CD3 Antibodies Research Articles

Absence of Vasoactive Intestinal Peptide Synthesis in Recipients in Allogeneic Bone Marrow Transplant Increases Graft Versus Host Diseases

Introduction: Vasoactive intestinal polypeptide (VIP) is an immunosuppressive neuropeptide. Activation of donor T cells leading to graft-versus-host disease (GvHD) in allogeneic bone marrow transplant (allo-BMT) occurs via early post transplant interactions between donor T cells and host antigen presenting cells and leads to upregulation of immune co-inhibitory pathways, including expression of VIP in T cells and dendritic cells. We have previously shown VIP signaling favorably regulates the activation status and enhances the graft-versus-leukemia (GvL) activities of donor T cells (Li et al., Cancer Research 2016). The role of the VIP produced by host antigen presenting cells in regulating GvHD in allo-BMT is unknown. We tested the hypothesis that absence of VIP expression by host cells would augment donor T cell activation and increase GvHD.Methods and Results: We used the B10BR->B6 MHC fully mismatched murine model of allogeneic BMT. Recipient wildtype (WT) and VIP knockout B6 (VIP-KO) mice were lethally irradiated with 11Gy and injected i.v. with 5×106 T cell depleted (TCD) bone marrow cells (BM) plus 0, 1 or 3 ×106 splenocytes from WT B10BR donors. Fifteen mice (males and females) were assigned to each splenocytes dosage group. The survival of recipients after BMT was recorded daily and the average clinical GvHD scores were obtained by combining weight-loss, activity, posture, fur texture and skin integrity data following standard published procedures. All three groups of WT recipients had better survival than VIP-KO recipients. VIP-KO mice transplanted with TCD BM alone had only 33.3% survival at day 75 compared with 73.3% survival among the WT recipients. Chimerism studies on day 10 post-BMT showed equivalent level of donor chimerism: 80% donor in WT recipients vs 72% donor in VIP-KO recipients, indicating increased mortality in the VIP-KO recipients was not due to early graft rejection. The addition of donor splenocytes to the graft resulted in significantly more GvHD-related mortality in VIP-KO recipients compared with WT recipients (Figure 1), with 80% vs 100% day 75 survival among recipients of 1x106 splenocytes (p=NS) and 0% vs 40% survival (p<0.01) among recipients of 3x106 donor splenocytes, respectively.To determine how the absence of VIP production in KO recipients modulates immune effector mechanisms that contributes to the differential survival rate post allo-BMT, we analyzed the recipients' splenocytes on day 30 post transplant with flow cytometry and staining with CD3, CD4, CD8, CD62L, CD44 and PD1 antibodies. VIP-KO recipients transplanted with 5M TCD BM plus 1M splenocytes had more CD4+ central memory and effector T cells, and CD8+ central memory and effector T cells. VIP-KO recipients transplanted with 5×106 TCD BM alone in addition had significantly more CD4 and CD8 effector T cells (p<0.01). VIP-KO recipients transplanted with 5×106 TCD BM plus 1x106 splenocytes had more PD1 expression on donor CD4+ central memory and effector T cells, and CD8+ central memory effector T cells, consistent with enhanced antigen-mediated activation.To visualize VIP produced by the host cells, we transplanted C57/BL6 recipients expressing a GFP transgene under the control of the VIP promoter (n=10) with 5×106 TCD BM plus 3 ×106 splenocytes from WT B10BR donors, and visualized VIP gene promoter activity as GFP expression in recipient lymphoid tissues and GvHD target organs. Only small number of CD45+ VIP-GFP+ cells were detected from in host liver, spleen and lymph nodes on day 10 post transplant (Figure 2), suggesting residual host leukocytes, particularly dendritic cells, expressed VIP.Conclusion: VIP production by recipient antigen presenting cells is a key pathway in limiting the activation of donor T cells. Physiological expression of VIP by host leukocytes regulate the GvHD activity of donor T cells. Novel methods to enhance VIP production in transplant recipients or the parenteral administration of VIP receptor agonists may limit GvHD, and may be particularly important in allo-BMT where GvL is not necessarily critical for success, such as transplants for hemoglobinopathy. [Display omitted] DisclosuresWaller:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; PRA: Consultancy; AMGEN: Consultancy; National Institutes of Health: Research Funding; Coulter Foundation: Research Funding; Helocyte: Consultancy; Cambium Medical Technologies: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Katz Foundation: Research Funding; Chimerix: Equity Ownership; Cerus: Equity Ownership; Celldex: Consultancy.

TNFAIP8 (TIPE) Deficiency Exacerbates Acute Graft Versus-Host Disease in Murine Model

Background: Acute graft-versus-host disease (GVHD) occurs after allogeneic hematopoietic cell transplant (alloHCT) and presents a donor immune cell response against host tissues associated with significant morbidity and mortality. Up to 60% of alloHCT recipients will develop acute GVHD. Damage to the gastrointestinal tract from the conditioning is a key step in its pathogenesis by allowing for systemic translocation of lipopolysaccharide (LPS) and other danger signals, hereby promoting host APC activation. Tumor necrosis factor (TNF)–induced protein 8 (TNFAIP8) family is a newly identified group of proteins consisting of TNFAIP8 (TIPE), TIPE1 (TNFAIP8L1, TNF–induced protein 8-like 1), TIPE2 (TNFAIP8L2), and TIPE3 (TNFAIP8L3). TIPE acts as a negative mediator of apoptosis via inhibition of caspase-3 activation and also induces proliferation. TIPE signaling is involved in activation of NF-kappa-B and AP-1 pathways. TIPE-deficient mice have been shown to develop more severe colitis than wild type mice due to TIPE deficiency in non-hematopoietic cells along with higher levels of TNF and IL-6. Given the potential roles of TIPE in regulating these processes and considering the importance of these cytokines and of epithelial injury in the pathophysiology of GVHD, we hypothesized, that TIPE deficiency of allogeneic HCT recipients results in worsened GVHD.Methods: 9 - 12 week old naive C57BL/6 or TIPE knockout C57BL/6 mice were conditioned with 1000cGy single dose TBI, followed by transplantation of 10 million bone marrow cells and 20 million splenocytes from either syngeneic C57BL/6 or allogeneic BALB/c donors. Animals were monitored for clinical GVHD and survival and analyzed at week 6 after HCT for serum cytokines, T cell expansion, whole spleen cell chimerism and cell proliferation marker.Results: Allogeneic wild type recipients demonstrated significantly better survival when compared to allogeneic TIPE knockout recipients by week 6 (66.7% vs 40.0%, p =0.024). All syngeneic animals survived. Clinical GVHD scores were significantly higher in TIPE-deficient allogeneic recipients at Day +28 (p=0.0166), +35 (p=0.0349), +42 (p=0.0241) compared to allogeneic wild type recipients. Day +42 serum cytokine analysis revealed increased IL-17A (p=0.0027), TNF (p=0.0396), IL-6 (p=0.0064) levels in TIPE-deficient allogeneic recipients compared to allogeneic control. This was associated with increased splenic CD4+ (p=0.04) but not CD8+ (p= 0.0898) cell expansion when compared to allogeneic control. TIPE-deficient and allogeneic control mice showed no differences in splenic donor T cell chimerism (84.4 versus 89.4, p=0.178), indicating comparable donor T cell engraftment. Ki-67 expression was significantly decreased in epithelial cells of small intestine of TIPE knockout allogeneic recipients when compared to allogeneic controls, and a similar nonsignificant trend was seen in the colon as well. Active caspase-3 expression was significantly increased in the small intestine of TIPE knockout allogeneic recipients when compared to allogeneic controls and a similar nonsignificant trend was seen in the colon as well. This observation was associated with significantly increased GI tract pathology scores of TIPE-deficient allogeneic recipients compared to allogeneic control. No differences were observed in pathology of lung and liver between TIPE-deficient allogeneic recipients and allogeneic control. Histopathologic analysis of T cell infiltration in small intestine and colon by immunohistochemistry for CD3 antibody revealed no difference between allogeneic groups, suggesting a role of TIPE-deficient nonhematopoietic cells in GVHD pathophysiology.Conclusion TIPE deficiency in allogeneic C57BL/6 recipients resulted in increased mortality after transplant, which was associated with significant increase in pro-inflammatory cytokines expression and decreased proliferation and increased apoptosis of intestinal epithelial cells, suggesting not only exaggerated inflammatory damage but also decreased epithelial regeneration. Therefore, TIPE seems to be a TNF receptor family member with rather protective function in GVHD gut pathophysiology and overexpression may be a potential future target to prevent or treat this complication after allo HCT. DisclosuresNo relevant conflicts of interest to declare.

T Cell Engagement without Cytokine Storm: A Novel Bcma x CD3 Antibody Killing Myeloma Cells with Minimal Cytokine Secretion

IntroductionMultiple myeloma (MM) is a plasma cell malignancy with an annual incidence of ~25,000. Improvements in standard of care have extended median survival to greater than 5 years; however, disease refractory to bortezomib and lenalidomide is still associated with a median life expectancy of less than 9 months. Novel therapies are critical to the treatment of these patients. Chimeric antigen receptor T cells (CAR-Ts) targeting B-cell maturation antigen (BCMA) -a protein found exclusively on the surface of plasma cells- have shown efficacy against relapsed/refractory MM in early phase clinical trials. However, toxicity from over-activation of T-cells and large-scale production of CAR-Ts still hinder this approach. Bispecific T-cell engaging antibodies redirecting T cells to BCMA circumvent the latter problem but to date have shown similar T-cell over-activation. Utilizing TeneoSeek, a next generation sequencing (NGS)-based discovery pipeline using in silico analysis of heavy chain only/fixed light chain antibody (HCA/Flic, respectively) sequences to enrich for antigen specific antibodies, we made a high affinity αBCMA HCA and a library of αCD3 Flic antibodies that show a >2 log range of EC50s for T cell activation in vitro . Of note, the library contained a low-activating αCD3 that induced T-cell dependent lysis of MM cells (when paired with an αBCMA HCA) with minimal cytokine secretion. To characterize the relative efficacy and potential therapeutic window of this unique molecule, we combined both a high- (TNB-384B) and the low-activating (TNB-383B) αCD3 moiety with a high-affinity bivalent αBCMA arm (αBCMA x αBCMA x CD3) and compared these constructs in vitro, in vivo, and ex vivo for T-cell activation, efficacy in killing MM cells, and toxicity.MethodsT-cell activation was measured via flow cytometry (CD69 expression) and cytokine ELISA (IL-2, INF-ɣ, TNFα, among others) in vitro. Lysis of MM cell lines (RPMI-8226, U266, and H929) was measured via flow cytometry in vitro . Ex vivo, survival of patient MM cells was measured via flow cytometry after incubation with TNB-383B, TNB-384B, or negative control antibody (NCA) with or without supplemental exogenous activated T cells. In vivo, NSG mice were engrafted with RPMI-8226 MM cells and human peripheral blood mononuclear cells (huPBMC), and the mice treated with TNB-383B, TNB-384B, or NCA. Tumor burden was evaluated via Bioluminescent Imaging (BLI). Pharmacokinetic (PK) studies were performed in mice and Cynomolgus monkeys using ELISA.ResultsBoth TNB-383B and TNB-384B bound to cell surface BCMA with nanomolar affinity. EC50s for cytotoxicity were in the single-digit nanomolar range for TNB-383B and single-digit picomolar for TNB-384B against MM cell lines in vitro . TNB-383B showed markedly reduced (ie IFN-ɣ) or absent (ie IL-2) cytokine release compared to TNB-384B even at saturating concentrations (Figure 1); these findings were consistent across 10 healthy huPBMC donors. Ex vivo, both TNB-383B and TNB-384B efficiently lysed primary MM cells in the presence and absence of supplementary T-cells. In vivo, both TNB-383B and TNB-384B mediated clearance of established MM tumor from NSG mice at doses as low as 10ng q1w x3 in the presence of huPBMC. PK profiles of both molecules were consistent with those of an IgG in mice (T1/2 ~5.5 days) and Cyno (T1/2 ~8.5 days). No activation of T-cells was observed in vitro (Figure 1) or in vivo in the absence of BCMA expressing target cells.ConclusionsBoth low-activating TNB-383B and high-activating TNB-384B αBCMA x αBCMA x αCD3 bispecific antibodies kill multiple myeloma cells in vitro and in vivo in a BCMA-dependent manner, and kill primary patient myeloma cells ex vivo . While TNB-384B showed T-cell activation comparable to other CD3-engaging bispecifics, TNB-383B induced significantly reduced cytokine secretion by T-cells without appreciable reduction of efficacy in vivo or ex vivo. In summary, both TNB-383B and TNB-384B show promise as MM therapeutics differentiated from current T-cell targeted therapies in clinical trials. [Display omitted] DisclosuresBuelow:TeneoBio: Employment. Pham:TeneoBio, Inc.: Employment. Dang:TeneoBio, Inc.: Employment. Pratap:TeneoBio, Inc.: Employment. Clarke:TeneoBio, Inc.: Employment. Harris:TeneoBio, Inc.: Employment. Jorgensen:TeneoBio, Inc.: Employment. Force Aldred:TeneoBio, Inc.: Employment. Schellenberger:TeneoBio, Inc.: Employment. Shah:Indapta Therapeutics: Other: Stock Ownership; Celgene: Research Funding; Celgene, Indapta Therapeutics, Takeda: Consultancy; TeneoBio, Inc.: Honoraria. Trinklein:TeneoBio: Employment. Wiita:Sutro Biopharma: Research Funding; TeneoBio, Inc.: Research Funding. Iyer:TeneoBio, Inc.: Employment. Van Schooten:TeneoBio, Inc.: Employment.

Tumor growth suppression by implantation of an anti-CD25 antibody-immobilized material near the tumor via regulatory T cell capture

ABSTRACT In this study, we designed and synthesized an implantable anti-CD25 antibody-immobilized polyethylene (CD25-PE) mesh to suppress tumor growth by removing regulatory T cells (Tregs). The PE mesh was graft-polymerized with poly(acrylic acid), and the anti-mouse CD25 antibody was then immobilized using the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide reaction. Immobilization of the antibody on the PE mesh was confirmed by immunostaining. The CD25-PE mesh could effectively and selectively capture CD25-positive cells through antigen-antibody interactions when the CD25-PE mesh was incubated with a suspension of mouse spleen cells, including CD25-positive cells. In addition, implantation of the CD25-PE mesh into mice subcutaneously demonstrated the Treg-capturing ability of the CD25-PE mesh with only a weak inflammatory reaction. In tumor-bearing mice, tumor growth was suppressed by subcutaneous implantation of the CD25-PE mesh near the tumor for 1 week. These results suggested that the anti-CD25 antibody-immobilized material could capture Tregs in vivo and inhibit tumor proliferation in a limited tumor-bearing mouse model. Further research is needed to facilitate cancer immunotherapy using implantable anti-CD25 antibody-immobilized material as a Treg-capturing device.

Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cell-homing mRNA-LNPs

Nucleoside-modified messenger RNA (mRNA)-lipid nanoparticles (LNPs) are the basis for the first two EUA (Emergency Use Authorization) COVID-19 vaccines. The use of nucleoside-modified mRNA as a pharmacological agent opens immense opportunities for therapeutic, prophylactic and diagnostic molecular interventions. In particular, mRNA-based drugs may specifically modulate immune cells, such as T lymphocytes, for immunotherapy of oncologic, infectious and other conditions. The key challenge, however, is that Tcells are notoriously resistant to transfection by exogenous mRNA. Here, we report that conjugating CD4 antibody to LNPs enables specific targeting and mRNA interventions to CD4+ cells, including Tcells. After systemic injection in mice, CD4-targeted radiolabeled mRNA-LNPs accumulated in spleen, providing ∼30-fold higher signal of reporter mRNA in Tcells isolated from spleen as compared with non-targeted mRNA-LNPs. Intravenous injection of CD4-targeted LNPs loaded with Cre recombinase-encoding mRNA provided specific dose-dependent loxP-mediated genetic recombination, resulting in reporter gene expression in about 60% and 40% of CD4+ Tcells in spleen and lymph nodes, respectively. Tcell phenotyping showed uniform transfection of Tcell subpopulations, with no variability in uptake of CD4-targeted mRNA-LNPs in naive, central memory, and effector cells. The specific and efficient targeting and transfection of mRNA to Tcells established in this study provides a platform technology for immunotherapy of devastating conditions and HIV cure.

OMIP 075: A 22-color panel for the measurement of antigen-specific T-cell responses in human and nonhuman primates.

OMIP 075: A 22-color panel for the measurement of antigen-specific T-cell responses in human and nonhuman primates.

Mathematical Modelling Based on In Vivo Imaging Suggests CD137-Stimulated Cytotoxic T Lymphocytes Exert Superior Tumour Control Due to an Enhanced Antimitotic Effect on Tumour Cells.

Simple SummaryCytotoxic T lymphocytes (CTLs) play an important role in controlling tumours, and an improved understanding of how they accomplish this will benefit immunotherapeutic cancer treatment strategies. Stimulation of CTLs by targeting their CD137 receptor is a strategy currently under investigation for enhancing responses against tumours, yet so far only limited quantitative knowledge regarding the effects of such stimulation upon CTLs has been obtained. Here, we develop mathematical models to describe dynamic in vivo two-photon imaging of tumour infiltrating CTLs, to characterise differences in their function either in the presence or absence of a CD137 agonist antibody. We showed that an increased antiproliferative effect and a more sustained presence of CTLs within the tumour were the most important effects associated with anti-CD137 treatment.Several immunotherapeutic strategies for the treatment of cancer are under development. Two prominent strategies are adoptive cell transfer (ACT) of CTLs and modulation of CTL function with immune checkpoint inhibitors or with costimulatory antibodies. Despite some success with these approaches, there remains a lack of detailed and quantitative descriptions of the events following CTL transfer and the impact of immunomodulation. Here, we have applied ordinary differential equation models to two photon imaging data derived from a B16F10 murine melanoma. Models were parameterised with data from two different treatment conditions: either ACT-only, or ACT with intratumoural costimulation using a CD137 targeted antibody. Model dynamics and best fitting parameters were compared, in order to assess the mode of action of the CTLs and examine how the CD137 antibody influenced their activities. We found that the cytolytic activity of the transferred CTLs was minimal without CD137 costimulation, and that the CD137 targeted antibody did not enhance the per-capita killing ability of the transferred CTLs. Instead, the results of our modelling study suggest that an antiproliferative effect of CTLs exerted upon the tumour likely accounted for the majority of the reduction in tumour growth after CTL transfer. Moreover, we found that CD137 most likely improved tumour control via enhancement of this antiproliferative effect, as well as prolonging the period in which CTLs were inside the tumour, leading to a sustained duration of their antitumour effects following CD137 stimulation.

Stem cell properties of GT101, a tumor infiltrating lymphocyte (TIL) product with multiple indications.

e14506 Background: Tumor infiltrating lymphocyte (TIL) is a kind of adoptive T cell therapy that has demonstrated clinical benefits in several types of solid tumors. GT101 is an autologous TIL therapy for the treatment of multiple solid tumors. Previous studies showed that in vitro expansion resulted in over 80% terminally differentiated effector T cells in the final TIL products, which may show good cytotoxicity but weak capability of persistence and proliferation in vivo. Herein we present phenotype and function characterization of our TIL product GT101 with a focus on its stemness attributes. Methods: We have produced more than 30 batches of GT101 TIL samples from 10 different cancer indications and analyzed their proliferation, cytokine production and phenotype at preREP and/or REP stage. Results: GT101 products keep a consistent T cell phenotype and good proliferation capacity, and are capable of secreting large amounts of cytokines after stimulation by anti-CD3/CD28 antibody, reflecting its good functionality. The residues in the final products, such as feeder cells and tumor cells, are below the detection limit. In contrast to equivalent TIL products previously reported across the world, we find that GT101 features a manufacturing process that preserves the stem cell characteristics of T cells. Analysis of all the 30 GT101 TIL samples demonstrates that the average percentage of central memory T cells (Tcm) and naïve T cells is 40-50% and 10-20% respectively in both CD8+ and CD4+ populations at the end of preREP stage. Meanwhile, CD8+TCF1+ cells are close to 50%, mainly in the form of PD-1-. In the phase of REP, the stemness of T cell is still well maintained—the average proportion of both Tcm and naïve T cells only decreases by about 5%, and that of CD8+TCF1+ cells down by about 10%. Conclusions: In general, GT101 demonstrates good stemness with a higher percentage of Tcm and TCF1+ than equivalent products previously reported across the world, suggesting better performance in persistence and proliferation in clinic.

Promising tolerability and efficacy results from dose-escalation in an ongoing phase Ib/II study of mosunetuzumab (M) with polatuzumab vedotin (Pola) in patients (pts) with relapsed/refractory (R/R) B-cell non-Hodgkin’s lymphoma (B-NHL).

7520 Background: Mosunetuzumab (M), a full-length, humanized, IgG1 bispecific antibody targeting CD20 and CD3, has shown promising efficacy and safety as monotherapy for R/R B-NHL (NCT02500407; Assouline, et al. ASH 2020). The combination of M with the anti-CD79b antibody-drug conjugate, Pola, showed synergistic anti-lymphoma activity in a mouse xenograft model. These data supported a Phase Ib/II, open-label, multicenter trial of M-Pola for R/R B-NHL (GO40516, NCT03671018). Here we present early clinical data from the Phase Ib cohort. Methods: Pts with R/R follicular lymphoma (FL, grade [Gr] 1–3a) or aggressive NHL (aNHL), including de novo diffuse large B-cell lymphoma (DLBCL), transformed FL (trFL) and FL Gr 3b (FL3b), received Cycle (C) 1 step-up doses of M on Day (D) 1 (1mg) and D8 (2mg), the target dose on C1D15, then continued at the target dose on C2D1 onwards. M was given every 21 days for eight cycles (or 17 cycles if stable disease or a partial response after C8). Pola (1.8mg/kg) was given with M on D1 of each cycle for six cycles. Results: As of November 17, 2020, 22 pts had received M-Pola (M target doses: 9mg, n=7; 20mg, n=3; 40mg, n=6; 60mg [with D1 dose of 30mg from C3 onwards], n=6). Pts had DLBCL (n=12), FL (n=3), FL3b (n=3) and trFL (n=4). Pt characteristics include: median age of 70 (38–81) years; median of 3 (1–10) prior lines of therapy; 7 (32%) pts had prior CAR-T therapy; 17 (77%) and 19 (86%) pts had disease refractory to last prior therapy and prior anti-CD20 therapy, respectively. Median follow-up duration was 9.6 (0.7–23.7) months. The most frequent treatment-related adverse events (AEs) were neutropenia (45.4%), fatigue, nausea and diarrhea (all 36.4%). Cytokine release syndrome (CRS) was observed in 2 pts (9.1%; both Gr 1 by ASTCT 2019 criteria). One dose-limiting toxicity (Gr 3 new onset atrial fibrillation) was observed in the 40mg cohort. The maximum tolerated dose was not exceeded. The most common Gr ≥3 and serious AEs were both neutropenia, observed in 8 (36.4%) and 3 (13.6%) pts, respectively. Two (9.3%) Gr 5 AEs occurred: sudden cardiac death (n=1) and respiratory failure (n=1); neither was deemed treatment related. No immune effector cell-associated neurotoxicity was observed. The Table shows preliminary efficacy data. Preliminary efficacy in the dose-escalation cohort. Conclusions: These data indicate that M-Pola has an acceptable safety profile, with no Gr ≥2 CRS observed, and promising efficacy in pts with R/R NHL with predominantly aggressive disease. The Phase II expansion cohort in R/R DLBCL is ongoing, with no mandatory hospitalization required. Clinical trial information: NCT03671018. [Table: see text]

Preliminary safety, pharmacokinetics (PK), pharmacodynamics (PD) and clinical efficacy of uliledlimab (TJ004309), a differentiated CD73 antibody, in combination with atezolizumab in patients with advanced cancer.

2511 Background: CD73 is implicated in tumor resistance to checkpoint immunotherapy (CPI) and plays a critical role in adenosine-mediated immune suppression. Uliledlimab, a differentiated CD73 antibody, inhibits the adenosine pathway in a non-competitive and unique intra-dimer binding mode. Uliledlimab suppresses tumor growth when combined with a PD-(L)1 inhibitor in multiple pre-clinical models. Methods: This 3+3 dose-escalation phase 1 study (NCT03835949) evaluated safety, tolerability, PK, PD and preliminary efficacy in cancer patients. Uliledlimab was administered intravenously at doses of 5, 10 or 15 mg/kg weekly (QW) or 15 or 20 mg/kg every 3 weeks (Q3W) alone in the first cycle and in combination with atezolizumab (1,200 mg Q3W) starting on week 4. Soluble CD73 in serum and CD73 receptor occupancy (RO) in circulating CD19+ B cells were measured. Expression of PD-L1, CD73 and A2A receptor was analyzed in baseline tumor specimens (n = 14). Tumor responses were assessed by RECIST/iRECIST. Results: As of 17 January 2021, 20 patients with advanced solid tumors were enrolled (M:F 8:12; mean age = 64; median prior regimens = 3 (range 1-9)). Uliledlimab was well-tolerated with no dose limiting toxicity. The most common treatment-related adverse events were first dose infusion related reactions (65%, n = 13) most commonly comprising chills/rigors, nausea, and vomiting (Grade 1 or 2) that resolved in subsequent infusions. PK appears linear at doses ≥ 10 mg/kg and modelling indicated a mean derived effective half-life of ̃19 days. Soluble CD73 was undetectable and complete RO was achieved in all patients after the first dose at ≥ 10 mg/kg. Anti-drug antibody was detected in 3/20 patients (15%). Among 13 efficacy-evaluable patients dosed at ≥ 10 mg/kg, complete response (CR = 1) and partial response (PR = 2) were observed in 3 patients (ORR = 23%) together with 3 stable disease (SD) patients (DCR = 46%). One PD-(L)1 inhibitor naïve patient with clear cell ovarian cancer achieved CR at 10 mg/kg QW and remains on study after 12 months. Two patients with NSCLC dosed at 15 mg/kg QW and 20 mg/kg Q3W, respectively, achieved PR. One patient failed nivolumab and the other received no prior PD-(L)1 inhibitor treatment. CD73 was expressed on 78% (mean) of malignant cells from archival tumor specimens in responders compared to 23% in non-responders. Conclusions: Uliledlimab is safe and well tolerated up to 20 mg/kg Q3W and 15 mg/kg QW. Full saturation of circulating and cell-bound CD73 was achieved at doses ≥ 10 mg/kg. Uliledlimab exhibited evidence of clinical activity in both PD-(L)1 treatment naïve and refractory cancer patients with high archival tumor expression of CD73. The results of this phase 1 study encourage further clinical investigation to evaluate the efficacy of uliledlimab in the treatment of solid tumors. Clinical trial information: NCT03835949.

Dendritic cell targeting with Fc-enhanced CD40 antibody agonists induces durable antitumor immunity in humanized mouse models of bladder cancer.

Intravesical immunotherapy using Bacille Calmette-Guérin (BCG) attenuated bacteria delivered transurethrally to the bladder has been the standard of care for patients with high-risk non-muscle-invasive bladder cancer (NMIBC) for several decades. BCG therapy continues to be limited by high rates of disease recurrence and progression, and patients with BCG-unresponsive disease have few effective salvage therapy options besides radical cystectomy, highlighting a need for new therapies. We report that the immune-stimulatory receptor CD40 is highly expressed on dendritic cells (DCs) within the bladder tumor microenvironment of orthotopic bladder cancer mouse models, recapitulating CD40 expression by DCs found in human disease. We demonstrate that local CD40 agonism in mice with orthotopic bladder cancer through intravesical delivery of anti-CD40 agonist antibodies drives potent antitumor immunity and induces pharmacodynamic effects in the bladder tumor microenvironment, including a reduction in CD8+ T cells with an exhausted phenotype. We further show that type 1 conventional DCs (cDC1) and CD8+ T cells are required for both bladder cancer immune surveillance and anti-CD40 agonist antibody responses. Using orthotopic murine models humanized for CD40 and Fcγ receptors, we demonstrate that intravesical treatment with a fully human, Fc-enhanced anti-CD40 agonist antibody (2141-V11) induces robust antitumor activity in both treatment-naïve and treatment-refractory settings, driving long-term systemic antitumor immunity with no evidence of systemic toxicity. These findings support targeting CD40-expressing DCs in the bladder cancer microenvironment through an intravesical agonistic antibody approach for the treatment of NMIBC.

PD-1 blockade prevents the progression of oral carcinogenesis.

Oral squamous cell carcinoma (OSCC) is one of the most common malignancies in the head and neck with a poor prognosis. Oral cancer development is a multistep process involving carcinogenesis. Though significant advances in cancer immunotherapy over the years, there is lack of evidence for T-cell exhaustion during oral carcinogenesis. Clinical specimens from healthy donors and patients diagnosed with oral leukoplakia (OLK) or OSCC were collected for immunohistochemical staining with PD-L1, CD86, CD8, PD-1 and CTLA-4 antibodies. Meanwhile, chemically induced mouse models of oral carcinogenesis were constructed with 4-nitroquinolone-N-oxide induction. Exhaustion status of T cells was measured by flow cytometry for spleens and by multiplex immunohistochemistry for formalin-fixed paraffin-embedded lesions in multiple stages of oral carcinogenesis. The efficacy of PD-1 blockade with or without cisplatin treatment was evaluated on the mice in precancerous and OSCC stages. We observed higher expression of PD-1 in the human OLK and OSCC tissues compared with the normal, while low expression CTLA-4 in all oral mucosa tissues. Animal experiments showed that the exhausted CD4+ T cells existed much earlier than exhausted CD8+ T cells, and an increased ratio of stem-like exhausted T cells and partially exhausted T cells were detected in the experimental groups. Besides, the expression of immune checkpoint markers (PDCD1, CTLA4 and HAVCR2) was strongly positively correlated with cytokines (IFNG and IL-2). In summary, T-cell exhaustion plays a vital role in oral carcinogenesis, and PD-1 blockade can prevent the progression of oral carcinogenesis.

Histopathological and immunohistochemical features of facial papules in frontal fibrosing alopecia.

Facial papules (FPs) are considered to be created by the inflammatory process, which involves facial vellus hairs, in frontal fibrosing alopecia. To demonstrate the histopathological features of FPs and the composition of the inflammatory infiltrate. In total, 18 patients with FPs were enrolled in the study after histopathological confirmation of lichen planopilaris. Histopathological evaluation of the specimens was performed by two dermatopathologists. The samples were immunostained with CD4, CD8 and CD123 monoclonal antibodies, and the percentage and proportion of cells stained with these markers were investigated. A follicular lichenoid reaction and perifollicular fibrosis were present in all cases. Vellus hairs were detected in 83.3% of biopsy specimens (15 cases), all of which were involved by the inflammation. The majority of the follicles (72%) revealed follicular plugs. Reduction and destruction of elastic fibres were visible in the perifollicular (adventitial) and the papillary dermis (100% and 78% of specimens, respectively). Prominent sebaceous glands and dilated ducts were detected in 78% and 72% of samples, respectively. CD4-positive T cells formed 67.72% and CD8-positive T cells 32.28% of the infiltrate, and the mean CD4/CD8 ratio was 2.48. In 13 (72.2%) biopsy specimens <10% of the infiltrate was positive for CD123 marker. Perifollicular inflammation, fibrosis and elastic-fibre destruction were constant histopathological features of FPs; furthermore, prominent sebaceous glands were present in the majority of samples. We also observed a CD4-positive predominance in the infiltrate.

Bispecific Antibody PD-L1 x CD3 Boosts the Anti-Tumor Potency of the Expanded Vγ2Vδ2 T Cells.

Vγ2Vδ2 T cell-based immunotherapy has benefited some patients in clinical trials, but the overall efficacy is low for solid tumor patients. In this study, a bispecific antibody against both PD-L1 and CD3 (PD-L1 x CD3), Y111, could efficiently bridge T cells and PD-L1 expressing tumor cells. The Y111 prompted fresh CD8+ T cell-mediated lysis of H358 cells, but spared this effect on the fresh Vδ2+ T cells enriched from the same donors, which suggested that Y111 could bypass the anti-tumor capacity of the fresh Vγ2Vδ2 T cells. As the adoptive transfer of the expanded Vγ2Vδ2 T cells was approved to be safe and well-tolerated in clinical trials, we hypothesized that the combination of the expanded Vγ2Vδ2 T cells with the Y111 would provide an alternative approach of immunotherapy. Y111 induced the activation of the expanded Vγ2Vδ2 T cells in a dose-dependent fashion in the presence of PD-L1 positive tumor cells. Moreover, Y111 increased the cytotoxicity of the expanded Vγ2Vδ2 T cells against various NSCLC-derived tumor cell lines with the releases of granzyme B, IFNγ, and TNFα in vitro. Meanwhile, the adoptive transferred Vγ2Vδ2 T cells together with the Y111 inhibited the growth of the established xenografts in NPG mice. Taken together, our data suggested a clinical potential for the adoptive transferring the Vγ2Vδ2 T cells with the Y111 to treat PD-L1 positive solid tumors.

Cord Blood T Cells Expressing High and Low PKCζ Levels Develop into Cells with a Propensity to Display Th1 and Th9 Cytokine Profiles, Respectively.

Low Protein Kinase C zeta (PKCζ) levels in cord blood T cells (CBTC) have been shown to correlate with the development of allergic sensitization in childhood. However, little is known about the mechanisms responsible. We have examined the relationship between the expression of different levels of PKCζ in CBTC and their development into mature T cell cytokine producers that relate to allergy or anti-allergy promoting cells. Maturation of naïve CBTC was initiated with anti-CD3/-CD28 antibodies and recombinant human interleukin-2 (rhIL-2). To stimulate lymphocyte proliferation and cytokine production the cells were treated with Phytohaemagglutinin (PHA) and Phorbol myristate acetate (PMA). Irrespective of the PKCζ levels expressed, immature CBTC showed no difference in lymphocyte proliferation and the production of T helper 2 (Th2) cytokine interleukin-4 (IL-4) and Th1 cytokine, interferon-gamma (IFN-γ), and influenced neither their maturation from CD45RA+ to CD45RO+ cells nor cell viability/apoptosis. However, upon maturation the low PKCζ expressing cells produced low levels of the Th1 cytokines, IFN-γ, IL-2 and tumour necrosis factor-alpha (TNF), no changes to levels of the Th2 cytokines, IL-4, IL-5 and IL-13, and an increase in the Th9 cytokine, IL-9. Other cytokines, lymphotoxin-α (LT-α), IL-10, IL-17, IL-21, IL-22 and Transforming growth factor-beta (TGF-β) were not significantly different. The findings support the view that low CBTC PKCζ levels relate to the increased risk of developing allergic diseases.

Adult T cell- leukemia/lymphoma associated with HTLV-1 presenting as giant mass of the face

A 30-year-old Arabic woman of low socio-economic status, without medical history of HIV infection, presented to our department for neglected giant mass of the face evolving for 2 years. She was treated for long time with herbal traditional treatments, which explains the delay of consultation. Physical examination revealed a bulky tumor, of reddish-brown color, occupying the entire face below the franc, more lateralised on the left side. The right eye had restricted movement, while the left eye was fixed (Panel A,B). CT scan showed a nasal cavity tumour with orbital and maxillary sinus invasion, without intra cranial extension or distant metastasis. Laboratory testing was positive for HTLV I/II and she was negative for HIV. Surgical biopsy with histo-pathological study established the diagnosis of HTLV-1 associated adult lymphoma/leukaemia, with strong CD4 and CD25 antibody staining. She received chemotherapy with CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) regimen, but she died after two cycles.

Immune signature of CCR7+ central memory T cells associates with disease severity and Immunoglobulin E in bronchial asthma.

Objective. CD4+T cell subtypes are the central orchestrators of airway inflammation in bronchialasthma (BA); however, the mechanisms that regulate their accumulation in asthmatic airways are still a challenging subject. In addition, neutrophils play a significant role in the development of airway remodeling and their presence may influence clinical presentation of BA being linked to the development of severe BA. Neutrophils have also been found to acquireantigen presenting functions, enabling them to directly activate T cells. The study aimed to evaluate the possible association of chemokine receptor 7 (CCR7)+ memory CD4+T cells andCCR4+ effector T cells with disease severity and immunoglobulin E (IgE) production as well as to explore the relationship between these cells and neutrophil function in both allergic andnon-allergic asthmatic patients. Methods. Flow cytometry was used to determine the expression of different T cell subset phenotypes (CCR7 memory CD4+ and CCR4+T cells using anti-human CD3, CD4, CD45RO, CCR4 and CCR7 monoclonal antibodies) utilizing peripheral blood mononuclear cells (PBMCs) isolated from 78 allergic asthmatic patients, 41 non-allergic asthmatic patients, and 40 healthy individuals. Moreover, neutrophils' phagocytic activity was assessed by ingestion of candida particles. Results. We demonstrated increased percentages of CCR7+ memory CD4+T cells and CCR4+CD4+T cells in patients compared to control, where this up regulation was significantly higher in allergic than non-allergic asthmatic patients. Additionally, these cells were negatively correlated with improved pulmonary tests and significantly associated with disease severity scores and IgE levels. The neutrophil phagocytic activity was markedly increased in patients compared to control, showing a significant positive correlation with disease severity. Conclusions. These findings suggest that increased CCR4+ CD4+ T cells and CCR7+ memory CD4+ T cells (Tcm) may be associated with BA severity, especially in allergic BA patients and can potentially contribute to the rational design of new therapeutic approaches for asthma in the future.

The capacity of CD4+ Vγ9Vδ2 T cells to kill cancer cells correlates with co-expression of CD56

Human Vγ9Vδ2 T cells are a unique T-cell type, and data from recent studies of Vγ9Vδ2 T cells emphasize their potential relevance to cancer immunotherapy. Vγ9Vδ2 T cells exhibit dual properties since they are both antigen-presenting cells and cytotoxic toward cancer cells. The majority of Vγ9Vδ2 T cells are double-negative for the co-receptors CD4 and CD8, and only 20–30% express CD8. Even though they are mostly neglected, a small fraction of Vγ9Vδ2 T cells also express the co-receptor CD4. Here the authors show that CD4+ Vγ9Vδ2 T cells comprise 0.1–7% of peripheral blood Vγ9Vδ2 T cells. These cells can be expanded in vitro using zoledronic acid, pamidronic acid or CD3 antibodies combined with IL-2 and feeder cells. Unlike most conventional CD4+ αβ T cells, CD4+ Vγ9Vδ2 T cells are potently cytotoxic and can kill cancer cells, which is here shown by the killing of cancer cell lines of different histological origins, including breast cancer, prostate cancer and melanoma cell lines, upon treatment with zoledronic acid. Notably, the killing capacity of CD4+ Vγ9Vδ2 T cells correlates with co-expression of CD56.

Controlling T cells spreading, mechanics and activation by micropatterning

We designed a strategy, based on a careful examination of the activation capabilities of proteins and antibodies used as substrates for adhering T cells, coupled to protein microstamping to control at the same time the position, shape, spreading, mechanics and activation state of T cells. Once adhered on patterns, we examined the capacities of T cells to be activated with soluble anti CD3, in comparison to T cells adhered to a continuously decorated substrate with the same density of ligands. We show that, in our hand, adhering onto an anti CD45 antibody decorated surface was not affecting T cell calcium fluxes, even adhered on variable size micro-patterns. Aside, we analyzed the T cell mechanics, when spread on pattern or not, using Atomic Force Microscopy indentation. By expressing MEGF10 as a non immune adhesion receptor in T cells we measured the very same spreading area on PLL substrates and Young modulus than non modified cells, immobilized on anti CD45 antibodies, while retaining similar activation capabilities using soluble anti CD3 antibodies or through model APC contacts. We propose that our system is a way to test activation or anergy of T cells with defined adhesion and mechanical characteristics, and may allow to dissect fine details of these mechanisms since it allows to observe homogenized populations in standardized T cell activation assays.

Agonistic CD40 Antibodies in Cancer Treatment.

Simple SummaryCD40 is a costimulatory molecule that is key for the activation of antigen-presenting cells and other innate immune cells. It plays an important role in anti-tumor immunity, and agonists of CD40 have been shown to eliminate tumors in both pre-clinical and clinical settings, alone and in combination with other treatment modalities. Here we assess the expression of CD40 and associations with other mediators of immunity in a variety of tumor types and review the potential of CD40 agonists for cancer treatment, given the promise of enhancing the interplay between innate and adaptive immunity.CD40 is expressed on a variety of antigen-presenting cells. Stimulation of CD40 results in inflammation by upregulation of other costimulatory molecules, increased antigen presentation, maturation (licensing) of dendritic cells, and activation of CD8+ T cells. Here we analyzed gene expression data from The Cancer Genome Atlas in melanoma, renal cell carcinoma, and pancreatic adenocarcinoma and found correlations between CD40 and several genes involved in antigen presentation and T cell function, supporting further exploration of CD40 agonists to treat cancer. Agonist CD40 antibodies have induced anti-tumor effects in several tumor models and the effect has been more pronounced when used in combination with other treatments (immune checkpoint inhibition, chemotherapy, and colony-stimulating factor 1 receptor inhibition). The reduction in tumor growth and ability to reprogram the tumor microenvironment in preclinical models lays the foundation for clinical development of agonistic CD40 antibodies (APX005M, ChiLob7/4, ADC-1013, SEA-CD40, selicrelumab, and CDX-1140) that are currently being evaluated in early phase clinical trials. In this article, we focus on CD40 expression and immunity in cancer, agonistic human CD40 antibodies, and their pre-clinical and clinical development. With the broad pro-inflammatory effects of CD40 and its ligand on dendritic cells and macrophages, and downstream B and T cell activation, agonists of this pathway may enhance the anti-tumor activity of other systemic therapies.