anti-CD4 mAb Research Articles

A phase 1 dose-escalation study of a PD-L1xCD27 bispecific antibody CDX-527 in patients with advanced malignancies.

2585 Background: CDX-527 is a bispecific antibody (BsAb) targeting PD-L1 and CD27 that is designed to block immune checkpoint PD-L1/PD-1 interactions while providing immune costimulation through CD27 signaling. CD27 is a key immunostimulatory molecule that enhances T cell activation, effector function, and survival. Combining anti-PD-L1 and anti-CD27 mAbs synergize in preclinical studies, activating complementary cytotoxic and proliferative gene expression profiles, respectively. Clinical studies demonstrated the safety and biological activity of combining varlilumab, an agonist anti-CD27 mAb, with nivolumab or atezolizumab, along with modest clinical activity of the combinations. CDX-527 is a novel human BsAb containing a neutralizing, high affinity IgG1k PD-L1 mAb and the single chain Fv fragment (scFv) of an agonist anti-CD27 mAb genetically attached to the C-terminus of each heavy chain, thereby making CDX-527 bivalent for each target. Pre-clinical studies demonstrated enhanced T cell activation by CDX-527 and anti-tumor activity of a surrogate bispecific compared to individual mAb combinations. Methods: CDX527-01 is a phase 1 first-in-human, open-label, multi-center, dose-escalation (DE) and expansion study evaluating safety, pharmacokinetics (PK), pharmacodynamics (PD), and clinical activity of CDX-527 in patient with advanced solid tumors that have progressed on standard-of-care therapy. The primary study objective is to characterize the safety and tolerability of CDX-527. CDX-527 is administered intravenously Q2W with doses ranging from 0.03 mg/kg up to 10.0 mg/kg or until the maximum tolerated dose. The first 2 cohorts of the DE phase initiate with single patients and subsequent DE cohorts will be conducted in 3+3 manner. Tumor-specific expansion cohorts may be enrolled to further characterize the safety, PK, PD, and efficacy of CDX-527. Tumor assessments are performed Q8W by the investigator per iRECIST. Biomarker assessments include characterizing the effects on peripheral blood immune cells and cytokines, and for the expansion cohorts, the impact of CDX-527 on the tumor microenvironment in paired tumor biopsies. Results: To date, 8 patients have received CDX-527 in doses ranging from 0.03 mg/kg to 1 mg/kg and 3 are still on treatment. There has been no drug related SAEs, DLTs or discontinuations due to an AE. Most common treatment related AEs were influenza-like illness, fatigue, and arthralgia (all at 25%). All drug related AEs have been grade 1 or 2. Conclusions: Preliminary results indicate that the novel anti-PD-L1xCD27 bispecific antibody CDX-527 up to and including the 1 mg/kg dose level has been well tolerated. Additional data will be presented, including the safety profile at higher dose levels along with clinical activity, as well as PK and PD data. Clinical trial information: NCT04440943.

Diversity of draining lymph node-tumor overlapping T cell receptor repertoire reflects the anti-tumor effect of cancer immunotherapies

Abstract CD8+ tumor-infiltrating lymphocytes, the main effector cells of anti-tumor immune responses, are composed of various T cell clones. Numerous studies have demonstrated the presence of tumor-reactive T cell clones with high frequency in tumors. However, it remains unclear as to which of the two mechanisms, the expansion of limited tumor-specific clones or the induction of diverse tumor-reactive ones, is more beneficial for the action of immune checkpoint inhibitors (ICIs). To address this question, we performed a T cell receptor repertoire analysis on B16F10 and on Lewis lung carcinoma tumor-bearing mice treated with immunotherapy including anti-PD-L1 monoclonal antibody (mAb) and anti-CD4 depletion mAb, which enhance the expansion of tumor-reactive CD8+ T cells. We focused on the T cell clones that overlapped between the tumor and draining lymph node (dLN) to analyze the T cell clones that were mobilized into the anti-tumor response after treatment. In anti-PD-L1 and anti-CD4 treated mice, the total frequency of CD8+ dLN-tumor overlapping clones in the tumor was increased. Surprisingly, although the expansion of the top10 overlapping clones did not differ, ICI treatment increased the total frequency of the 11th or lower overlapping clones, indicating the broadening of the tumor-reactive T cell repertoire. Moreover, the administration of FTY720 to tumor-bearing mice, which inhibits egress of T cells from the LNs to circulation, reduced the total frequency of the 11th or lower overlapping clones in the tumor and reduced the long-term anti-tumor effect of anti-PD-L1 treatment. These results suggest that mobilization of diverse tumor-reactive clones is essential for enhancing and persistent anti-tumor effect of ICIs.

Mechanism of differentiation and unique repertoire of IgM secreting B-1 plasma cells

Abstract Natural antibodies are broadly reactive, mostly IgM antibodies that provide a first line of defense against pathogens. Distinct populations of fetal/neonatal-derived IgM-secreting B-1 cells, including non-terminally differentiated B-1 and B-1 plasma cells (PC) are present in bone marrow (BM) and spleen. B-1PC are transcriptionally and phenotypically similar to foreign antigen-induced, long-lived conventional PC, prompting us to hypothesize that both receive similar differentiation signals. To determine the extent to which natural IgM-producing B-1PC depend on T cell help, we studied TCRβδ−/− mice lacking either all T cells, or depleted of CD4 T cells by anti-CD4 mAb treatment. Both showed significantly reduced numbers of BM but not spleen B-1PC and slightly increased populations of B-1 cells. Consistent with those data, reconstitution of RAG-deficient mice with B-1 and CD4+ T cells, but not with B-1 cells alone, restored the BM B-1PC compartment and significantly enhanced serum IgM levels. B-1PC did not bind to PtC-containing liposomes and depletion of B-1 PC had minimal effect on the number of anti-PtC IgM ASC. In contrast, most anti-influenza natural IgM ASC were B-1PC, consistent with reduced serum levels influenza-binding natural IgM in TCRβδ−/− mice. Together, the study reveals that in contrast to previous assumptions, some natural IgM production is dependent on CD4 T cell help, suggesting that multiple mechanisms regulate induction of natural IgM secretion.

Using Selective Bcl2 Inhibition to Induce Cardiac Allograft Tolerance

<h3>Purpose</h3> Cardiac allograft tolerance in non-human primates (NHPs) has been achieved by our group using a mixed chimerism model with allogeneic bone marrow transplant after non-myeloablative conditioning but not without kidney co-transplantation. B cell lymphoma 2 (Bcl2) inhibition has allowed for durable mixed chimerism with reduced conditioning in the murine skin transplant model. We investigated whether the Bcl2 inhibitor ABT199 could enhance mixed chimerism and induce cardiac allograft tolerance in the absence of kidney cotransplantation. <h3>Methods</h3> Fifteen cynomolgus recipients underwent mixed chimerism conditioning (ATGAM, total body and thymic irradiation) then organ and bone marrow transplantation. Group 1 recipients received heart alone (n=5), Group 2 recipients received heart and kidney cotransplants (n=7), and in Group 3, heart recipients received ABT199 in lieu of kidney cotransplants (n=3).Group 1 and 2 received 3Gy total body irradiation (TBI) while Group 3 received 1.5Gy TBI. All recipients underwent a 28-day course of immunosuppression with a calcineurin inhibitor and anti-CD154 mAb after which all immunosuppression was stopped. <h3>Results</h3> All five Group 1 recipients demonstrated cellular rejection (ISHLT 3R) by 96 days post bone marrow transplant (pBMTx). In contrast, no Group 2 recipients showed early rejection and 6/7 survived >400 days pBMTx. The first two Group 3 recipients developed GvHD and succumbed by days 59 and 68 pBMTx without evidence of rejection. However, after reducing the size of the donor BMT, the third recipient is now 76 days pBMTx with excellent allograft contractility and no evidence of GVHD. Mean peak lymphoid chimerism was 10% on day 26 pBMTx for Group 1, 26% on day 33 pBMTx for Group 2, and 57% on day 54 pBMTx for Group 3. (Figure 1) <h3>Conclusion</h3> After further refinement of the conditioning regimen, ABT199 may allow for the induction of heart allograft tolerance without the need for kidney co-transplantation and with a reduction in TBI thereby improving safety.

Effects of IL-2 and/ or Anti-IL6R Therapy on Long-Term Cardiac Allograft Survival in Non-Human Primates

Purpose Cardiac allograft tolerance in non-human primates (NHPs) has been achieved by our group using a mixed chimerism model with allogeneic bone marrow transplant after non-myeloablative conditioning but not without kidney co-transplantation. Transplantation of the heart alone results in graft rejection within 150-180 days. Our results suggest that the ability of the kidney to confer tolerance on the co-transplanted heart relates to its ability to induce the expansion/activation of host regulatory T cells (Tregs). We investigated whether IL-2 and/ or anti-IL6R therapy could substitute for the kidney and induce cardiac allograft tolerance by augmenting Tregs. Methods Sixteen cynomolgus NHPs underwent heart and donor bone marrow transplant with mixed chimerism conditioning including total body and thymic irradiation, ATGAM, anti-CD154 mAb, and cyclosporine until post-operative day (POD) 28. Recipients in Group A (n=5) received low dose IL-2 therapy (1M IU/m2 SC, POD -6 to 5) and an anti-IL6R mAb (tocilizumab, 10 mg/kg IV, POD 0, 7, 14, 21, 28, 56, 84, 112). Recipients in Group B (n=11) received tocilizumab alone (10 mg/kg IV, POD 0, 7, 14, 21, 28, 56, 84). Results In Group A, 3/5 recipients rejected at POD 127, 198, and 254 with acute cellular rejection (ACR), antibody mediated rejection (AMR), and donor specific antibodies (DSA). 2/5 died due to sepsis (POD11) or technical complications (POD7). In Group B, 4/11 recipients are ongoing, of which 2 are >430 days post-transplant without ACR. 1/11 rejected at POD 621 but only after donor skin transplant on POD 526. 1/11 failed to develop chimerism and rejected at POD 169. Graft failure/ death occurred in 5/11 due to technical or sedation complications (POD3, POD5 [n=2], POD9) and pancytopenia (POD65). Group A recipients exhibited an average 15-fold expansion in percent CD25+FoxP3+ of peripheral CD3+CD4+ cells compared to an average 3-fold expansion in Group B. Conclusion Despite its ability to promote Tregs, combined IL-2/ aIL6R treatment failed to achieve long-term allograft survival. We surmise that this was due to IL-2 stimulation of alloreactive memory T cells. In contrast, aIL6R mAb treatment alone achieved long-term allograft survival (>1 year) off immunosuppression in 3 recipients and represents a promising strategy towards clinical cardiac allograft tolerance.

Antibody-mediated depletion of viral reservoirs is limited in SIV-infected macaques treated early with antiretroviral therapy.

The effectiveness of virus-specific strategies, including administered HIV-specific mAbs, to target cells that persistently harbor latent, rebound-competent HIV genomes during combination antiretroviral therapy (cART) has been limited by inefficient induction of viral protein expression. To examine antibody-mediated viral reservoir targeting without a need for viral induction, we used an anti-CD4 mAb to deplete both infected and uninfected CD4+ T cells. Ten rhesus macaques infected with barcoded SIVmac239M received cART for 93 weeks starting 4 days after infection. During cART, 5 animals received 5 to 6 anti-CD4 antibody administrations and CD4+ T cell populations were then allowed 1 year on cART to recover. Despite profound CD4+ T cell depletion in blood and lymph nodes, time to viral rebound following cART cessation was not significantly delayed in anti-CD4-treated animals compared with controls. Viral reactivation rates, determined based on rebounding SIVmac239M clonotype proportions, also were not significantly different in CD4-depleted animals. Notably, antibody-mediated depletion was limited in rectal tissue and negligible in lymphoid follicles. These results suggest that, even if robust viral reactivation can be achieved, antibody-mediated viral reservoir depletion may be limited in key tissue sites.

A cell-penetrating CD40-TRAF2,3 blocking peptide diminishes inflammation and neuronal loss after ischemia/reperfusion.

While the administration of anti‐CD154 mAbs in mice validated the CD40‐CD154 pathway as a target against inflammatory disorders, this approach caused thromboembolism in humans (unrelated to CD40 inhibition) and is expected to predispose to opportunistic infections. There is a need for alternative approaches to inhibit CD40 that avoid these complications. CD40 signals through TRAF2,3 and TRAF6‐binding sites. Given that CD40‐TRAF6 is the pathway that stimulates responses key for cell‐mediated immunity against opportunistic pathogens, we examined the effects of pharmacologic inhibition of CD40‐TRAF2,3 signaling. We used a model of ischemia/reperfusion (I/R)‐induced retinopathy, a CD40‐driven inflammatory disorder. Intravitreal administration of a cell‐penetrating CD40‐TRAF2,3 blocking peptide impaired ICAM‐1 upregulation in retinal endothelial cells and CXCL1 upregulation in endothelial and Müller cells. The peptide reduced leukocyte infiltration, upregulation of NOS2/COX‐2/TNF‐α/IL‐1β, and ameliorated neuronal loss, effects that mimic those observed after I/R in Cd40−/− mice. While a cell‐penetrating CD40‐TRAF6 blocking peptide also diminished I/R‐induced inflammation, this peptide (but not the CD40‐TRAF2,3 blocking peptide) impaired control of the opportunistic pathogen Toxoplasma gondii in the retina. Thus, inhibition of the CD40‐TRAF2,3 pathway is a novel and potent approach to reduce CD40‐induced inflammation, while likely diminishing the risk of opportunistic infections that would otherwise accompany CD40 inhibition.

Long‐term porcine islet graft survival in diabetic non‐human primates treated with clinically available immunosuppressants

Although pancreatic islet transplantation is becoming an effective therapeutic option for patients with type 1 diabetes (T1D) who suffer from a substantially impaired awareness of hypoglycemia, its application is limited due to the lack of donors. Thus, pig-to-human islet xenotransplantation has been regarded as a promising alternative due to the unlimited number of "donor organs." Long-term xenogeneic islet graft survival in pig-to-non-human primate (NHP) models has mainly been achieved by administering the anti-CD154 mAb-based immunosuppressant regimen. Since the anti-CD154 mAb treatment has been associated with unexpected fatal thromboembolic complications in clinical trials, the establishment of a new immunosuppressant regimen that is able to be directly applied in clinical trials is an urgent need. We assessed an immunosuppressant regimen composed of clinically available agents at porcine islet transplantation in consecutive diabetic NHPs. Porcine islet graft survival in consecutive diabetic NHPs (n=7; >222, >200, 181, 89, 62, 55, and 34 days) without severe adverse events. We believe that our study could contribute greatly to the initiation of islet xenotransplantation clinical trials.

Effective control of Staphylococcus aureus lung infection despite tertiary lymphoid structure disorganisation.

Tertiary lymphoid structures (TLS) are triggered by persistent bronchopulmonary infection with Staphylococcus aureus, but their roles remain elusive. The present study sought to examine the effects of B- and/or T-cell depletion on S. aureus infection and TLS development (lymphoid neogenesis) in mice. C57Bl/6 mice were pre-treated with 1) an anti-CD20 monoclonal antibody (mAb) (B-cell depletion) or 2) an anti-CD4 and/or an anti-CD8 mAb (T-cell depletion) or 3) a combination of anti-CD20, anti-CD4 and anti-CD8 mAbs (combined B- and T-cell depletion) or 4) isotype control mAbs. After lymphocyte depletion, mice were infected by intratracheal instillation of agarose beads containing S. aureus (106 CFU per mouse). 14 days later, bacterial load and lung inflammatory cell infiltration were assessed by cultures and immunohistochemistry, respectively. 14 days after S. aureus-bead instillation, lung bacterial load was comparable between control and lymphocyte-depleted mice. While TLS were observed in the lungs of infected mice pre-treated with control mAbs, these structures were disorganised or abolished in the lungs of lymphocyte-depleted mice. The absence of CD20+ B-lymphocytes had no effect on CD3+ T-lymphocyte infiltration, whereas CD4+/CD8+ T-cell depletion markedly reduced CD20+ B-cell infiltration. Depletion of CD4+ or CD8+ T-cells separately had limited effect on B-cell infiltration, but led to the absence of germinal centres. TLS disorganisation is not associated with loss of infection control in mice persistently infected with S. aureus.

Novel Approaches to Improve Myeloma Cell Killing by Monoclonal Antibodies.

The monoclonal antibodies (mAbs) have significantly changed the treatment of multiple myeloma (MM) patients. However, despite their introduction, MM remains an incurable disease. The mAbs currently used for MM treatment were developed with different mechanisms of action able to target antigens, such as cluster of differentiation 38 (CD38) and SLAM family member 7 (SLAMF7) expressed by both, MM cells and the immune microenvironment cells. In this review, we focused on the mechanisms of action of the main mAbs approved for the therapy of MM, and on the possible novel approaches to improve MM cell killing by mAbs. Actually, the combination of anti-CD38 or anti-SLAMF7 mAbs with the immunomodulatory drugs significantly improved the clinical effect in MM patients. On the other hand, pre-clinical evidence indicates that different approaches may increase the efficacy of mAbs. The use of trans-retinoic acid, the cyclophosphamide or the combination of anti-CD47 and anti-CD137 mAbs have given the rationale to design these types of combinations therapies in MM patients in the future. In conclusion, a better understanding of the mechanism of action of the mAbs will allow us to develop novel therapeutic approaches to improve their response rate and to overcome their resistance in MM patients.

IN VIVO ANTI-CD45RB ANTIBODY-INDUCED REGULATORY T CELLS DEMONSTRATE SPECIES-SPECIFIC TOLERANCE TO DISCORDANT HUMAN XENOGENIC ISLETS

Organ transplantation-Sichuan Youth Science and Technology Innovation Research Team. Background: Although islet xenotransplantation has been proven to be a promising strategy for treatment of type 1 diabetes, it’s application in clinical setting would have to overcome acute cellular rejection and challenging species barrier. The aim of this study was to determine the role of Foxp3+ regulatory T cells (Treg) in anti-CD45RB induced dominant and specific tolerance to human islet cells in mice. Methods: One thousand and five hundred IEQ human islets were isolated from deceased donors and transplanted under the kidney capsule of B6 diabetic mice, B6µMT-/- mice and thymectomized µMT-/- mice. The recipients were then temporarily treated with low dose anti-CD45RB. CD4+Foxp3+Tregs were identified in the blood, spleen, draining lymph nodes and within the graft of tolerant and rejecting mice by FCM and immunohistochemistry. Additional anti-CD25 mAb was applied in the tolerant B6µMT-/-mice, which received another human islets simultaneously. The CD4+CD25+ Tregs sorted from the tolerant mice were adoptively transferred to diabetic Scid mice which were grafted with another human islets to assess the suppressive function. Results: In vitro, CD4+CD25-Foxp3-T cells which were activated with anti-CD3/anti-CD28 beads and cultured with anti-CD45RB demonstrated significant increase in Foxp3+ expression. In vivo, an increase of Foxp3+ T cells was observed in treated mice and more significant increase in tolerant mice, compared with a negligible increase in rejected mice. Anti-CD45RB alone allowed indefinite graft survival in 26.6% of B6 mice and 88.9% in µMT-/- mice, whereas none of xenografts were accepted in these mice treated simultaneously with anti-CD25. µMT-/-mice which were thymectomized and treated by anti-CD45 yielded 100% of grafts with long-term survival, which indicated that the transplant tolerance was thymus-derived Tregs independent. Moreover, the µMT-/-mice which tolerated to the first human islets accepted the re-transplanted second party of islets from another human, whereas the tolerance was hampered by anti-CD25 treatment. Adoptive cell transfer model showed the CD4+CD25+ Tregs from the mice tolerant to the first human islets prolonged survival of the second human islets, compared to natural Tregs (p < 0.05). Conclusions: These data suggest that anti-CD45RB-induced tolerance in islet xenotransplantation is mediated by Tregs expansion. These tolerant Tregs had the potent capacity to transfer dominant and species-specific tolerance. The National Natural Science Foundation of China (No. 81172832, 81771723). Sichuan Youth Science and Technology Foundation (2013JQ0020). Special Program for Sichuan Youth Science and Technology Innovation Research Team (2014TD0010).

Abstract 4538: A safe and potent agonist ADG106 targeting a unique epitope of CD137 with novel mechanism of actions

Abstract Antagonist antibodies targeting immune checkpoint co-inhibitory receptor such as PD-1/PD-L1 have achieved great clinical success in cancer immunotherapy. However, agonistic antibodies targeting the co-stimulatory T cell receptors in the TNFR superfamily, and CD137 in particular, has suffered significant setback due to either dose-dependent severe liver toxicity by Urelumab, or modest clinical anti-tumor activity by Utomilumab. ADG106, a safe and potent antibody targeting a unique epitope of CD137, is shown to hold the great promise to achieve much better efficacy and safety profile for immunotherapy in preclinical and phase 1 studies. ADG106, a fully human agonistic anti-CD137 IgG4 mAb, binds to a unique and conserved epitope with multiple cross-species reactivity, and overlaps with CD137 ligand binding site. This unique antibody acts in a novel mechanism with the following attributes: 1) it agonizes CD137 in a natural ligand-like fashion; 2) it blocks ligand to disable its reverse signaling; 3) it exhibits strong but specific Fc receptor mediated crosslinking in comparison with Utomilumab. ADG106 shows robust dose-dependent single agent anti-tumor activity in multiple syngeneic mouse tumor models and induces durable antigen-specific memory immunity that protects animals from the same re-challenged tumor cells. Consistent with the preclinical observations, ongoing phase I trial shows that the same antibody, ADG106, is well-tolerated in cancer patients in dose escalation from 0.03 to 3 mg/kg; and it also shows single agent activity on circulating T cell activation and clonal expansion in anti-tumor activity. Furthermore, ADG106 has also induced complete remission of the liver metastatic lesion in a lymphoma patient at 1mg/kg dose. Preclinical studies demonstrate that ADG106 enhances activation and inflammatory cytokine release of primed T cells alone or together with other immunomodulatory agents in vitro, as well as exhibits synergistic in vivo anti-tumor activity in combination with variety of other cancer therapies, including checkpoint inhibitors, chemotherapies and targeted agents. Mechanistic analyses suggest that ADG106 stimulates tumor infiltration and expansion of CD4+ and CD8+ T cells, thereby promoting the antitumor responses. These findings support that ADG106-boosted immune response could offer an effective but alternative solution for cancer immunotherapy in single and combination therapies, especially for non-responders to current PD-1/PD-L1 based immunotherapies. Citation Format: Guizhong Liu, Felix Du, Xiaohong She, Yi Zhu, Anthony W. Tolcher, Peter Luo. A safe and potent agonist ADG106 targeting a unique epitope of CD137 with novel mechanism of actions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4538.

Therapeutic Effects of Anti-Bone Morphogenetic Protein and Activin Membrane-Bound Inhibitor Treatment in Psoriasis and Arthritis.

The transforming growth factor β (TGFβ) inhibitor BAMBI (bone morphogenetic protein and activin membrane-bound inhibitor) has been shown to control differentiation of CD4+ T lymphocytes into either tolerogenic Treg cells or pathogenic Th17 cells, through the regulation of TGFβ and interleukin-2 (IL-2) signaling strength. The present study was undertaken to explore the potential beneficial effects of this strategy of pharmacologic inhibition using novel anti-BAMBI monoclonal antibodies (mAb) in different experimental murine models of chronic skin and joint inflammatory/autoimmune disease. Development of Saccharomyces cerevisiae mannan-induced psoriatic arthritis (MIP) (n = 18-30 mice per group), imiquimod-induced skin psoriasis (n = 20-30 mice per group), or type II collagen-induced arthritis (CIA) (n = 13-16 mice per group) was analyzed in a total of 2-5 different experiments with either wild-type (WT) or BAMBI-deficient B10.RIII mice that were left untreated or treated with mAb B101.37 (mouse IgG1 anti-BAMBI), a mouse IgG1 anti-TNP isotype control, anti-CD25, or anti-TGFβ mAb. Treatment of normal mice with IgG1 anti-BAMBI mAb clone B101.37 led to expansion of Treg cells in vivo, and had both preventive and therapeutic effects in mice with MIP (each P < 0.05 versus controls). The conferred protection against disease progression was found to be mediated by Treg cells, which controlled the activation and expansion of pathogenic IL-17-producing cells, and was dependent on the level of TGFβ activity. Furthermore, treatment with B101.37 mAb blocked both the development of skin psoriasis induced by imiquimod and the development of CIA in mice (each P < 0.05 versus controls). Finally, pharmacologic inhibition of BAMBI with the IgM anti-BAMBI mAb B143.14 also potentiated the suppressive activity of Treg cells in vitro (P < 0.001 versus controls). These results in murine models identify BAMBI as a promising new therapeutic target for chronic inflammatory diseases and other pathologic conditions modulated by Treg cells.

An Agonistic Anti-CD137 Antibody Disrupts Lymphoid Follicle Structure and T-Cell-Dependent Antibody Responses

SummaryCD137 is a costimulatory receptor expressed on natural killer cells, T cells, and subsets of dendritic cells. An agonistic monoclonal antibody (mAb) against CD137 has been used to reduce tumor burden or reverse autoimmunity in animal models and clinical trials. Here, we show that mice treated with an agonistic anti-CD137 mAb have reduced numbers of germinal center (GC) B cells and follicular dendritic cells (FDCs) in lymphoid tissues, which impair antibody responses to multiple T-cell-dependent antigens, including infectious virus, viral proteins, and conjugated haptens. These effects are not due to enhanced apoptosis or impaired proliferation of B cells but instead correlate with changes in lymphoid follicle structure and GC B cell dispersal and are mediated by CD137 signaling in CD4+ and CD8+ T cells. Our experiments in mice suggest that agonistic anti-CD137 mAbs used in cancer and autoimmunity therapy may impair long-term antibody and B cell memory responses.

Myeloid-derived suppressor cells expand after transplantation and their augmentation increases graft survival.

Myeloid-derived suppressor cells (MDSCs) expand in an inflammatory microenvironment such as cancer and autoimmunity. To study if transplantation induces MDSCs and these cells regulate allograft survival, C57BL/6 donor hearts were transplanted into BALB/c recipients and endogenous MDSCs were characterized. The effects of adoptive transfer of transplant (tx), tumor (tm), and granulocyte-colony stimulating factor (g-csf)-expanded MDSCs or depletion of MDSC were assessed. MDSCs expanded after transplantation (1.7-4.6-fold) in the absence of immunosuppression, homed to allografts, and suppressed proliferation of CD4 T cells in vitro. Tx-MDSCs differed phenotypically from tm-MDSCs and g-csf-MDSCs. Among various surface markers, Rae-1 expression was notably low and TGF-β receptor II was high in tx-MDSCs when compared to tm-MDSCs and g-csf-MDSCs. Adoptive transfer of these three MDSCs led to differential graft survival: control (6days), tx-MDSCs (7.5days), tm-MDSCs (9.5days), and g-csf-MDSCs (19.5days). In combination with anti-CD154 mAb, MDSCs synergistically extended graft survival from 40days (anti-CD154 alone) to 86days with tm-MDSCs and 132days with g-csf-MDSCs. Early MDSC depletion (day 0 or 20), however, abrogated graft survival, but late depletion (day 25) did not. In conclusion, MDSCs expanded following transplantation, migrated to cardiac allografts, prolonged graft survival, and were synergistic with anti-CD154 mAb.

Recipient expression of C-type lectin receptor Mincle promotes rapid T cell homeostatic recovery following antibody mediated lymphoablation

Abstract Antibody-mediated lymphocyte depletion is widely used in transplantation. We recently showed that T cell recovery in mouse heart allograft recipients treated with anti-thymocyte globulin (mATG) depends on B cells activated via CD40 and is mediated by B cell-derived IL-1β and IL-6. Mincle (Clec-4e) is a FcRγ-coupled C-type lectin receptor that binds ligands released by pathogens or damaged cells and signals through the Card-9 pathway. While Mincle functions are well characterized in myeloid cells, its significance in B cells remains to be determined. Our goal was to investigate the role of Mincle in initiating B cell cytokine production in lymphopenic heart allograft recipients. The NanoString and RT-PCR analyses of B cells showed that mATG depletion resulted in B cell up-regulation of Mincle and Card-9 gene expression. Flow cytometry confirmed cell surface Mincle expression in B cells from mATG-treated recipients but not from naïve mice. Treatment with anti-CD154 mAb in addition to mATG decreased the B cell expression of Mincle, IL-1β, IL-6 and TNF-α. Moreover, B cell IL-1β and TNF-α expression were reduced in mATG treated Mincle KO allograft recipients compared to WT controls. Most importantly, Mincle KO recipients treated with mATG had severely impaired T cell recovery and significantly prolonged allograft survival compared to WT recipients. We propose that B cells activation via CD40 results in up-regulation of cell surface Mincle. Endogenous ligands released from necrotic graft cells bind to Mincle thus inducing B cells to secrete proinflammatory cytokines and facilitate CD8+ T cell proliferation. These data identify Mincle as a novel therapeutic target to improve the efficacy of lymphoablation in allograft recipients.

Characterization of novel CD25 antibodies that deplete human regulatory T cells in humanized mouse model

Abstract Regulatory T cells (Tregs) are key players in the suppression of immune responses and in the maintenance of immune homeostasis. Due to their central role in pathogenesis of multiple conditions, Tregs are an attractive target for immunomodulatory drug development. Tregs exhibit an activated phenotype and share the expression of many activation antigens with activated conventional T cells (Tconv). Our goal in this project was to generate and characterize monoclonal antibodies (mAbs) that would specifically react with human Tregs. mAbs were raised in mice by immunization with purified, in vitro expanded human Tregs. In contrast to widely available anti-CD25 mAbs that equally bound both activated Tregs and activated Tconv, we identified several mAbs that bound CD25 on expanded Tregs, but failed to bind to CD25 on expanded Tconv. To determine whether a similar selective binding could be demonstrated in vivo, we used the xeno-GVHD model in which NOD-SCID-γc−/− (NSG) mice are engrafted with human peripheral blood mononuclear cells (PBMCs). Several of the mAbs bound to CD25 expressed on Tregs, but not to CD25 expressed on activated Tconv. To test the effect of in vivo administration of the Treg specific mAbs, one of the clones, 2B010, was selected and injected intravenously (i.v) 2 weeks after PBMCs engraftment. Injection of 2B010 resulted in markedly reduced Tregs frequencies and numbers, much less depletion of CD25+ activated Tconv, and no reduction of CD25+CD8+ T cells. Further studies will address the effect of these mAbs at reversing the Treg-mediated protection of xeno-GVHD and enhancing tumor immunity in reconstituted NSG recipients bearing transplanted tumors.

Vascularized composite allotransplantation combined with costimulation blockade induces mixed chimerism and reveals intrinsic tolerogenic potential.

Vascularized composite allotransplantation (VCA) has become a valid therapeutic option to restore form and function after devastating tissue loss. However, the need for high-dose multidrug immunosuppression to maintain allograft survival is still hampering more widespread application of VCA. In this study, we investigated the immunoregulatory potential of costimulation blockade (CoB; CTLA4-Ig and anti-CD154 mAb) combined with nonmyeoablative total body irradiation (TBI) to promote allograft survival of VCA in a fully MHC-mismatched mouse model of orthotopic hind limb transplantation. Compared with untreated controls (median survival time [MST] 8 days) and CTLA4-Ig treatment alone (MST 17 days), CoB treatment increased graft survival (MST 82 days), and the addition of nonmyeloablative TBI led to indefinite graft survival (MST > 210 days). Our analysis suggests that VCA-derived BM induced mixed chimerism in animals treated with CoB and TBI + CoB, promoting gradual deletion of alloreactive T cells as the underlying mechanism of long-term allograft survival. Acceptance of donor-matched secondary skin grafts, decreased ex vivo T cell responsiveness, and increased graft-infiltrating Tregs further indicated donor-specific tolerance induced by TBI + CoB. In summary, our data suggest that vascularized BM-containing VCAs are immunologically favorable grafts promoting chimerism induction and long-term allograft survival in the context of CoB.

CD137, an attractive candidate for the immunotherapy of lung cancer.

Immunotherapy has become a hotspot in cancer therapy in recent years. Several immune checkpoints inhibitors have been used to treat lung cancer. CD137 is a kind of costimulatory molecule that mediates T cell activation, which regulates the activity of immune cells in a variety of physiological and pathological processes. Targeting CD137 or its ligand (CD137L) has been studied, aiming to enhance anticancer immune responses. Accumulating studies show that anti‐CD137 mAbs alone or combined with other drugs have bright antitumor prospects. In the following, we reviewed the biology of CD137, the antitumor effects of anti‐CD137 Ab monotherapy and the combined therapy in lung cancer.

Inducing Donor MHC Chimerism with Bone Marrow Derived Exosomes in Non-Human Primates

Cardiac allograft tolerance in non-human primates (NHPs) has been achieved by our group via hematopoietic mixed chimerism consisting of an allogeneic bone marrow (BM) and kidney co-transplantation from the same donor after non-myeloablative conditioning. In this setting, T cell tolerance is believed to rely on expression of donor MHC molecules in the host's primary and secondary lymphoid organs. Here, we investigated whether donor MHC chimerism could be achieved using donor bone marrow derived extracellular vesicles (exosomes) instead of cells. Exosomes were isolated through serial ultracentrifugation from supernatants of donor BM cells cultured for 48 hours (expressing H38 MHC class I epitope). Two H38- cynomolgus macaque recipients received conditioning consisting of ATGAM and thymic irradiation, with or without total body irradiation (TBI) before a heart and kidney co-transplantation. Both recipients underwent a short course of immunosuppression with a calcineurin inhibitor and co-stimulation blockade with anti-CD154 mAb. A single dose of H38+ donor bone marrow derived exosomes was infused into H38- recipients on post-operative day 2. Image flow cytometry was used to detect the presence of recipient cells displaying donor MHC class I on their surface (cross-dressed) in the blood, thymus, spleen and lymph nodes. Infusion of donor bone marrow derived exosomes resulted in the presence of recipient peripheral blood leukocytes displaying donor MHC class I molecules in both animals. Flow cytometric analysis showed that MHC class I chimerism was restricted to the myeloid cell compartment. Tissues collected from the total body irradiated NHP showed that H38+ cross-dressed cells were also present in the lymph nodes, thymus, and spleen. In a separate study, a NHP recipient treated with an exosome protocol without TBI is currently 54 days post-transplantation with a well-functioning cardiac allograft. Donor MHC chimerism in the form of MHC cross-dressing can be achieved in a NHP model using donor bone marrow derived exosomes. These findings suggest that bone marrow derived exosomes could be used in place of whole bone marrow to induce allograft tolerance. The use of exosomes would allow for the reduction of recipient conditioning and would eliminate the risk of GVHD making this approach clinically safer than traditional BMT.