vaccination with EMD640744 elicited T-cell responses against survivin peptides in the majority of patients, demonstrating the immunologic efficacy of EMD640744.

Hyperclustering of Death Receptor 5 (DR5) after binding of its ligand TRAIL induces apoptosis. Targeting DR5 with agonistic antibodies has been evaluated for the treatment of cancer, however clinical efficacy of conventional DR5-targeting monoclonal antibodies (mAbs) has been disappointing. We applied the HexaBody® technology to improve antibody-mediated DR5 clustering on cancer cells. This technology is based on the natural concept that, upon binding to antigens on a cell surface, immunoglobulin G (IgG) molecules can organize into ordered hexamers through intermolecular Fc-Fc interactions. HexaBody molecules are IgG1 molecules with a single point mutation in the Fc domain that enhances these Fc-Fc interactions upon binding to membrane-bound targets, while retaining solution-monomericity. HexaBody-DR5/DR5 (Hx-DR5-01/05) is a 1:1 mixture of two humanized non-competing DR5-specific mAbs, each carrying an E430G hexamerization-enhancing mutation. We previously demonstrated that both dual epitope targeting and enhanced hexamerization through Fc-Fc interactions are required for DR5 agonist activity of Hx-DR5-01/05 in vitro. Here, we confirmed that Hx-DR5-01/05 showed superior anti-tumor activity compared to the single HexaBody molecules or a 1:1 mixture of their wild type counterparts in vivo, using a mouse xenograft model. Furthermore, we screened the potency of Hx-DR5-01/05 in vitro in a broad panel of human tumor cells lines using a cell viability assay, and in vivo in xenograft models. As IgG hexamers are known to provide an optimal docking site for complement component C1q, we studied if there was a role for C1q in Hx-DR5-01/05-dependent DR5 agonist activity. Hx-DR5-01/05 induced potent cytotoxicity in 104 tumor cell lines and in more than ten xenograft models representing many solid cancer lineages. For optimal cytotoxicity in vitro, Hx-DR5-01/05 required the presence of serum or purified C1q. In contrast, polyclonal IgG crosslinking, a mimic for FcγR-mediated antibody crosslinking, did not enhance potency. These data were confirmed in vivo. A Hx-DR5-01/05 variant deficient for both C1q and FcγR binding, showed significantly reduced anti-tumor activity in a colon cancer xenograft model, while a Hx-DR5-01/05 variant deficient in FcγR but not C1q binding showed anti-tumor activity comparable to Hx-DR5-01/05. In summary, Hx-DR5-01/05 is a mixture of two DR5-specific HexaBody molecules that shows potent DR5 agonist activity in a multitude of preclinical models through enhanced IgG hexamerization upon binding to two different DR5 epitopes on the cell surface. Cytotoxicity of Hx-DR5-01/05 was most optimal in the presence of C1q and completely independent of FcγR-mediated antibody crosslinking or effector functions in vitro and in vivo. A clinical trial to assess clinical safety of Hx-DR5-01/05 in patients is currently ongoing. Citation Format: Marije B. Overdijk, Kristin Strumane, Antonio Ortiz Buijsse, Claudine Vermot-Desroches, Thessa Kroes, Bart de Jong, Naomi Hoevenaars, Frank J. Beurskens, Rob N. de Jong, Andreas Lingnau, Paul W. Parren, Ulf Forssmann, A Kate Sasser, Janine Schuurman, Esther C. Breij. DR5 agonist activity of HexaBody®-DR5/DR5 (GEN1029) is potentiated by C1q and independent of Fc-gamma receptor binding in preclinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2391.

Purpose We evaluated the safety, tolerability, and pharmacokinetics (PK) of EMD 525797 (DI17E6), a humanized monoclonal antibody targeting αv-integrins, in healthy subjects. Methods In this first-in-human, double-blind, placebo-controlled, randomized Phase 1 study, healthy male volunteers were consecutively assigned to 6 ascending single-dose cohorts of 35, 100, 250, 500, 1000, or 1500 mg. Per dose cohort, EMD 525797 or placebo was administered over 1 h as an intravenous 250-mL infusion to 6 and 3 volunteers, respectively. Escalation to the next dose level was based on evaluation of safety, tolerability, and PK data. Results Fifty-five subjects (aged 18–45 years) were randomized. Twenty-seven of 37 (73 %) subjects receiving EMD 525797 reported a total of 61 adverse events (AEs), including 38 events (in 17 subjects) considered by the investigator to be treatment related. A total of 35 AEs were reported by 14 of 18 (78 %) placebo-treated subjects. The most commonly occurring AEs were gastrointestinal disorders, abnormal laboratory values, and increased or decreased biochemistry and/or hematology values, as well as headaches, which occurred at a slightly higher frequency in the EMD 525797 group compared with placebo. There were no serious AEs or deaths. EMD 525797 PK appeared to be dose dependent, especially at lower doses. Conclusion Ascending single doses of EMD 525797 were shown to be safe and well tolerated. No safety concerns were identified. This study supports the ongoing investigation of EMD 525797.

Background/Aims: Parathyroid hormone (PTH) derivatives exert pronounced renal and osteoanabolic properties when given intermittently. The current study was performed to assess the pharmacokinetic and pharmacodynamic properties as well as safety of subcutaneously applied PTH-1-37 after repeated dosing in healthy subjects. Methods: This randomized, double-blind, dose-escalating, placebo and active comparator controlled study was conducted in 33 healthy postmenopausal women. Subjects were allocated to one of five treatment options: 10, 20, or 40 µg PTH-1-37, 20 µg PTH-1-34 or placebo, administered as once daily subcutaneous doses for three days. Plasma drug concentrations and serum levels of endogenous PTH-1-84, and calcium as markers of biological activity were monitored during the treatment. Results: PTH was absorbed rapidly from the subcutaneous tissue with a median tmax of 30 minutes for 20 and 40 µg of PTH-1-37. tmax was 45 minutes for 20 µg PTH-1-34. Elimination half-lives were estimated as 76 ± 34 min and 70 ± 13 min for 20 µg and 40 µg PTH-1-37 (mean ± SD), and 78 ± 34 for 20 µg PTH-1-34. Both PTH fragments (PTH-1-37 and PTH-1-34) increased serum calcium. For PTH-1-37 the effect on serum calcium was dose-dependent. Suppression of endogenous PTH-1-84 was seen after the application of both PTH-1-37 and PTH-1-34. During the study period, the subjects experienced no unexpected or serious adverse events. Conclusions: PTH-1-37 is rapidly absorbed after s.c. injection, has a short plasma elimination half-life, and does not accumulate during multiple dosing. Biological activity was demonstrated by rising serum calcium and decreasing endogenous PTH-1-84 in blood plasma. The study drugs were well tolerated and safe. Our investigation presents data that PTH-1-37 is an excellent drug candidate for intervening with syndromes of dysregulation of calcium metabolism.

Background: Modulation of leukocyte recruitment through blocking of chemokine receptors has been proposed as an attractive therapeutic strategy. We have previously demonstrated that n-Nonanoyl-CC chemokine ligand 14 (NNY-CCL14), a modified analog of the naturally occurring chemokine CCL14(9-74) internalizes and desensitizes human CCR3 resulting in the inactivation of eosinophils. However, inhibitory effects of NNY-CCL14 in murine models of allergic airway inflammation are assigned to its interaction with CCR1 and CCR5. Aim of the study: As CCL2 and its receptor CCR2 have been shown to play important roles in the development of Th2 inflammation, we further evaluated the effects of NNY-CCL14 treatment on CCL2-mediated activation of CCR2. Methods: Effects of NNY-CCL14 treatment were studied on cell lines transfected with human CCR2 and primary leukocytes. Functional effects were assessed by calcium efflux assays, flow cytometry and chemotaxis. Results: Prestimulation with NNY-CCL14 desensitized CCR2-mediated responses to further stimulation with its selective ligand CCL2. No significant internalization of CCR2 was observed when the cells were stimulated with NNY-CCL14, even at concentrations eliciting maximal [Ca2+]i mobilization. Above all, NNY-CCL14 pretreatment blocked CCL2-induced chemotaxis of monocytes. Conclusions: This study demonstrates that NNY-CCL14 is a partial agonist of CCR2, inhibiting responses of monocytes to the CCR2-selective ligand CCL2. NNY-CCL14 attenuates CCR2-mediated responses by rapidly desensitizing the receptor and preventing chemotaxis, although it is able to induce calcium mobilization but does not lead to CCR2 internalization. Hence this study provides further insights into the possible mechanisms of action of NNY-CCL14, which interacts with multiple chemokine receptors inhibiting the migration and activation of different cell populations involved, thus acting as a potential therapeutic compound to alleviate allergic inflammation.

Chemokines mediate the recruitment of leukocytes to the sites of inflammation. N-terminal truncation of chemokines by the protease dipeptidyl peptidase IV (DPPIV) potentially restricts their activity during inflammatory processes such as allergic reactions, but direct evidence in vivo is very rare. After demonstrating that N-terminal truncation of the chemokine CCL11/eotaxin by DPPIV results in a loss of CCR3-mediated intracellular calcium mobilization and CCR3 internalization in human eosinophils, we focused on the in vivo role of CCL11 and provide direct evidence for specific kinetic and rate-determining effects by DPPIV-like enzymatic activity on CCL11-mediated responses of eosinophils. Namely, it is demonstrated that i.v. administration of CCL11 in wild-type F344 rats leads to mobilization of eosinophils into the blood, peaking at 30 min. This mobilization is significantly increased in DPPIV-deficient F344 rats. Intradermal administration of CCL11 is followed by a dose-dependent recruitment of eosinophils into the skin and is significantly more effective in DPPIV-deficient F344 mutants as well as after pharmacological inhibition of DPPIV. Interestingly, CCL11 application leads to an up-regulation of DPPIV, which is not associated with negative feedback inhibition via DPPIV-cleaved CCL11(3–74). These findings demonstrate regulatory effects of DPPIV for the recruitment of eosinophils. Furthermore, they illustrate that inhibitors of DPPIV have the potential to interfere with chemokine-mediated effects in vivo including but not limited to allergy.

Modulation of leukocyte recruitment through intervention with chemokine receptors is an attractive, therapeutic strategy. Recently, we have shown that n-Nonanoyl (NNY)-CCL14 internalizes and desensitizes human (h)CCR3, resulting in the inactivation of eosinophils. In this study, we investigated the interaction of NNY-CCL14 with CCR1 and CCR5 and the relevance of these NNY-CCL14 receptors on its in vivo effects in allergic airway inflammation. NNY-CCL14 has inactivating properties on CCR1+ and CCR5+ cell lines and primary leukocytes. It desensitizes hCCR1- and hCCR5-mediated calcium release and internalizes these receptors from the cellular surface. Treatment of OVA-sensitized BALB/c mice with NNY-CCL14 resulted in reduced pulmonary inflammation. Above all, it is demonstrated that systemic treatment with NNY-CCL14 down-modulates CCR5 from the surface of lymphocytes in vivo. Although NNY-CCL14 acts on murine lymphocytes and internalizes CCR5, it does not internalize CCR3 on mouse eosinophils, showing species selectivity regarding this particular receptor. Therefore, the inhibitory effects of NNY-CCL14 in murine models of allergic airway inflammation can be assigned to its interaction with CCR5. The presented results substantiate the relevance of CCR5 as a target for allergic airway inflammation.

Human defensins are natural peptide antibiotics. On the basis of the position and bonding of six conserved cysteine residues, they are divided into two families, designated α- and β-defensins. Human α-defensins are expressed predominantly in neutrophils (human neutrophil peptides (HNP) 1–4) or intestinal Paneth cells (human defensins (HD) 5 and 6). Although α-defensins have been implicated in the pathogenesis of inflammatory bowel disease, their immunomodulatory functions are poorly understood. In the present study, HNP-1, HNP-3, and HD5 were found to be potent chemotaxins for macrophages but not dendritic cells using Gαi proteins and MAPK as signal transducers. α-Defensins were also chemoattractive for the human mast cell line HMC-1 but lacked, in contrast to β-defensins, the ability to induce intracellular calcium fluxes. Furthermore, HNP-1, HNP-3, and HD5 comparably mobilized naive as well as memory T lymphocytes. Using the protein kinase C (PKC) inhibitors GF109 and Gö6976, we observed a PKC-independent functional desensitization to occur between human α-defensins, which suggests a common receptor for HNP-1, HNP-3, and HD5 on immune cells. This α-defensin receptor was subject to heterologous desensitization by the PKC activator PMA and to PKC-dependent cross-desensitization by human β-defensins. Conversely, α-defensins desensitized β-defensin-mediated migration of immune cells in a PKC-dependent manner, suggesting unique receptors for both defensin families. Taken together, our observations indicate that chemoattraction of macrophages, T lymphocytes, and mast cells represents an immunomodulatory function which is evolutionarily conserved within the human α-defensin family and tightly regulated by β-defensins.

Previous cancer vaccination trials often aimed to activate CD8+ cytotoxic T-cell (CTL) responses with short (8–10mer) peptides and targeted CD4+ helper T cells (TH) with HLA class II–binding longer peptides (12–16 mer) that were derived from tumor antigens. Accordingly, a study of immunomonitoring focused on the detection of CTL responses to the short, and TH responses to the long, peptides. The possible induction of concurrent TH responses to short peptides was widely neglected. In a recent phase I vaccination trial, 53 patients with different solid cancers were vaccinated with EMD640744, a cocktail of five survivin-derived short (9- or 10-mer) peptides in Montanide ISA 51VG. We monitored 49 patients and found strong CD8+ T-cell responses in 63% of the patients. In addition, we unexpectedly found CD4+ TH cell responses against at least two of the five short peptides in 61% (23/38) of the patients analyzed. The two peptides were recognized by HLA-DP4– and HLA-DR–restricted TH1 cells. Some short peptide–reactive (sp)CD4 T cells showed high functional avidity. Here, we show that a short peptide vaccine is able to activate a specific CD4+ T-cell repertoire in many patients, facilitating a strong combined CD4+/CD8+ T-cell response. Cancer Immunol Res; 4(1); 18–25. ©2015 AACR.

Despite the several advances in the last few years into treatment of advanced lung cancer, the 5-year survival remains extremely low. New therapeutic strategies are currently under investigation, and immunotherapy seems to offer a promising treatment alternative. In the last decade, therapeutic cancer vaccines in lung cancer have been rather disappointing, mainly due to the lack of efficient predictive biomarkers. A better refinement of the patient population that might respond to treatment might finally lead to a success story. For the first time, the immune checkpoint inhibitors are demonstrating sustained antitumor response and improved survival and they may be the first immunotherapeutics available for patients with lung cancer.

Monocyte infiltration into inflammatory sites is generally preceded by neutrophils. We show here that neutrophils may support this process by activation of CCL15, a human chemokine circulating in blood plasma. Neutrophils were found to release CCL15 proteolytic activity in the course of hemofiltration of blood from renal insufficiency patients. Processing of CCL15 immunoreactivity (IR) in the pericellular space is suggested by a lack of proteolytic activity in blood and blood filtrate, but a shift of the retention time (tR) of CCL15-IR, detected by chromatographic separation of CCL15-IR in blood and hemofiltrate. CCL15 molecules with N-terminal deletions of 23 (Δ23) and 26 (Δ26) aa were identified as main proteolytic products in hemofiltrate. Neutrophil cathepsin G was identified as the principal protease to produce Δ23 and Δ26 CCL15. Also, elastase displays CCL15 proteolytic activity and produces a Δ21 isoform. Compared with full-length CCL15, Δ23 and Δ26 isoforms displayed a significantly increased potency to induce calcium fluxes and chemotactic activity on monocytes and to induce adhesiveness of mononuclear cells to fibronectin. Thus, our findings indicate that activation of monocytes by neutrophils is at least in part induced by quantum proteolytic processing of circulating or endothelium-bound CCL15 by neutrophil cathepsin G.

The aim of this review is to give an overview of the role of chemokines, particularly ligands of the CC chemokine receptor CCR3, in allergic diseases and to show the new concept in the treatment of allergies using chemokine receptor antagonists. Allergic diseases such as allergic asthma, allergic rhinitis and atopic dermatitis are characterized by a complex interaction of different cell types and mediators. Among this, Th2 cells, mast cells, basophils and eosinophils are found in the inflamed tissue due to the attraction of chemokines. Of all the known chemokine receptors, the chemokine receptor CCR3 seems to play the major role in allergic diseases which is supported by the detection of this receptor on the cell types mentioned above. Therefore, academic and industrial research focus on compounds to block this receptor. To date, certain chemokine receptor antagonists derived from peptides and small molecules exist to block the chemokine receptor CCR3. However, the in vivo data about these compounds and the mechanisms of receptor interaction are poorly understood, as yet. For the development of additional chemokine receptor antagonists, more details about the interaction between the ligands and their receptors are required. Therefore, additional studies will lead to the identification of novel CCR3 chemokine receptor antagonists, which can be therapeutically used in allergic asthma, allergic rhinitis, and atopic dermatitis.

TPS2605 Background: HuMax-AXL-ADC is an antibody-drug conjugate (ADC) composed of an Axl-specific human monoclonal immunoglobulin G1 (IgG1κ) conjugated via a protease-cleavable valine-citrulline linker to the microtubule disrupting agent monomethyl auristatin E (MMAE). In vivo, HuMax-AXL-ADC demonstrated therapeutic anti-tumor efficacy in patient-derived xenograft models representing a variety of solid cancers, including pancreas, thyroid, lung, esophageal, cervical cancers and malignant melanoma. The non-clinical safety profile and pharmacokinetics (PK) of a once every 3 weeks (1Q3W) dosing schedule were established in cynomolgus monkeys. Methods: The primary objective of this trial is to determine the MTD and to establish the safety profile of HuMax-AXL-ADC in a mixed population of patients with specified solid tumors: ovarian, cervical, endometrial, thyroid cancer, NSCLC, and malignant melanoma. The trial consists of two parts, a phase I dose escalation part and a phase IIa expansion part. The dose escalation part explores two different dosing regimens: the first investigates doses from 0.3 up to 2.8 mg/kg to be administered 1Q3W. The second investigates doses in the range of 0.45 to 1.4 mg/kg to be administered weekly for 3 weeks followed by one treatment-free week (3Q4W dosing schedule). The second arm has a delayed start to inform a safe starting dose: when at least 8 patients have been evaluated for dose limiting toxicities, the 1.5 mg/kg cohort of the 1Q3W arm has been declared safe, and the predicted PK parameters of the starting dose in the 3Q4W arm are below pre-defined limits, the 3Q4W arm will be initiated. The 1Q3W arm follows a modified Bayesian Continuous Reassessment Method including escalation with overdose control in up to 41 patients on up to 7 main and 4 intermediate dose levels while the 3Q4W arm is run as a standard 3+3 trial design on up to 5-6 dose levels. In the phase IIa expansion part, further safety and biological activity data will be generated in selected indications using cohorts of 22 patients (11+11 patients in each cohort applying the Simon’s two-stage design). Clinical trial information: NCT 02988817.

Patients (n = 34) with previously untreated, slowly progressive asymptomatic stage I/II multiple myeloma or with stage II/III multiple myeloma in stable response/plateau phase following conventional anti-tumor therapy were immunized repeatedly with the antigen-specific cancer immunotherapeutic agent tecemotide (L-BLP25). Additionally, patients were randomly allocated to either single or multiple low doses of cyclophosphamide to inhibit regulatory T cells (Treg). Immunization with tecemotide resulted in the induction/augmentation of a mucin 1-specific immune response in 47% of patients. The immune responses appeared to involve a Th1-like cellular immune response involving CD4 and CD8 T cells. The rate of immune responses was similar with single versus multiple dosing of cyclophosphamide and in patients with vs. without pre-existing mucin 1 immunity. On-treatment reductions in the slope of M-protein concentration over time (but not fulfilling clinical criteria for responses with conventional anti-tumor agents) were observed in 45% of evaluable patients, predominantly in those without versus with pre-existing mucin 1 immunity and in patients with early stage disease. No differences were seen in patients receiving single or multiple cyclophosphamide dosing. Treatment with tecemotide was generally well tolerated. Repeated vs. single dosing of cyclophosphamide had no impact on Treg numbers and was stopped after a case of fatal encephalitis that was assessed as possibly study-related. Tecemotide immunotherapy induces mucin 1-specific cellular immune responses in a substantial proportion of patients, with preliminary evidence of changes in the M-protein concentration time curve in a subset of patients.

Lung cancer is the most common cause of cancer mortality globally, accounting for 1.2 million deaths per year (Ferlay et al. 2010). The majority of lung cancer patients present with advanced disease (stage IIIb/IV), and despite recent treatment advances, the overall 5-year survival rate is 12–15 %. The development of new therapeutic strategies is therefore essential, and immunotherapy offers promising treatment alternatives that may help fight disease death with minimal impact on normal tissues.

Mobilization of hematopoietic stem and progenitor cells (HPCs) is induced by treatment with granulocyte-colony stimulating factor, chemotherapy, or irradiation. We observed that these treatments are accompanied by a release of chemotactic activity into the blood. This plasma activity is derived from the bone marrow, liver, and spleen and acts on HPCs via the chemokine receptor CXCR4. A human blood peptide library was used to characterize CXCR4-activating compounds. We identified CXCL12[22-88] and N-terminally truncated variants CXCL12[24-88], CXCL12[25-88], CXCL12[27-88], and CXCL12[29-88]. Only CXCL12[22-88] could effectively bind to CXCR4 and induce intracellular calcium flux and chemotactic migration of HPCs. CXCL12[25-88] and CXCL12[27-88] revealed neither agonistic nor antagonistic activities in vitro, whereas CXCL12[29-88] inhibited CXCL12[22-88]-induced chemotactic migration. Since binding to glycosaminoglycans (GAG) modulates the function of CXCL12, binding to heparin was analyzed. Surface plasmon resonance kinetic analysis showed that N-terminal truncation of Arg22-Pro23 increased the dissociation constant KD by one log10 stage ([22-88]: KD: 5.4 ± 2.6 μM; [24-88]: KD: 54 ± 22.4 μM). Further truncation of the N-terminus decreased the KD ([25-88] KD: 30 ± 4.8 μM; [27-88] KD: 23 ± 1.6 μM; [29-88] KD: 19 ± 5.4 μM), indicating increasing competition for heparin binding. Systemic in vivo application of CXCL12[22-88] as well as CXCL12[27-88] or CXCL12[29-88] induced a significant mobilization of HPCs in mice. Our findings indicate that plasma-derived CXCL12 variants may contribute to the regulation of HPC mobilization by modulating the binding of CXCL12[22-88] to GAGs rather than blocking the CXCR4 receptor and, therefore, may have a contributing role in HPC mobilization.

Despite the several advances in the last few years into treatment of advanced lung cancer, the 5-year survival remains extremely low. New therapeutic strategies are currently under investigation, and immunotherapy seems to offer a promising treatment alternative. In the last decade, therapeutic cancer vaccines in lung cancer have been rather disappointing, mainly due to the lack of efficient predictive biomarkers. A better refinement of the patient population that might respond to treatment might finally lead to a success story. For the first time, the immune checkpoint inhibitors are demonstrating sustained antitumor response and improved survival and they may be the first immunotherapeutics available for patients with lung cancer.

Previous studies have shown the implication of β-defensins in host defense of the human body. The human β-defensins 1 and 2 (hBD-1, hBD-2) have been isolated by biochemical methods. Here we report the identification of a third human β-defensin, called human β-defensin 3 (hBD-3; cDNA sequence, Genbank accession no. AF295370), based on bioinformatics and functional genomic analysis. Expression of hBD-3 is detected throughout epithelia of many organs and in non-epithelial tissues. In contrast to hBD-2, which is upregulated by microorganisms or tumor necrosis factor-α (TNF-α), hBD-3 expression is increased particularly after stimulation by interferon-γ. Synthetic hBD-3 exhibits a strong antimicrobial activity against gram-negative and gram-positive bacteria and fungi, including Burkholderia cepacia. In addition, hBD-3 activates monocytes and elicits ion channel activity in biomembranes, specifically in oocytes of Xenopus laevis. This paper also shows that screening of genomic sequences is a valuable tool with which to identify novel regulatory peptides. Human β-defensins represent a family of antimicrobial peptides differentially expressed in most tissues, regulated by specific mechanisms, and exerting physiological functions not only related to direct host defense.

The hemofiltrate CC chemokines CCL14a (formerly HCC-1), CCL14b (formerly HCC-3), and CCL15 (formerly HCC-2) are encoded by mono- as well as bicistronic transcripts from a tandem gene arrangement on human chromosome 17q11.2. The transcription and splicing into several mono- and bicistronic transcripts of this gene complex are unique for human genes. No corresponding mechanism is known in nonprimate mammalian species such as mice and rats. The extremely high concentration of CCL14a in human plasma is exceptional for chemokines and led to the identification of this chemokine. Several molecular forms of CCL14a have been isolated and investigated. The mature propeptide CCL14a(1–74) is a low-affinity agonist of CCR1 which is converted to a high-affinity agonist of CCR1 and CCR5 on proteolytic processing by serine proteases. In contrast, CCL15 is characterized using molecular forms deduced from the mRNA/cDNA and shown to activate cells via CCR1 and CCR3, also dependent on the amino-terminal length. Hemofiltrate CC chemokines are chemoattractants for different types of leukocytes including monocytes, eosinophils, T cells, dendritic cells, and neutrophils. In this review, we emphasize the genomic organization, expression patterns, and biochemical properties of CCL14a, CCL14b, and CCL15. We report results of significance for the development of therapeutic strategies, especially concerning HIV infection and inflammatory diseases.

Hemofiltrate CC chemokine (HCC)-1 is a recently described human chemokine that is constitutively expressed in numerous tissues and is present at high concentrations in normal plasma. Using a cell line expressing CC chemokine receptor (CCR)5 as a bioassay, we isolated from human hemofiltrate an HCC-1 variant lacking the first eight amino acids. HCC-1[9–74] was a potent agonist of CCR1, CCR3, and CCR5 and promoted calcium flux and chemotaxis of T lymphoblasts, monocytes, and eosinophils. It also blocked entry of HIV-1 strains using CCR5 as coreceptor. Limited tryptic digestion of HCC-1 generated the active variant. Conditioned media from several tumor cell lines activated HCC-1 with a high efficiency, and this activity could be inhibited by serine protease inhibitors. Our results indicate that HCC-1 represents a nonfunctional precursor that can be rapidly converted to the active chemokine by proteolytic processing. This process represents an additional mechanism by which tumor cells might generate chemoattractant molecules and recruit inflammatory cells. It might also affect HIV-1 replication in infected individuals and play an important role in AIDS pathogenesis.

The immunologic mechanisms involved in the development of chronic pancreatitis (CP) are poorly understood. Chronically inflamed tissues contain increased numbers of mononuclear cells expressing the CC chemokine receptor 5 (CCR5), which is also a coreceptor for HIV entry of macrophagetropic strains. However, whether this receptor is involved in the inflammatory process in CP is not known. In the current study, we analyzed the expression of CCR5 in CP. The detection of chemokine receptors on inflammatory cells would strongly suggest their involvement in the pathogenesis of CP (i.e., attraction and activation of these cells). To further evaluate this, we consecutively analyzed the expression of 2 ligands of CCR5: RANTES and MIP-alpha.Pancreatic tissue samples of 22 patients with CP and of 7 healthy pancreas were evaluated. CCR5, RANTES, and MIP-1alpha were analyzed by Northern blot analysis. Consecutive tissue sections were stained for CCR5, CD3, and CD68 to define the leukocyte subtype expressing CCR5 in CP.By Northern blot analysis, CCR5, RANTES, and MIP-1alpha messenger RNA (mRNA) levels were 12.9-fold, 13.3-fold and 9.2-fold higher in CP specimens compared with healthy controls, respectively (P.01). Immunostaining for CCR5 revealed a 30-fold increase of CCR5-positive cells in CP tissue compared with the healthy pancreas. Staining of consecutive tissue sections revealed that the majority of CCR5-positive cells were also CD68-positive (macrophages).Our data indicate that a remarkable portion of CCR5-positive cells in CP are macrophages. CCR5 is most likely involved in the attraction and activation of these macrophages, since the CCR5 ligands RANTES and MIP-1alpha are concomitantly upregulated.

Background: Peritonitis is, even today, a significant source of death and complications. The objective of this study was to determine the morbidity and mortality rates, the incidence of reoperations, and the need for additional treatment strategies (on demand) in patients with diffuse peritonitis. Methods: Prospective analysis including all patients (n = 258) with diffuse peritonitis admitted to our surgical service between November 1993 and April 1998 who underwent a uniform surgical treatment concept of peritonitis including early intervention, source control, and extensive intraoperative lavage. Results: The 258 patients with diffuse peritonitis averaged a mean Mannheim Peritonitis Index of 27.1 points (range, 11-43 points). Source control at the initial operation was possible in 230 of the patients (89%), of those, 21 patients (9%) needed reintervention. In 28 patients (11%), source control was not possible at the initial operation. Twenty of these patients (71%) had to undergo additional treatment strategies (on demand) such as continuous lavage and/or laparostomy. Overall 228 of the 258 patients (88%) needed just 1 initial surgical intervention. The overall morbidity rate was 41%; the rate of reoperation was 12%, and the hospital mortality rate was 14%. Conclusions: A conservative surgical treatment concept supplemented with “extensive” intraoperative lavage reduces the reoperation rate compared with other treatment standards of peritonitis and achieves a low mortality rate in patients with diffuse peritonitis. (Surgery 2000;127:178-84.)

Cloning and sequencing of the upstream region of the gene of the CC chemokine HCC-1 led to the discovery of an adjacent gene coding for a CC chemokine that was named “HCC-2.” The two genes are separated by 12-kbp and reside in a head-to-tail orientation on chromosome 17. At variance with the genes for HCC-1 and other human CC chemokines, which have a three-exon-two-intron structure, the HCC-2 gene consists of four exons and three introns. Expression of HCC-2 and HCC-1 as studied by Northern analysis revealed, in addition to the regular, monocistronic mRNAs, a common, bicistronic transcript. In contrast to HCC-1, which is expressed constitutively in numerous human tissues, HCC-2 is expressed only in the gut and the liver. HCC-2 shares significant sequence homology with CKβ8 and the murine chemokines C10, CCF18/MRP-2, and macrophage inflammatory protein 1γ, which all contain six instead of four conserved cysteines. The two additional cysteines of HCC-2 form a third disulfide bond, which anchors the COOH-terminal domain to the core of the molecule. Highly purified recombinant HCC-2 was tested on neutrophils, eosinophils, monocytes, and lymphocytes and was found to exhibit marked functional similarities to macrophage inflammatory protein 1α. It is a potent chemoattractant and inducer of enzyme release in monocytes and a moderately active attractant for eosinophils. Desensitization studies indicate that HCC-2 acts mainly via CC chemokine receptor CCR1.

A novel human CC chemokine complementary DNA was identified in a library constructed from human fetal RNA, cloned into a baculovirus vector, and expressed in Sf9 insect cells. The mature recombinant protein that was released had the NH2-terminal sequence pyro-QPDALNVPSTC...and consisted of 75 amino acids. Minor amounts of two variants of 77 and 82 residues (NH2 termini: LAQPDA...and FNPQGLAQPDA...) were released as well. The novel chemokine was designated monocyte chemotactic protein 4 (MCP-4) and the variants were designated (LA)MCP-4 and (FNPQGLA)MCP-4. MCP-4 shares the pyroglutamic acidproline NH2-terminal motif and 56-61% sequence identity with the three known monocyte chemotactic proteins and is 60% identical to eotaxin. It has marked functional similarities to MCP-3 and eotaxin. Like MCP-3, MCP-4 is a chemoattractant of high efficacy for monocytes and T lymphocytes. On these cells, it binds to receptors that recognize MCP-1, MCP-3, and RANTES. On eosinophils, MCP-4 has similar efficacy and potency as MCP-3, RANTES, and cotaxin. It shares receptors with eotaxin and shows full cross-desensitization with this cosinophil-selective chemokine. Of the two variants, only (LA)MCP-4 could be purified in sufficient quantities for testing and was found to be at least 30-fold less potent than MCP-4 itself. This suggests that the 75-residue form with the characteristic NH2 terminus of an MCP is the biologically relevant species.

Background This Phase Ib trial assessed the maximum tolerated dose (MTD) and safety of the Toll-like receptor 9 agonist IMO-2055 combined with 5-fluorouracil, cisplatin, and cetuximab (PFE) as first-line palliative treatment in patients with relapsed and/or metastatic squamous cell carcinoma of the head and neck (R/M SCCHN). Methods A standard 3 + 3 study design was used. Patients were sequentially enrolled to be treated with IMO-2055 (0.16, 0.32, or 0.48 mg/kg/day; days 1, 8, 15), 5-fluorouracil (1,000 mg/m2/day; days 1–4), cisplatin (100 mg/m2/day; day 1) and cetuximab (400 mg/m2/day first dose; then 250 mg/m2/day; days 1, 8, 15) every 3 weeks. Results Thirteen patients received IMO-2055. Dose-limiting toxicities (DLTs; ie, any Grade [G]3/4 treatment-related adverse events [TEAEs] in cycle 1) occurred in 2/4 patients treated with IMO-2055 0.32 mg/kg (G4 hypokalemia and hypomagnesemia [n = 1]; G4 septicemia, hyperthermia, febrile neutropenia, and G3 hypotension [n = 1]). In the IMO-2055 0.16-mg/kg expansion cohort, 1 patient experienced DLTs of G3 sepsis, bacteremia, and hyperthermia. The most common G ≥ 3 TEAEs were neutropenia (n = 9; not including febrile neutropenia [n = 1]), hypokalemia (n = 5), and hypomagnesemia (n = 4). Serious adverse events (SAEs) occurred in 8 patients, including 4 with SAEs considered IMO-2055 related; 1 of these patients died. Best response achieved overall was partial response in 3 patients and stable disease in 9 patients. The overall safety profile led to early trial termination; the safety monitoring committee did not confirm the MTD (formally IMO-2055 0.16 mg/kg). Conclusions Regimens combining IMO-2055 and PFE cannot be recommended for further development in R/M SCCHN patients.