Development of ICT01, a first-in-class, anti-BTN3A antibody for activating Vγ9Vδ2 T cell-mediated antitumor immune response.

Gamma delta T (γδ T) cells are among the most potent cytotoxic lymphocytes. Activating anti­butyrophilin 3A (BTN3A) antibodies prime diverse tumor cell types to be killed by Vγ9Vδ2 T cells, the predominant γδ T cell subset in peripheral circulation, by mechanisms independent of tumor antigen­major histocompatibility complex (MHC) complexes. In this report, we describe the development of a humanized monoclonal antibody, ICT01, with subnanomolar affinity for the three isoforms of BTN3A. We demonstrate that ICT01-activated Vγ9Vδ2 T cells kill multiple tumor cell lines and primary tumor cells, but not normal healthy cells, in an efficient process requiring approximately 20% target occupancy. We show that ICT01 activity is dependent on BTN3A and BTN2A but independent of the phosphoantigen (pAg)­binding B30.2 domain. ICT01 delays the growth of hematologic and solid tumor xenografts and prolongs survival of NOD/SCID/IL2rγnull (NSG) mice adoptively transferred with human Vγ9Vδ2 T cells. In single- and multiple-dose safety studies in cynomolgus macaques that received up to 100 mg/kg once weekly, ICT01 was well tolerated. With respect to pharmacodynamic endpoints, ICT01 selectively activated Vγ9Vδ2 T cells without affecting other BTN3A-expressing lymphocytes such as αß T or B cells. A first-in-human, phase 1/2a, open-label, clinical study of ICT01 was thus initiated in patients with advanced-stage solid tumors (EVICTION: NCT04243499; EudraCT: 2019-003847-31). Preliminary results show that ICT01 was well tolerated and pharmacodynamically active in the first patients. Digital pathology analysis of tumor biopsies of a patient with melanoma suggests that ICT01 may promote immune cell infiltration within the tumor microenvironment.

A humanized monoclonal antibody specific for invariant Natural Killer T (iNKT) cells for in vivo depletion.

Invariant Natural Killer T (iNKT) cells are a subset of T cells recognizing glycolipid antigens presented by CD1d. Human iNKT cells express a conserved T cell receptor (TCR)-α chain (Vα24-Jα18) paired with a specific beta chain, Vß11. The cells are both innate-like, with rapid cytokine release, and adaptive-like, including thymic positive selection. Over activation of iNKT cells can mediate tissue injury and inflammation in multiple organ systems and play a role in mediating the pathology associated with clinically important inflammatory diseases. At the same time, iNKT cell activation can play a role in protecting against infectious disease and cancer or modulate certain autoimmune diseases through its impact on both the innate and adaptive immune system. This suggests that approaches to cause iNKT cell reduction and/or depletion could treat inflammatory diseases while approaches to promote activation may have therapeutic potential in certain infections, cancer or autoimmune disease. This report summarizes the characterization of a humanized monoclonal depleting antibody (NKTT120) in the cynomolgus macaque. NKTT120 is being developed to treat iNKT mediated inflammation that is associated with chronic inflammatory conditions like sickle cell disease and asthma. NKTT120 binds to human iTCRs and to FCγRI and FCγRIII and has been shown to kill target cells in an ADCC assay at low concentrations consistent with the FCγR binding. iNKT cells were depleted within 24 hours in cynomolgus macaques, but T cell, B cell, and NK cell frequencies were unchanged. iNKT cell recovery was dose and time dependent. T cell dependent antigen responses were not impaired by NKTT120 mediated iNKT depletion as measured by response to KLH challenge. NKTT120 administration did not induce an inflammatory cytokine release at doses up to 10 mg/kg. These data support the use of NKTT120 as an intervention in inflammatory diseases where iNKT reduction or depletion could be beneficial.

A heat shock protein based polyvalent vaccine targeting HSV-2: CD4(+) and CD8(+) cellular immunity and protective efficacy.

Efforts to develop a subunit vaccine against genital herpes have been hampered by lack of knowledge of the protective antigens of HSV-2, the causative agent of the disease. Vaccines based either on selected antigens or attenuated live virus approaches have not demonstrated meaningful clinical activity. We present here results of a therapeutic vaccine candidate, HerpV (formerly called AG-707), consisting of 32 HSV-2 peptides derived from 22 HSV-2 proteins, complexed non-covalently to the HSP70 chaperone and formulated with QS-21 saponin adjuvant. HerpV is observed to be immunogenic, generating CD4(+) and CD8(+) T cell responses in three mouse strains including HLA-A2 transgenic mice. Optimal T cell stimulation was dependent on the synergistic adjuvant properties of QS-21 with hsp70. The vaccine provided significant protection from viral challenge in a mouse prophylaxis model and showed signals of activity in a guinea pig therapeutic model of existing infection. Peripheral blood mononuclear cells from human HSV-2(+) subjects also showed reactivity in vitro to a subset of individual peptides and to the pool of all 32 peptides. Recombinant human Hsc70 complexed with the 32 peptides also stimulated the expansion of CD8(+) T cells from HSV-2(+) subjects in vitro. These studies demonstrate that HerpV is a promising immunotherapy candidate for genital herpes, and provide a foundation for evaluating HerpV in human HSV-2(+) subjects with the intent of eliciting CD4(+) and CD8(+) T cell responses to a broad array of viral antigens.

B and T lymphocyte attenuator is highly expressed on CMV-specific T cells during infection and regulates their function.

B and T lymphocyte attenuator (BTLA), like its relative programmed cell death-1 (PD-1), is a receptor that negatively regulates murine T cell activation. However, its expression and function on human T cells is currently unknown. We report in this study on the expression of BTLA in human T cell subsets as well as its regulation on virus-specific T cells during primary human CMV infection. BTLA is expressed on human CD4(+) T cells during different stages of differentiation, whereas on CD8(+) T cells, it is found on naive T cells and is progressively downregulated in memory and differentiated effector-type cells. During primary CMV infection, BTLA was highly induced on CMV-specific CD8(+) T cells immediately following their differentiation from naive cells. After control of CMV infection, BTLA expression went down on memory CD8(+) cells. Engagement of BTLA by mAbs blocked CD3/CD28-mediated T cell proliferation and Th1 and Th2 cytokine secretion. Finally, in vitro blockade of the BTLA pathway augmented, as efficient as anti-PD-1 mAbs, allogeneic as well as CMV-specific CD8(+) T cell proliferation. Thus, our results suggest that, like PD-1, BTLA provides a potential target for enhancing the functional capacity of CTLs in viral infections.

PD-L1 and PD-L2 differ in their molecular mechanisms of interaction with PD-1.

The programmed death-1 (PD-1) molecule is involved in peripheral tolerance and in the immune escape mechanisms during chronic viral infections and cancer. PD-1 interacts with two ligands, PD-L1 and PD-L2. We have investigated the molecular mechanisms of PD-1 interactions with its ligands by surface plasmon resonance and cell surface binding as well as the ability of the two ligands to compete for PD-1 binding. PD-L1 and PD-L2 bound PD-1 with comparable affinities, but striking differences were observed at the level of the association and dissociation characteristics. PD-L1, but not PD-L2, had a delayed interaction reminiscent of a phenomenon of conformational transition. These mechanisms were confirmed by using PD-L1 mAbs that delayed the dissociation of PD-L1 from PD-1. This mechanism was not restricted to PD-1 binding since PD-L1 behaved in a similar manner with its second ligand, CD80. Finally, we could demonstrate that PD-L1 and PD-L2 competed for PD-1 binding and conversely, an antagonist PD-1 mAb blocked both PD-L1 and PD-L2 binding to PD-1 and strongly enhanced T-cell proliferation. These data further emphasize the differential molecular mechanisms of interaction of PD-L1 and PD-L2 with PD-1, and suggest possible new approach for the therapy of chronic infection, cancer and transplantation.

Cosignaling molecules around LIGHT-HVEM-BTLA: from immune activation to therapeutic targeting.

The regulation of the immune system at the cell surface is primarily controlled by two families of cosigaling molecules: the immunoglobulin (Ig) superfamily, or "CD28 and B7 family", and the tumor necrosis factor receptor (TNFR) family. Here, we summarized the principal structural and functional characteristics of both families. In this respect, the interaction between HVEM, a TNF receptor, and BTLA, an Ig family member, has provided a new perspective and an additional level of complexity in the crosstalk between these two regulatory systems. This review will present a summary of the recent advances in the immunobiology of the LIGHT-HVEM-LTbetaR-BTLA network. The LIGHT-HVEM-BTLA system has emerged as a major regulator of immune responses and lymphocyte activation, whereas LIGHT-LTbetaR participates in lymphoid tissue development and cell death. Moreover, recent studies have provided encouraging new insights into the roles of the LIGHT-HVEM-LTbetaR-BTLA axis as a potential target for controlling anti-tumor responses.

Current vaccine adjuvants: an overview of a diverse class.

Numerous compounds are under evaluation as immunological adjuvants for improvement of vaccine performance. This review will briefly summarize some of the many diverse substances that are currently being utilized as vaccine adjuvants in preclinical and clinical studies.

LIGHT, a new TNF superfamily member, is essential for memory T helper cell-mediated activation of dendritic cells.

LIGHT is a recently identified member of the TNF superfamily that is up-regulated on activated T cells and can cooperate with CD40L to condition DC for the priming of CTL. In this report, we show that an intracellular pool of LIGHT is found selectively in some CD45RA-CD27+CD4+ "central" memory T cells and to a greater extent in some CD45RA-CD27-CD4+ effector memory T cells. LIGHT, like CD40L, is rapidly up-regulated on memory CD4+ T cells upon activation. Unlike CD40L, LIGHT up-regulation on naive T cells is delayed. Stimulation of DC with each subset of memory T cells in the presence of superantigen revealed that CD40L and LIGHT are required for optimal secretion of IL-12. Moreover, effector memory T cells, which can interact with DC in peripheral tissues, contribute to CCR7 induction on DC. LIGHT and CD40L can also regulate IL-12 production by CCR7+ DC capable of migration to lymph nodes. These data suggest that both LIGHT and CD40L may be involved in the maintenance or reactivation of secondary Th1 responses.

Inhibition of pro-inflammatory cytokine generation by CTLA4-Ig in the skin and colon of mice adoptively transplanted with CD45RBhi CD4+ T cells correlates with suppression of psoriasis and colitis.

Transfer of CD45RBhi CD4 + naïve T cells into severe combined immunodeficient (SCID) mice induces colitis and skin lesions. Recipients treated with cyclosporin A (CsA), CTLA4-Ig, or vehicle were evaluated for weight loss, skin lesions, and cutaneous blood flow. Necropsy, histological, hematological and cytokine analyses were performed at the conclusion of the experiment to confirm the clinical findings. Vehicle-treated mice lost weight and had 100% incidence of skin lesions by 46-days. CsA-treated mice also lost weight, but only 3/8 mice developed mild, clinically evident skin lesions. In contrast, all CTLA4-Ig-treated mice gained weight and did not develop skin lesions. Increase in cutaneous blood flow correlated with the development of skin lesions. Granulocyte numbers, which were high or moderately high in the vehicle- or CsA-treated mice, respectively, remained as low in the CTLA4-Ig-treated group as in untreated mice. IFN-gamma, IL-1beta, and TNF-alpha levels in the gut and skin correlated with the extent of inflammation in both organs. Histology revealed that CTLA4-Ig but not CsA effectively prevented both autoimmune disorders. The ability of CTLA4-Ig to prevent both colitis and skin lesions suggests that CD28-dependent co-stimulation of T cells is critical for generation of pro-inflammatory cytokines and induction of clinical disease in such autoimmune disorders.

CD4 coating, but not CD4 depletion, is a predictor of efficacy with primatized monoclonal anti-CD4 treatment of active rheumatoid arthritis.

OBJECTIVE: Double blind studies were conducted with the anti-CD4 monoclonal antibody (Mab) keliximab in patients with active, stable rheumatoid arthritis (RA), to confirm preliminary evidence of efficacy and safety from open. uncontrolled studies. METHODS: We enrolled 136 and 186 patients into 2 consecutive, randomized, double blind trials, with similar populations [apart from inclusion of disease modifying antirheumatic drug (DMARD)-naïve patients in Study 2]. Patients received 4 weeks intravenous placebo or keliximab [40, 80, 120, or 140 mg twice weekly (bw), or 240 mg once weekly (ow)]. The primary endpoint was the American College of Rheumatology (ACR) 20 response criteria, one week after the end of treatment. RESULTS: ACR 20 response rates in Study I were 19%, 42%, 51%*, and 69%* (*p < 0.05 compared to placebo), with placebo, 40, 80, or 140 mg keliximab bw, respectively. The response rates in Study 2 were 30%, 39%, 46% and 47% with placebo, 80 or 120 mg bw, or 240 mg keliximab ow, respectively. In the 2 studies, there was a dose dependent increase in peripheral blood CD4+ T cell coating with keliximab, but a different pattern of CD4 depletion was seen. While only 12% of keliximab treated patients in Study I had CD4 counts below 250 cells/mm3 at the end of the treatment period, 47% fell below this level in Study 2. Clinical response was not correlated with CD4 depletion, but was correlated with CD4+ T cell coating with keliximab. CONCLUSION: Coating of peripheral blood CD4+ T cells with keliximab, but not CD4 depletion, is a determinant of clinical response.