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Background and aims: Dickkopf-1 (DKK1) is associated with poor prognosis in intrahepatic cholangiocarcinoma (iCCA), but the mechanisms behind this are unclear. Here, we show that DKK1 plays an immune regulatory role in vivo and inhibition reduces tumour growth.Methods: Various in vivo GEMM mouse models and patient samples were utilized to assess the effects of tumour specific DKK1 overexpression in iCCA. DKK1-driven changes to the tumour immune microenvironment were characterized by immunostaining and gene expression analysis. DKK1 overexpressing and damage induced models of iCCA were used to demonstrate the therapeutic efficacy of DKK1 inhibition in these contexts using the anti-DKK1 therapeutic, DKN-01.Results: DKK1 overexpression in mouse models of iCCA drives an increase in chemokine and cytokine signalling, the recruitment of regulatory macrophages, and promotes the formation of a tolerogenic niche with higher numbers of regulatory T cells. We show a similar association of DKK1 with FOXP3 and regulatory T cells in patient tissue and gene expression data, demonstrating these effects are relevant to human iCCA. Finally, we demonstrate that inhibition of DKK1 with the monoclonal antibody mDKN-01 is effective at reducing tumour burden in two distinct mouse models of the disease.Conclusion: DKK1 promotes tumour immune evasion in iCCA through the recruitment of immune suppressive macrophages. Targeting DKK1 with a neutralizing antibody is effective at reducing tumour growth in vivo. As such, DKK1 targeted and immune modulatory therapies may be an effective strategy in iCCA patients with high DKK1 tumour expression or tolerogenic immune phenotypes.

Flow Cytometry Gating Strategy

Dickkopf-1 (DKK1), a secreted modulator of Wnt signaling, is overexpressed in many cancers, is often associated with worse clinical outcomes, and has been shown to have immunosuppressive effects. DKN-01 is an IgG4 clinical stage antibody that potently and specifically neutralizes human and murine DKK1 and has recently completed a promising study in combination with pembrolizumab in patients with gastric/gastroesophageal junction cancer. The purpose of this study is to characterize a murine version of DKN-01 (mDKN-01) and to better understand its mechanism of action. We examined the efficacy of mDKN-01 in both melanoma and metastatic breast cancer models. Immune depletion experiments revealed a requirement for natural killer (NK) but not B and T cells for tumor growth inhibition. mDKN-01 treatment promotes the induction of the NK-activating cytokines IL15 and IL33 as well as an enhanced recruitment of CD45+ cells. Other treatment-related changes include a reduction of Gr-1+CD11b+ myeloid-derived suppressor cells (MDSC) in the tumor and spleen and the upregulation of PD-L1 on MDSCs. In addition, mDKN-01 has a marked effect at reducing pulmonary metastases in the mouse 4T1 breast cancer model. Finally, the mDKN-01/anti-PD-1 combination was more effective at inhibiting melanoma growth than mDKN-01 alone. Taken together, our data demonstrate that mDKN-01 has efficacy by blocking the immunosuppressive effects of DKK1 in the tumor microenvironment (TME) and provides insight into the clinical activity observed with DKN-01–based treatment.Implications:mDKN-01 reverses a DKK1-mediated innate immune suppression in the TME and has additive efficacy with a PD-1 inhibitor.

mDKN-01 has Additive Activity with an Anti-PD-1 Antibody

Supplementary Table from Phase IB Study of GITR Agonist Antibody TRX518 Singly and in Combination with Gemcitabine, Pembrolizumab, or Nivolumab in Patients with Advanced Solid Tumors

Purpose:TRX518 is a mAb engaging the glucocorticoid-induced TNF receptor−related protein (GITR). This open-label, phase I study (TRX518-003) evaluated the safety and efficacy of repeated dose TRX518 monotherapy and in combination with gemcitabine, pembrolizumab, or nivolumab in advanced solid tumors.Patients and Methods:TRX518 monotherapy was dose escalated (Part A) and expanded (Part B) up to 4 mg/kg loading, 1 mg/kg every 3 weeks. Parts C–E included dose-escalation (2 and 4 mg/kg loading followed by 1 mg/kg) and dose-expansion (4 mg/kg loading) phases with gemcitabine (Part C), pembrolizumab (Part D), or nivolumab (Part E). Primary endpoints included incidence of dose-limiting toxicities (DLT), serious adverse events (SAE), and pharmacokinetics. Secondary endpoints were efficacy and pharmacodynamics.Results:A total of 109 patients received TRX518: 43 (Parts A+B), 30 (Part C), 26 (Part D), and 10 (Part E), respectively. A total of 67% of patients in Parts D+E had received prior anti–PD(L)1 or anti–CTLA-4. No DLTs, treatment-related SAEs, and/or grade 4 or 5 AEs were observed with TRX518 monotherapy. In Parts C–E, no DLTs were observed, although TRX518-related SAEs were reported in 3.3% (Part C) and 10.0% (Part E), respectively. Objective response rate was 3.2%, 3.8%, 4%, and 12.5% in Parts A+B, C, D, and E, respectively. TRX518 affected peripheral and intratumoral regulatory T cells (Treg) with different kinetics depending on the combination regimen. Responses with TRX518 monotherapy+anti–PD1 combination were associated with intratumoral Treg reductions and CD8 increases and activation after treatment.Conclusions:TRX518 showed an acceptable safety profile with pharmacodynamic activity. Repeated dose TRX518 monotherapy and in combination resulted in limited clinical responses associated with immune activation.See related commentary by Hernandez-Guerrero and Moreno, p. 3905

Supplementary Data from Phase IB Study of GITR Agonist Antibody TRX518 Singly and in Combination with Gemcitabine, Pembrolizumab, or Nivolumab in Patients with Advanced Solid Tumors

Supplementary Figure from Phase IB Study of GITR Agonist Antibody TRX518 Singly and in Combination with Gemcitabine, Pembrolizumab, or Nivolumab in Patients with Advanced Solid Tumors

Dickkopf-1 (DKK1), a secreted modulator of Wnt signaling, is overexpressed in many cancers, is often associated with worse clinical outcomes, and has been shown to have immunosuppressive effects. DKN-01 is an IgG4 clinical stage antibody that potently and specifically neutralizes human and murine DKK1 and has recently completed a promising study in combination with pembrolizumab in patients with gastric/gastroesophageal junction cancer. The purpose of this study is to characterize a murine version of DKN-01 (mDKN-01) and to better understand its mechanism of action. We examined the efficacy of mDKN-01 in both melanoma and metastatic breast cancer models. Immune depletion experiments revealed a requirement for natural killer (NK) but not B and T cells for tumor growth inhibition. mDKN-01 treatment promotes the induction of the NK-activating cytokines IL15 and IL33 as well as an enhanced recruitment of CD45+ cells. Other treatment-related changes include a reduction of Gr-1+CD11b+ myeloid-derived suppressor cells (MDSC) in the tumor and spleen and the upregulation of PD-L1 on MDSCs. In addition, mDKN-01 has a marked effect at reducing pulmonary metastases in the mouse 4T1 breast cancer model. Finally, the mDKN-01/anti-PD-1 combination was more effective at inhibiting melanoma growth than mDKN-01 alone. Taken together, our data demonstrate that mDKN-01 has efficacy by blocking the immunosuppressive effects of DKK1 in the tumor microenvironment (TME) and provides insight into the clinical activity observed with DKN-01–based treatment. Implications: mDKN-01 reverses a DKK1-mediated innate immune suppression in the TME and has additive efficacy with a PD-1 inhibitor.

Purpose: TRX518 is a mAb engaging the glucocorticoid-induced TNF receptor−related protein (GITR). This open-label, phase I study (TRX518-003) evaluated the safety and efficacy of repeated dose TRX518 monotherapy and in combination with gemcitabine, pembrolizumab, or nivolumab in advanced solid tumors. Patients and Methods: TRX518 monotherapy was dose escalated (Part A) and expanded (Part B) up to 4 mg/kg loading, 1 mg/kg every 3 weeks. Parts C–E included dose-escalation (2 and 4 mg/kg loading followed by 1 mg/kg) and dose-expansion (4 mg/kg loading) phases with gemcitabine (Part C), pembrolizumab (Part D), or nivolumab (Part E). Primary endpoints included incidence of dose-limiting toxicities (DLT), serious adverse events (SAE), and pharmacokinetics. Secondary endpoints were efficacy and pharmacodynamics. Results: A total of 109 patients received TRX518: 43 (Parts A+B), 30 (Part C), 26 (Part D), and 10 (Part E), respectively. A total of 67% of patients in Parts D+E had received prior anti–PD(L)1 or anti–CTLA-4. No DLTs, treatment-related SAEs, and/or grade 4 or 5 AEs were observed with TRX518 monotherapy. In Parts C–E, no DLTs were observed, although TRX518-related SAEs were reported in 3.3% (Part C) and 10.0% (Part E), respectively. Objective response rate was 3.2%, 3.8%, 4%, and 12.5% in Parts A+B, C, D, and E, respectively. TRX518 affected peripheral and intratumoral regulatory T cells (Treg) with different kinetics depending on the combination regimen. Responses with TRX518 monotherapy+anti–PD1 combination were associated with intratumoral Treg reductions and CD8 increases and activation after treatment. Conclusions: TRX518 showed an acceptable safety profile with pharmacodynamic activity. Repeated dose TRX518 monotherapy and in combination resulted in limited clinical responses associated with immune activation. See related commentary by Hernandez-Guerrero and Moreno, p. 3905

GITR is a TNF receptor, and its activation promotes immune responses and drives antitumor activity. The receptor is activated by the GITR ligand (GITRL), which is believed to cluster receptors into a high-order array. Immunotherapeutic agonist antibodies also activate the receptor, but their mechanisms are not well characterized. We solved the structure of full-length mouse GITR bound to Fabs from the antibody DTA-1. The receptor is a dimer, and each subunit binds one Fab in an orientation suggesting that the antibody clusters receptors. Binding experiments with purified proteins show that DTA-1 IgG and GITRL both drive extensive clustering of GITR. Functional data reveal that DTA-1 and the anti-human GITR antibody TRX518 activate GITR in their IgG forms but not as Fabs. Thus, the divalent character of the IgG agonists confers an ability to mimic GITRL and cluster and activate GITR. These findings will inform the clinical development of this class of antibodies for immuno-oncology.

Dickkopf-1 (DKK1) is a secreted modulator of Wnt signaling that is frequently overexpressed in tumors and associated with poor clinical outcomes. DKN-01 is a humanized monoclonal therapeutic antibody that binds DKK1 with high affinity and has demonstrated clinical activity in gastric/gastroesophageal junction (G/GEJ) patients with elevated tumoral expression of DKK1. Here we report on the validation of a DKK1 RNAscope chromogenic in situ hybridization assay to assess DKK1 expression in G/GEJ tumor tissue. To reduce pathologist time, potential pathologist variability from manual scoring and support pathologist decision making, a digital image analysis algorithm that identifies tumor cells and quantifies the DKK1 signal was developed. Following CLIA guidelines the DKK1 RNAscope chromogenic in situ hybridization assay and digital image analysis algorithm were successfully validated for sensitivity, specificity, accuracy, and precision. The DKK1 RNAscope assay in conjunction with the digital image analysis solution is acceptable for prospective screening of G/GEJ adenocarcinoma patients. The work described here will further advance the companion diagnostic development of our DKK1 RNAscope assay and could generally be used as a guide for the validation of RNAscope assays with digital image quantification.

PURPOSE Metastatic castration-resistant prostate cancer (mCRPC) with low androgen receptor (AR) and without neuroendocrine signaling, termed double-negative prostate cancer (DNPC), is increasingly prevalent in patients treated with AR signaling inhibitors and is in need of new biomarkers and therapeutic targets. METHODS Candidate genes enriched in DNPC were determined using differential gene expression analysis of discovery and validation cohorts of mCRPC biopsies. Laboratory studies were carried out in human mCRPC organoid cultures, prostate cancer (PCa) cell lines, and mouse xenograft models. Epigenetic studies were carried out in a rapid autopsy cohort. RESULTS Dickkopf-1 (DKK1) expression is increased in DNPC relative to prostate-specific antigen (PSA)–expressing mCRPC in the Stand Up to Cancer/Prostate Cancer Foundation discovery cohort (11.2 v 0.28 reads per kilobase per million mapped reads; q < 0.05; n = 117) and in the University of Washington/Fred Hutchinson Cancer Research Center cohort (9.2 v 0.99 fragments per kilobase of transcript per million mapped reads; P < .0001). DKK1 expression can be regulated by activated Wnt signaling in vitro and correlates with activating canonical Wnt signaling mutations and low PSA mRNA in mCRPC biopsies ( P < .05). DKK1 hypomethylation was associated with increased DKK1 mRNA expression (Pearson r = −0.66; P < .0001) in a rapid autopsy cohort (n = 7). DKK1-high mCRPC biopsies are infiltrated with significantly higher numbers of quiescent natural killer (NK) cells ( P < .005) and lower numbers of activated NK cells ( P < .0005). Growth inhibition of the human PCa model PC3 by the anti-DKK1 monoclonal antibody DKN-01 depends on the presence of NK cells in a severe combined immunodeficient xenograft mouse model. CONCLUSION These results support DKK1 as a contributor to the immunosuppressive tumor microenvironment of DNPC. These data have provided the rationale for a clinical trial targeting DKK1 in mCRPC (ClinicalTrials.gov identifier: NCT03837353 ).

Modulating T cell homeostatic mechanisms with checkpoint blockade can efficiently promote endogenous anti-tumor T cell responses1-11. However, many patients still do not benefit from checkpoint blockade12, highlighting the need for targeting of alternative immune pathways13. Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) is an attractive target for immunotherapy, owing to its capacity to promote effector T cell (Teff) functions14,15 and hamper regulatory T cell (Treg) suppression16-20. On the basis of the potent preclinical anti-tumor activity of agonist anti-GITR antibodies, reported by us and others16,21,22, we initiated the first in-human phase 1 trial of GITR agonism with the anti-GITR antibody TRX518 ( NCT01239134 ). Here, we report the safety profile and immune effects of TRX518 monotherapy in patients with advanced cancer and provide mechanistic preclinical evidence to rationally combine GITR agonism with checkpoint blockade in future clinical trials. We demonstrate that TRX518 reduces circulating and intratumoral Treg cells to similar extents, providing an easily assessable biomarker of anti-GITR activity. Despite Treg reductions and increased Teff:Treg ratios, substantial clinical responses were not seen. Similarly, in mice with advanced tumors, GITR agonism was not sufficient to activate cytolytic T cells due to persistent exhaustion. We demonstrate that T cell reinvigoration with PD-1 blockade can overcome resistance of advanced tumors to anti-GITR monotherapy. These findings led us to start investigating TRX518 with PD-1 pathway blockade in patients with advanced refractory tumors ( NCT02628574 ).

Over the past decade, blockade of the immune checkpoints CTLA-4, PD-1, and PD-L1 has been demonstrated to significantly extend survival of cancer patients across multiple tumor types, including metastatic melanoma, formally proving that immunotherapy is a viable option for the treatment of cancer. These successes have paved the way for the development of additional immune-modulatory antibodies, blocking alternative inhibitory receptors, or engaging costimulatory receptors such as the TNF receptor family member GITR. However, the clinical experience accumulated thus far with checkpoint blockade has clearly shown that only a limited fraction of patients achieve durable clinical benefit with these treatments. This highlights the need to deepen our understanding of the molecular mechanisms underlying response and resistance to immunotherapy and design more personalized and rational combinations based on these therapies. Immune-regulatory mechanisms are one of the major barriers limiting efficacy of immunotherapy. CTLA-4 blockade and GITR costimulation are two immunotherapeutic strategies known to interfere with conventional immunosuppressive regulatory T cells (Tregs). We thus investigated the effects of CTLA-4 blockade and GITR costimulation on suppressive T cells in in vivo mouse melanoma models resistant to these therapies with the aim to clarify the molecular mechanisms underlying refractoriness and provide the rationale to develop more effective therapeutic combinations. To understand the clinical relevance of these findings, we explored the same effects in cancer patients treated with CTLA-4 blockade or GITR costimulation. We found that CTLA-4 blockade, while counteracting conventional Tregs, promotes the expansion of a subset of CD4+Foxp3-T cells expressing high levels of PD-1 (4PD1hi), which constitute a new immunosuppressive T-cell population with T-follicular-helper-like features. Importantly, we observed that anti-CTLA-4 increases the frequency of circulating 4PD1hi in a dose-dependent manner. In contrast, PD-1 blockade decreases 4PD1hi in function of its clinical activity, underscoring the relevance of this cell subset as a pharmacodynamic and prognostic biomarker of checkpoint blockade. These findings indicate that optimizing checkpoint blockade doses and combination regimens so as to keep 4PD1hi in the right balance may favor a positive outcome. In mouse melanoma models of response (early tumors) and refractoriness (advanced tumors) to GITR agonism, we found that anti-GITR efficiently reduces Tregs and increases effector:Treg ratios in both curative and refractory treatment conditions. However, T-cell activation and cytotoxic functions are favored selectively in the presence of low tumor burden. Counteracting exhaustion with PD-1 blockade in combination with GITR agonism restored responsiveness of advanced tumors and CD8+ T-cell functionality. Aligned with the effects of anti-GITR in mice, we found that the agonist anti-human GITR antibody TRX518 decreases Tregs in peripheral blood and tumor to similar extents in patients treated in the first-in-human single-dose monotherapy trial (NCT01239134). However, coincident downregulation of Tregs in the peripheral blood and at the tumor site upon TRX518 was not sufficient to achieve substantial clinical responses in this population of advanced solid cancer patients. This suggests that Treg elimination from advanced tumors may not be sufficient to activate cytotoxic T-cell responses unless the T-cell exhaustion process is concurrently blocked. Based on these preclinical and clinical observations, we have started to explore anti-GITR in combination with PD-1 pathway blockade in patients with advanced solid tumors (NCT02628574). Taken as a whole, these findings illustrate the value of conventional and nonconventional immune-suppressive T cells as biomarkers of biologic and therapeutic activity of immunotherapy in melanoma and other tumor types. In addition, these results indicate that inhibition of immune-regulatory mechanisms, such as immunosuppressive T cells, may need to be associated with strategies able to positively activate T-cell responses to achieve significant clinical benefit. Citation Format: Roberta Zappasodi, Sadna Budhu, Cynthia Sirard, Jingjing Qi, Cailian Liu, Yanyun Li, Yasin Senbabaoglu, Sasikanth Manne, Billel Gasmi, Hong Zhong, Xia Yang, Moshen Abu-Akeel, David Schaer, Alexander Huang, Walter Newman, Philip Wong, Katherine S. Panageas, Michael A. Postow, Henry Koon, Vamsidhar Velcheti, Margaret K. Callahan, Matthew D. Hellmann, E. John Wherry, Taha Merghoub, Jedd D. Wolchok. Overcoming immune resistance with rationally designed combination immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr IA04.

The clinical successes of checkpoint blockade have demonstrated that proper modulation of T cell co-inhibitory/co-stimulatory pathways can achieve substantial anti-tumor activity. However, many patients are or become refractory to checkpoint blockade. Additional forms of immunotherapy targeting alternative immune pathways are thus needed. Antibodies (Abs) engaging the TNF receptor GITR can enhance T cell functions and counteract regulatory T cell (Treg) suppression and have shown potent anti-tumor activity in animal models. Based on this evidence, we initiated the first in-human phase-I trial with the humanized aglycosylated anti-GITR Ab TRX518 (NCT01239134). Here, we report the immune effects of a single ascending dose of TRX518 monotherapy in 37 advanced cancer patients in this phase-I trial and provide mechanistic preclinical evidence to rationally combine GITR agonism with checkpoint blockade. We found that TRX518 frequently reduces circulating Tregs. In 8 patients for whom pre- and post-treatment tumor biopsies were available, reductions in intra-tumor and circulating Tregs after TRX518 were positively correlated. Yet, these patients did not experience substantial clinical responses. To explain this outcome, we modeled sensitivity and refractoriness to anti-GITR by treating B16F10-melanoma-bearing mice with the Ab DTA-1 on day 4 (curative regimen) or day 7 (refractory regimen) after tumor implantation respectively. We found that Tregs were significantly reduced and CD8+:Treg and Teff:Treg ratios increased in both responding and refractory tumors. Interestingly, CD8+ T cells from refractory tumors overexpressed T cell exhaustion markers and did not up-regulate memory and functional markers. We thus tested whether counteracting exhaustion could overcome resistance of advanced tumors to anti-GITR. PD-1 blockade in combination with anti-GITR starting on day 7 after tumor implantation controlled tumor growth similar to the curative anti-GITR monotherapy regimen (day 4 treatment) and achieved 50% complete response rate associated with long-lasting anti-tumor immunological memory. This was associated with more activated and less exhausted profiles of intra-tumor CD8+ T cells, which displayed enhanced tumor-lytic capacity compared to CD8+ T cells from non-responding tumors treated with each agent alone. These results indicate that Treg reduction can serve as a pharmacodynamic biomarker of anti-GITR in patients. However, Treg elimination from advanced tumors may not be sufficient to activate cytotoxic CD8+ T cell responses unless the T cell exhaustion process is concurrently blocked. This provides the rationale to combine immunotherapies targeting Tregs with strategies able to counteract exhaustion, such as anti-PD-1, to regress advanced tumors. Based on these observations, we have started to investigate TRX518 in combination with PD-1 pathway blockade in patients with advanced solid tumors (NCT02628574). Citation Format: Roberta Zappasodi, Cynthia Sirard, Yanyun Li, Sadna Budhu, Moshen Abu-Akeel, Cailian Liu, Xia Yang, Hong Zhong, Walter Newman, Jingjing Qi, Philip Wong, David Schaer, Henry Koon, Vamsidhar Velcheti, Margaret K. Callahan, Jedd Wolchok, Taha Merghoub. Rational combination of GITR agonism with PD-1 blockade [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 2711.

Wnt signaling is a fundamental pathway that is dysregulated in oncology. The Wnt signaling modulator DKK1 is expressed in a variety of tumor types and elevated levels frequently correlate with poor survival. DKK1 promotes tumor growth by stimulating proliferation, metastasis, and angiogenesis, and has been implicated in contributing to an immune suppressive tumor microenvironment. DKN-01 is a humanized monoclonal therapeutic antibody that binds DKK1 with high affinity and selectivity. It is currently being evaluated clinically as a monotherapy and in combination with chemotherapies and other immunotherapies in a variety of solid tumors. Here we demonstrate that efficacy from a murine version of DKN-01 (mDKN-01) in a syngeneic tumor model requires NK cells. mDKN-01 also reduces lung metastasis, further supporting a model where DKK1 neutralization stimulates a NK mediated anti-cancer response. In addition, DKN-01 has efficacy in a human prostate xenograft model that expresses high levels of DKK1 and retains functioning NK cells. Taken together, these data support an innate immunotherapeutic mechanism of action of DKN-01 and suggest that tumor expression of DKK1 may predict response. As such, evaluation of peripheral blood and tumor biopsies from patients undergoing treatment with DKN-01 in combination with chemotherapy or other immunotherapies are underway to demonstrate innate immune mediated anti-tumor activity and validate DKK1 expression as a biomarker. Citation Format: Michael Haas, Heidi Heath, Franziska Schurpf-Huber, Yinyuan Wu, Xinjun Zhang, Xi He, Min Yang, Walter Newman, Michael Kagey. Mechanistic insight into anti-tumor activity of DKN-01, a therapeutic antibody with immune modulatory activity [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 2404.

Introduction: At 52.5% identity in their extracellular domains (ECDs), mouse and human GITR (CD357) sequences are only modestly conserved. Thus transitioning promising murine anti-tumor activity data with the anti-GITR DTA-1 and 2F8 Mabs to clinical trials with the non-crossreactive anti-human GITR Mab TRX518 poses a considerable limitation. Here we investigated three elements of the GITR mouse and human antibodies to address this limitation: 1) the functional epitopes targeted by anti-human GITR (TRX-518) and anti-murine GITR (DTA-1 and 2F8) antibodies; 2) their biological activities on primary T cells; and 3) the role of anti-GITR Fc receptor engagement. Results and Conclusions: Alanine scanning, amino acid substitutions and flow cytometry experiments were performed to define critical residues important for the binding of all three Mabs to their respective GITR targets. Swaps of 5-8 of these critical residues showed both the loss of murine and the gain of human directed Mab binding when residues critical for TRX18 binding were substituted into murine GITR. Likewise, the substitution of residues critical for DTA-1/2F8 binding into human GITR resulted in loss of TRX518 binding and the gain of 2F8/DTA-1 binding. We then mapped these critical residues to 3D structural models of the murine and human GITR ECDs built by contact-map guided fragment assembly simulations. Regions defined by these critical residues showed a high degree of overlap, indicating structural conservation even without amino acid identity. Thus the epitopes defined by both DTA-1 and 2F8 are homologous to those defined on human GITR by TRX518. In in vitro standard proliferation/suppression assays with effector T cells (Teff) and regulatory T cells (Tregs) isolated from human healthy donors and naïve mice respectively, TRX518, similarly to DTA-1, significantly increased proliferation, activation and pro-inflammatory cytokine production of human and murine Teff cultured alone or in the presence of Tregs. These observations indicate that Mabs directed to similar mouse and human GITR ECD epitopes result in similar biological activities. Lastly, we questioned the importance of a functional Fc domain in anti-GITR Mabs in order to translate these in vitro effects into anti-tumor activity in vivo. To this end, we compared the anti-tumor activity of DTA-1 and its aglycosyl version in the aggressive and poorly immunogenic B16 melanoma model. Both Fc and non-Fc binding DTA-1 were able to control tumor growth. This suggests that the aglycosyl-anti-GITR Mab TRX518 has the potential to be effective in the clinic despite the lack of Fc receptor engagement. These results align with our preliminary results in patients treated with TRX518 monotherapy showing reductions in Treg frequencies both in tumor biopsies and in the periphery. TRX518 would therefore appear to be well-poised for use in the treatment of solid tumor malignancies. Note: This abstract was not presented at the meeting. Citation Format: Roberta Zappasodi, Heidi Heath, Yang Zhang, Michael Haas, Min Yang, Christopher Mirabelli, Jedd D. Wolchok, Cyndi Sirard, Walter Newman, Taha Merghoub. GITR cancer immunotherapy: Epitope swapping of anti-GITR TRX518 to inform functional translatability from mouse to human [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 2402.

Immune checkpoint blockade has evidenced the therapeutic activity of modulating T-cell co-inhibition/co-stimulation processes. However, many patients are refractory to these therapies, highlighting the need for developing additional forms of immunotherapy targeting alternative immune pathways. In this regard, the T-cell co-stimulatory receptor glucocorticoid-induced TNFR-related protein (GITR, TNFRSF18) is an attractive target for agonist antibodies (Abs). By promoting effector T-cell (Teff) function and hampering regulatory T-cell (Treg) suppression, GITR engagement may exert a dual positive effect on anti-tumor immune responses. We and others have reported potent antitumor effects of anti-GITR Abs in preclinical mouse models. Based on this rationale, we initiated the first-in-human phase-I trial of GITR stimulation with the GITR agonist monoclonal Ab (mAb) TRX518 (NCT01239134). TRX518 is a humanized aglycosylated IgG1κ mAb that binds and stimulates human GITR without engaging Fc effector functions. Here, we report the immune effects of a single ascending dose of TRX518 monotherapy in advanced cancer patients and provide mechanistic preclinical evidence to rationally combine GITR agonism with checkpoint blockade in future clinical trials. Analysis of peripheral blood mononuclear cells (PBMCs) from 37 advanced refractory solid cancer patients treated with >/= 0.005 mg/kg TRX518 (cohorts 3-9) revealed frequent reductions in circulating Tregs after treatment, with GITR+ Tregs and activated CD45RA-Foxp3hi effector Tregs (eTregs) being preferentially affected. In 8 patients for whom pre- and post-treatment PBMC samples and tumor biopsies were available, reductions in intratumor and circulating Tregs after TRX518 were positively correlated. However, coincident down-regulation of circulating and intratumor Tregs upon TRX518 was not sufficient to achieve a clinical benefit. To clarify the mechanisms underlying this outcome, we modeled tumor sensitivity and refractoriness to anti-GITR therapy by treating B16F10-melanoma-bearing mice with the mAb DTA-1 on day 4 (curative regimen, early tumors) or day 7 (refractory regimen, advanced/established tumors) after tumor implantation respectively. Time course analysis of T-cell infiltrates revealed that intratumor Tregs were significantly reduced and Teff:Treg ratios increased in both responding and refractory tumors. However, in responding tumors, Tregs completely failed to accumulate, suggesting that the presence of Tregs during tumor formation and progression could affect T-cell functionality. Gene expression analysis of intratumor CD8+ T-cells showed up-regulation of activation/memory T-cell markers and down-regulation of exhaustion markers in responding but not in refractory tumors. To overcome resistance to anti-GITR, we thus combined the anti-GITR refractory regimen (day 7 treatment) with PD-1 blockade starting on day 7 after tumor implantation. This combination controlled tumor growth similar to the curative anti-GITR monotherapy (day 4 treatment) and achieved 50% long-lasting complete response. This was associated with more activated and less exhausted profiles of intratumor CD8+ T-cells, which showed enhanced anti-tumor cytotoxicity compared to CD8+ T-cells from nonresponding tumors treated with each agent alone. These results indicate for the first time that Treg reduction may be a pharmacodynamic biomarker of anti-GITR therapy in patients. However, Treg elimination from advanced tumors is not sufficient to activate cytotoxic CD8+ T-cell responses unless the T-cell exhaustion process is concurrently blocked. This underscores the need to combine Treg-inhibiting/depleting immunotherapies with strategies able to counteract exhaustion, such as PD-1 blockade, to regress advanced tumors. Based on these observations, we have initiated a clinical trial exploring TRX518 in combination with PD-1 pathway blockade in patients with advanced solid tumor malignancies (NCT02628574). Citation Format: Roberta Zappasodi, Cynthia Sirard, Yanyun Li, Sadna Budhu, Moshen Abu-Akeel, Cailian Liu, Xia Yang, Hong Zhong, Walter Newman, Jinjin Qi, Phillip Wong, David Schaer, Henry Koon, Vamsidhar Velcheti, Michael Postow, Margaret K Callahan, Jedd D. Wolchok, Taha D. Merghoub. Mechanistic rationale to combine GITR agonism with PD-1 blockade in cancer patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr PR01.

Background— Acute coronary syndrome is the leading cause of mortality worldwide. However, treatment of acute coronary occlusion inevitably results in ischemia-reperfusion injury. Circulating natural IgM has been shown to play a significant role in mouse models of ischemia-reperfusion injury. A highly conserved self-antigen, nonmuscle myosin heavy chain II, has been identified as a target of pathogenic IgM. We hypothesized that a monoclonal antibody (m21G6) directed against nonmuscle myosin heavy chain II may inhibit IgM binding and reduce injury in a preclinical model of myocardial infarction. Thus, our objective was to evaluate the efficacy of intravenous m21G6 treatment in limiting infarct expansion, troponin release, and left ventricular dysfunction in a swine myocardial infarction model. Methods and Results— Massachusetts General Hospital miniature swine underwent occlusion of the midleft anterior descending coronary artery for 60 minutes, followed by 1 hour, 5-day, or 21-day reperfusion. Specificity and localization of m21G6 to injured myocardium were confirmed using fluorescently labeled m21G6. Treatment with m21G6 before reperfusion resulted in a 49% reduction in infarct size ( P P P Conclusions— Treatment with m21G6 significantly reduced infarct size and troponin-T release, and led to marked preservation of cardiac function in our study. Overall, these findings suggest that pathogenic IgM blockade represents a valid therapeutic strategy in mitigating myocardial ischemia-reperfusion injury.

Stimulation of glucocorticoid-induced tumor necrosis factor receptor (GITR) has been shown to enhance antitumor immunity by stimulating effector CD4+ and CD8+ T cells and attenuating suppression and depleting by CD4+Foxp3+ regulatory T cells (Treg). However, GITR monotherapy does not effectively control tumor growth. We hypothesize that cyclophosphamide (CTX), a cytotoxic chemotherapeutic agent with key immunomodulatory properties, can enhance the potency of GITR engagement anti-tumor effects. Further, we hypothesize that CTX can induce neoantigen formation by increasing tumor mutation burden. In this study, we administered high-dose CTX (250 mg/kg) followed the next day by an agonist anti-GITR antibody (DTA-1) to C56BL/6 mice bearing poorly immunogenic B16 melanoma tumors. We found that the combination therapy can cause regression of established tumors whereas the single agents did not. Mechanistically, we found that CTX administration results in tumor antigen release, which enhances the antitumor T cell response after treatment with DTA-1. This effect is lost when lower doses of CTX are given or CTX is administered prior to tumor inoculation, suggesting that tumor cell death and cross-presentation of tumor antigens to T cells is necessary. Furthermore, Treg cells are an important target of combination therapy. Intratumoral Treg levels were substantially reduced in treated mice, which greatly increased the ratio of T effector/Treg in the tumor. In conclusion, CTX and agonist anti-GITR antibody slows tumor progression and can induce regression of palpable, poorly immunogenic B16 melanoma tumors, likely by enhancing cross-priming and increasing the T effector/Treg ratio in the tumor. Thus, this combination warrants further investigation as a novel immunotherapy approach and provides rational for exploring combination of GITR agonist antibodies with chemotherapeutic treatments in clinical settings. Citation Format: Allison Betof Warner, Daniel Hirschhorn, Adam Cohen, Cailian Liu, Walter Newman, Cyndi Sirard, Jedd Wolchok, Taha Merghoub. Treatment with agonist anti-GITR antibody after chemotherapy enhances tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1699.

Wnt signaling is a fundamental pathway that is dysregulated in oncology. The Wnt signaling modulator DKK1 is expressed in a variety of tumor types and elevated levels frequently correlate with poor survival. DKK1 promotes tumor growth by stimulating proliferation, metastasis, and angiogenesis, and has been implicated in contributing to an immune suppressive tumor microenvironment. DKN-01 is a humanized monoclonal therapeutic antibody that binds DKK1 with high affinity and selectivity. It is currently being evaluated clinically as a monotherapy and in combination in a variety of solid tumors. Here we describe further characterization of the mechanism of action of DKN-01 and demonstrate immune mediated anti-tumor activity in nonclinical models. A murine version of DKN-01 (mDKN-01) has efficacy in a syngeneic melanoma B16 tumor model. However, mDKN-01 is unable to impede B16 tumor growth in NSG immunodeficient mice, indicating that a functioning immune system is required for antibody activity. Furthermore, preliminary data suggest that mDKN-01 is targeting a myeloid derived suppressor cell population in the tumor microenvironment. These data support an immune mediated mechanism of action of DKN-01 and provide a rationale for clinical development in combination with immunotherapy agents. The first clinical study evaluating DKN-01 in combination with pembrolizumab has initiated enrollment in patients with relapse/refractory esophagogastric malignancies. Citation Format: Michael Haas, Heidi Heath, Yinyuan Wu, Xinjun Zhang, Cynthia Sirard, Xi He, Walter Newman, Michael Kagey. DKN-01, a therapeutic DKK1 neutralizing antibody, has immune modulatory activity in nonclinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1710.

Severe sepsis, a lethal syndrome after infection or injury, is the third leading cause of mortality in the United States. The pathogenesis of severe sepsis is characterized by organ damage and accumulation of apoptotic lymphocytes in the spleen, thymus, and other organs. To examine the potential causal relationships of apoptosis to organ damage, we administered Z-VAD-FMK, a broad-spectrum caspase inhibitor, to mice with sepsis. We found that Z-VAD-FMK–treated septic mice had decreased levels of high mobility group box 1 (HMGB1), a critical cytokine mediator of organ damage in severe sepsis, and suppressed apoptosis in the spleen and thymus. In vitro, apoptotic cells activate macrophages to release HMGB1. Monoclonal antibodies against HMGB1 conferred protection against organ damage but did not prevent the accumulation of apoptotic cells in the spleen. Thus, our data indicate that HMGB1 production is downstream of apoptosis on the final common pathway to organ damage in severe sepsis.

Background: The tumor necrosis factor receptor GITR is expressed at high levels on regulatory T cells (Tregs) and up-regulated on activated T cells. Evidence from preclinical studies has shown the dual activity of GITR stimulation to counteract Tregs functionally and quantitatively and enhance T-cell effector functions, thus providing the rationale to develop GITR agonist antibodies for cancer immunotherapy. These agents have demonstrated potent therapeutic effects associated with both Treg reduction and modulation in tumor-bearing mice. Here we investigate Treg modulation as a biomarker of activity of the fully humanized agonist anti-GITR antibody TRX-518 in advanced solid cancer patients and the potential mechanisms underlying this effect. Methods: Patients were accrued to 9 cohorts (up to 6 patients/cohort) to receive a single dose of TRX518 (dose range: 0.0001-8 mg/kg). Pharmacodynamic analyses included flow cytometric evaluation of frequency and phenotype of circulating T cells at different time points up to 12 weeks after treatment. Relevant changes observed with these analyses were monitored in pre- and post-treatment tumor biopsies by immunofluorescence staining. Effects of GITR stimulation with TRX-518 on human Tregs were studied in in vitro cultures with CD4+ T cells from healthy donors. Results: Analysis of peripheral blood mononuclear cells from 37 advanced refractory solid cancer patients treated with >=0.005 mg/kg TRX-518 (cohorts 3-9) revealed frequent reduction in circulating Tregs after treatment with TRX-518. Proliferation potential of Tregs was not altered by TRX-518, as indicated by their persistent Ki67 expression. We then asked whether a subset of Tregs was preferentially affected and found that GITR+ Tregs and CD45RA-Foxp3hi effector Tregs (eTregs) were specifically targeted by TRX-518 in a dose-dependent manner. Accordingly, we observed that eTregs express GITR at higher levels than naïve CD45RA+Foxp3+ Tregs. In a subset of patients for whom pre- and post-treatment tumor biopsies were available (n=8; 3 melanoma, 2 lung, 2 colorectal and 1 bladder cancer patients), we tested whether intra-tumor Tregs, which are typically enriched in eTregs, were also affected. Noteworthy, we observed that changes in intra-tumor and circulating Tregs after TRX-518 were positively correlated. As a potential mechanism responsible for this effect, our initial in vitro analyses indicate that GITR stimulation with TRX-518 destabilizes Treg phenotype by down-regulating Foxp3 and enhancing expression of the prototype Th1-lineage transcription factor T-bet. This suggests that TRX-518 may favor Treg to effector-T-cell conversion, thus explaining the effect of Treg loss observed in patients. Conclusions: Circulating Treg reduction is a potential pharmacodynamic biomarker of TRX518 biological activity. This parameter may allow predictive correlation with changes in intratumoral Treg infiltration. We plan to further investigate this effect and its relevance for the association with clinical responses in our recently opened TRX518 multi-dose study. Clinical trial information: NCT01239134. Citation Format: Roberta Zappasodi, Yanyun Li, Mohsen Abu-Akeel, Jingjing Qi, Philip Wong, Cynthia Sirard, Michael Postow, David A. Schaer, Walter Newman, Henry Koon, Vamsidhar Velcheti, Margaret K. Callahan, Jedd D. Wolchok, Taha Merghoub. Intratumor and peripheral Treg modulation as a pharmacodynamic biomarker of the GITR agonist antibody TRX-518 in the first in-human trial [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 CT018. doi:10.1158/1538-7445.AM2017-CT018

OBJECTIVE HIV-1 infection in humans has been reported to lead to a shift in the cytokine balance, with a relative decrease in T helper 1 type cytokines, especially IL-2. On the basis of the expression of CD45RA, in combination with homing markers CD62L or alpha4beta7, T helper cells can be sub-divided into naive, activated naive, central memory and effector memory cells as well as gut-homing subpopulations. In addition, each subset may have the potential to express distinct cytokines. At present it is unclear whether the changes in cytokine expression observed in HIV-1-infected individuals are secondary to changes within the composition of CD4 T cell subsets or are caused by changes in cytokine expression within each subset. MATERIALS AND METHODS A new technique was developed to detect cytokine expression in phorbol 12-myristate 13-acetate/ionomycin-activated CD62L and alpha4beta7-expressing CD4 T cell subsets, using the protease inhibitor KD-IX-73-4. RESULTS In SIV-infected macaques that develop AIDS a marked decrease in IL-2 expression was found within central, effector, or gut-homing memory cell subsets, whereas the expression of IL-2 in naive T cell subsets remained unaffected. This reduced IL-2 expression by memory cells and not a loss of the frequency of CD4 memory cells accounted for the reduced expression of IL-2 by CD4 T cells during SIV infection. CONCLUSION As defined by the cell surface markers utilized, it appears that progression to AIDS is associated with functional impairment of memory cells, but not changes in lymphocyte circulation patterns.

Barley contains high levels of soluble dietary fibre, including mixed linked 1→3, 1→4β-D-glucans (β-glucan). An extract of β-glucan from waxy, hulless barley containing 56% total dietary fibre (TDF) was incorporated into flour tortillas, cornstarch pudding and apple granola bars to provide 2 g soluble fibre as β-glucan per serving. The foods were tested for objective functional properties. Flour tortillas with β-glucan were incorporated into rat diets and compared to diets containing an equivalent amount of cellulose, to test the fibre effect on growth and lipid metabolism parameters. Rats fed β-glucan tortillas had lower feed consumption and body weight (P 0·05). Plasma LDL-cholesterol of rats fed β-glucan was lower (P 0·05). Rats fed β-glucan tortillas had higher (P

The CC chemokine eotaxin, originally purified from bronchoalveolar lavage fluid of sensitized guinea pigs following allergen challenge, is a potent eosinophil-selective chemoattractant. In the present study, we have used 111In-eosinophils and human eotaxin to characterize the profile of chemokine-induced eosinophil accumulation in vivo in rat skin. Intradermally injected eotaxin caused a dose-dependent accumulation of 111In-eosinophils. Time course studies indicated that the response was rapid, since all the accumulation occurred within the first 1 to 2 h of eotaxin injection. The i.v. administration of anti-intercellular adhesion molecule-1, anti-vascular cell adhesion molecule-1, or anti-α4 integrin mAbs significantly inhibited the eosinophil accumulation induced by 100 pmol of human eotaxin by 73, 43, and 67%, respectively. Further, when 111In-eosinophils were pretreated in vitro with anti-α4 integrin or anti-β2 integrin mAbs, or with a combination of both mAbs, eotaxin-induced responses in vivo were reduced by 52, 49, and 68%, respectively. Eosinophil accumulation induced by intradermal IL-4, but not that induced by TNF-α or leukotriene B4, appeared to be mediated in part by endogenously generated eotaxin. Anti-eotaxin Abs significantly inhibited (54%) the later phases (24–28 h) but not the early phase (0–4 h) of the response to IL-4. This was consistent with eotaxin mRNA expression peaking at 18 h after IL-4 injection. Our findings show that human eotaxin is a potent inducer of eosinophil accumulation in vivo, this response being dependent on α4 integrin/vascular cell adhesion molecule-1 and β2 integrin/intercellular adhesion molecule-1 adhesion pathways. Further, the eosinophil accumulation in response to IL-4 is partly mediated by endogenously generated eotaxin.

The glucose and lipid responses of barley diets were investigated with chicks based on either flour or red dog (RD) from each of three barley cultivars, Glacier, covered high-amylose Glacier (HAG) and hull-less high-amylose Glacier (HHAG), with two maize control diets. Mean time interval blood glucose levels were higher (P < 0.05) for chicks fed uncooked barley flour or RD, compared with controls. Total and LDL plasma cholesterol were lower (P < 0.05) for chicks fed the HAG and HHAG flour and all barley RD diets. HDL cholesterol values for chicks fed Glacier and HHAG flour and HHAG RD diets were higher than controls. The LDL/HDL cholesterol ratios for chicks fed HAG and HHAG flour and all barley RD diets were lower (P < 0.05) than controls. Lower body weights were observed in all chickens fed barley diets. The results suggest that the soluble fibre, mainly β-glucan and starch structures, in barley may be responsible for the hypocholesterolaemic effect.

Chemokine receptors are coupled to G-proteins and their activation results in prominent changes in cell migration and growth. The downstream signaling pathways that mediate these effects of chemokines are largely uncharacterized. Macrophage inflammatory protein 1β (MIP 1β) binding to its cognate receptor CCR5 resulted in activation of the related adhesion focal tyrosine kinase (RAFTK), with subsequent activation of the cytoskeletal protein paxillin and the downstream transcriptional activators, c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) and p38 mitogen-activated protein (MAP) kinase. Inhibition of RAFTK by a dominant-negative kinase mutant markedly attenuated JNK/SAPK activity. Thus, RAFTK appears to provide a functional “bridge” for the transmission of CCR5 receptor signaling to the cytoskeleton and nucleus, primary sites of chemotaxis and growth regulation.

Forty adult rats were used in an experiment to evaluate the effect of feeding a high viscosity barley milling fraction on plasma and liver lipids and intestinal content viscosity. Shonkin barley was milled into 14 fractions utilizing an 8-roller dry mill. The fraction selected for this experiment was classified as break shorts containing 8.4% β-glucan. Rats were randomly allotted to diets containing 0.5% cholesterol and barley shorts at either 0% (control), 30, 60, or 90% of the diet. Experimental duration was 21 d at which time rats were sacrificed, small intestine contents removed and viscosities determined. The animals were not fasted prior to sacrifice. Response criteria measured were plasma lipids and glucose, liver lipids, wet digesta weight and viscosity of intestinal contents. Plasma lipids and glucose were not different (P > .05) for rats fed any of the diets. There were no differences in liver weights among groups. Rats fed different levels of shorts had significantly lower (P

Barley, oats, and wheat were stored anaerobically for three months after adjusting the dry matter content to 60%. The content of true protein was reduced when grains were high‐moisture stored, but the amino acid content was not affected to the same extent. High‐moisture storage decreased the soluble dietary fibre and soluble β‐glucan content in barley and oats, and simultaneously the extract viscosity of the grain was reduced to the same level as for wheat. In two experiments with two to five week old broiler chickens given diets with 70% dried or high‐moisture stored grain, feed:gain ratio was reduced (P < 0.05) to the same level as for the wheat diets when high‐moisture stored barley instead of dried barley was fed, and digestibility of dry matter, organic matter, fat, and minerals increased (P < 0.05). Weight gain and cleanness of cages also increased (P < 0.05), and the relative size of the digestive tract was reduced (P < 0.05). Digestibility of dry matter, organic matter, and minerals increased (P

The cholesterol-lowering effect of barley and more specifically the contribution of barley fiber and barley oil to cholesterol-lowering was studied in hamsters. This was achieved by comparing a semi-purified diet containing cellulose and peanut oil (control) to a whole barley diet and barley diets from which barley fiber, barley oil, or barley oil and barley fiber were removed. The barley fiber was removed enzymatically and the oil was extracted with hexane. The cholesterol-lowering effect of the barley oil was also tested by adding it in place of peanut oil to the control semi-purified diet. Plasma triglycerides and HDL cholesterol levels were not influenced by dietary treatments. Total plasma cholesterol was lower (P Copyright © 1996 Elsevier Science Inc.

Chemokine receptors serve as coreceptors for HIV entry into CD4+ cells. Their expression is thought to determine the tropism of viral strains for different cell types, and also to influence susceptibility to infection and rates of disease progression. Of the chemokine receptors, CCR5 is the most important for viral transmission, since CCR5 is the principal receptor for primary, macrophage-tropic viruses, and individuals homozygous for a defective CCR5 allele (delta32/delta32) are highly resistant to infection with HIV-1. In this study, CCR5-specific mAbs were generated using transfectants expressing high levels of CCR5. The specificity of these mAbs was confirmed using a broad panel of chemokine receptor transfectants, and by their non-reactivity with T cells from delta32/delta32 individuals. CCR5 showed a distinct pattern of expression, being abundant on long-term activated, IL-2-stimulated T cells, on a subset of effector/memory T cells in blood, and on tissue macrophages. A comparison of normal and CCR5 delta32 heterozygotes revealed markedly reduced expression of CCR5 on T cells from the heterozygotes. There was considerable individual to individual variability in the expression of CCR5 on blood T cells, that related to factors other than CCR5 genotype. Low expression of CCR5 correlated with the reduced infectability of T cells with macrophage-tropic HIV-1, in vitro. Anti-CCR5 mAbs inhibited the infection of PBMC by macrophage-tropic HIV-1 in vitro, but did not inhibit infection by T cell-tropic virus. Anti-CCR5 mAbs were poor inhibitors of chemokine binding, indicating that HIV-1 and ligands bind to separate, but overlapping regions of CCR5. These results illustrate many of the important biological features of CCR5, and demonstrate the feasibility of blocking macrophage-tropic HIV-1 entry into cells with an anti-CCR5 reagent.

For efficient entry into target cells, primary macrophage-tropic and laboratory-adapted human immunodeficiency viruses type 1 (HIV-1) require particular chemokine receptors, CCR-5 and CXCR-4, respectively, as well as the primary receptor CD4 (refs 1-6). Here we show that a complex of gp120, the exterior envelope glycoprotein, of macrophage-tropic primary HIV-1 and soluble CD4 interacts specifically with CCR-5 and inhibits the binding of the natural CCR-5 ligands, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta (refs 7, 8). The apparent affinity of the interaction between gp120 and CCR-5 was dramatically lower in the absence of soluble CD4. Additionally, in the absence of gp120, an interaction between a two-domain CD4 fragment and CCR-5 was observed. A gp120 fragment retaining the CD4-binding site and overlapping epitopes was able to interact with CCR-5 only if the V3 loop, which can specify HIV-1 tropism and chemokine receptor choice, was also present on the molecule. Neutralizing antibodies directed against either CD4-induced or V3 epitopes on gp120 blocked the interaction of gp12O-CD4 complexes with CCR-5. These results suggest that HIV-1 attachment to CD4 creates a high-affinity binding site for CCR-5, leading to membrane fusion and virus entry.

BACKGROUND & AIMS: Integrins play diverse roles in cellular actions and signalling in the immune system. In the context of mucosal immune responses, the integrin alpha 4 beta 7 has received particular attention because of its intimate involvement in lymphocyte recruitment to normal gastrointestinal mucosa and associated lymphoid tissue. The aim of this study was to determine the functional relevance of alpha 4 beta 7 in the pathogenesis of colonic inflammatory disease using the colitic cotton-top tamarin, an animal model of human ulcerative colitis. METHODS: Chronically colitic cotton-top tamarins were given either a cross-reactive monoclonal antibody to human alpha 4 beta 7 or an irrelevant control monoclonal antibody. The animals were then evaluated clinically and mucosal biopsy specimens assessed by histological and quantitative morphometric analysis. RESULTS: A blocking monoclonal antibody to alpha 4 beta 7 integrin ameliorated inflammatory activity and rapidly improved stool consistency when administered to chronically colitic animals. Furthermore, using morphometric analysis of biopsy specimens, antibody therapy reduced the mucosal density of alpha 4 beta 7+ lymphocytes and alpha 4 beta 7 neutrophils and macrophages. CONCLUSIONS: These results suggest that the alpha 4 beta 7 integrin represents a novel, potentially organ- specific therapeutic target for the treatment of inflammatory bowel disease. (Gastroenterology 1996 Nov;111(5):1373-80)

The chemokine eotaxin is unusual in that it appears to be a highly specific chemoattractant for eosinophils. Ligand-binding studies with radiolabeled eotaxin demonstrated a receptor on eosinophils distinct from the known chemokine receptors CKR-1 and -2. The distinct eotaxin binding site on human eosinophils also bound RANTES (regulated on activation T expressed and secreted) and monocyte chemotactic protein (MCP)3. We have now isolated a cDNA from eosinophils, termed CKR-3, with significant sequence similarity to other well characterized chemokine receptors. Cells transfected with CKR-3 cDNA bound radiolabeled eotaxin specifically and with high affinity, comparable to the binding affinity observed with eosinophils. This receptor also bound RANTES and MCP-3 with high affinity, but not other CC or CXC chemokines. Furthermore, receptor transfectants generated in a murine B cell lymphoma cell line migrated in transwell chemotaxis assays to eotaxin, RANTES, and MCP-3, but not to any other chemokines. A monoclonal antibody recognizing CKR-3 was used to show that eosinophils, but not other leukocyte types, expressed this receptor. This pattern of expression was confirmed by Northern blot with RNA from highly purified leukocyte subsets. The restricted expression of CKR-3 on eosinophils and the fidelity of eotaxin binding to CKR-3, provides a potential mechanism for the selective recruitment and migration of eosinophils within tissues.

Soluble adhesion protein intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule (E-selectin) were measured in serum and cerebrospinal fluid (CSF) of patients with relapsing-remitting multiple sclerosis (RRMS) in remission and in exacerbation, as well as patients with chronic progressive MS, stable MS, and in patients with other neurological and inflammatory diseases (ONDs). Serum ICAM-1 and E-selectin were significantly elevated in patients with MS over those with ONDs and controls. CSF VCAM-1 and E-selectin were found to be elevated over control and disease control samples. No increase in CSF ICAM-1 was observed. Results were analyzed longitudinally and by MS category. In paired CSF and serum samples from patients in exacerbation, elevated VCAM-1 correlated with increased serum VCAM-1 in 5 of 7 patients. Elevated CSF E-selectin did not correlate with elevations in serum E-selectin.

The demonstration of soluble isoforms of adhesion molecules has added a layer of complexity of our understanding of lymphoid-endothelial cell interactions. This is especially true in the light of observations which show levels of these isoforms to be raised during disease processes. Here, Andrew Gearing and Walter Newman review the evidence that increased levels of circulating, soluble adhesion molecules may be a key to understanding the prognosis and pathology of certain diseases.

Using a panel of anti-CD3-TCR monoclonal antibodies (OKT3 A-E), it appears possible to separate the ability to cause surface antigen modulation from inhibition of MLR or induction of mitosis. OKT3D, an antibody that recognizes the CD3 antigen at a site that can be differentiated from the epitopes recognized by other members of this panel by competition binding, does not cause antigen modulation when incubated with human T cells for up to 3 days. Despite this, OKT3D is mitogenic and is capable of blocking MLR. Two different isotypes were produced from the OKT3D clone, IgG1 and IgG2b. The IgG2b isotype of OKT3D blocked MLR even in individuals unable to respond mitogenically to this antibody. Use of members of this panel may now permit dissection of the types of signals delivered by the CD3-TCR complex inducing mitosis, receptor modulation, and other T-cell responses.

Cytokines such as interleukin 1 (IL-1) promote adhesiveness in human umbilical vein endothelial cells for leukocytes including basophils, eosinophils, and neutrophils, and induce expression of adherence molecules including ICAM-1 (intercellular adhesion molecule-1), ELAM-1 (endothelial-leukocyte adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1). In the present study, blocking monoclonal antibodies (mAb) recognizing ICAM-1, ELAM-1, and VCAM-1 have been used to compare their roles in IL-1-induced adhesion of human basophils, eosinophils, and neutrophils. IL-1 treatment of endothelial cell monolayers for 4 hours induced a four- to eight-fold increase in adhesion for each cell type. Treatment of endothelial cells with either anti-ICAM-1 or anti-ELAM-1 mAb inhibited IL-1-induced adherence of each cell type. In contrast, treatment with anti-VCAM-1 mAb inhibited basophil and eosinophil (but not neutrophil) adhesion, and was especially effective in blocking eosinophil adhesion. The effects of these mAb were at least additive. Indirect immunofluorescence and flow cytometry demonstrated expression of VLA-4 alpha (very late activation antigen-4 alpha, a counter-receptor for VCAM-1) on eosinophils and basophils but not on neutrophils. These data document distinct roles for ICAM-1, ELAM-1, and VCAM-1 during basophil, eosinophil, and neutrophil adhesion in vitro, and suggest a novel mechanism for the recruitment of eosinophils and basophils to sites of inflammation in vivo.

T cell adhesion to endothelium is critical to lymphocyte recirculation and influx into sites of inflammation. We have systematically analyzed the role of four receptor/ligand interactions that mediate adhesion of peripheral human CD4+ T cells to cultured human umbilical vein endothelial cells (HUVEC): T cell LFA-1 binding to ICAM-1 and an alternative ligand ("ICAM-X"), T cell VLA-4 binding to VCAM-1, and T cell binding to ELAM-1. Contributions of these four pathways depend on the activation state of both the T cell and HUVEC, and the differentiation state of the T cell. ELAM-1 plays a significant role in mediating adhesion of resting CD4+ T cells to activated HUVEC. LFA-1 adhesion dominates with PMA-activated T cells but the strength and predominant LFA-1 ligand is determined by the activation state of the HUVEC; while ICAM-1 is the dominant ligand on IL-1-induced HUVEC, "ICAM-X" dominates binding to uninduced HUVEC. Adhesion via VLA-4 depends on induction of its ligand VCAM-1 on activated HUVEC; PMA activation of T cells augments VLA-4-mediated adhesion, both in the model of T/HUVEC binding and in a simplified model of T cell adhesion to VCAM-1-transfected L cells. Unlike LFA-1 and VLA-4, ELAM-1-mediated adhesion is not increased by T cell activation. Differential expression of adhesion molecules on CD4+ T cell subsets understood to be naive and memory cells also regulates T/HUVEC adhesion. Naive T cell adhesion to HUVEC is mediated predominantly by LFA-1 with little or no involvement of the VLA-4 and ELAM-1 pathways. In contrast, memory T cells bind better to HUVEC and utilize all four pathways. These studies demonstrate that there are at least four molecular pathways mediating T/HUVEC adhesion and that the dominance/hierarchy of these pathways varies dramatically with the activation state of the interacting cells and the differentiation state of the T cell.