Phase 1, first-in-human study of TYRP1-TCB (RO7293583), a novel TYRP1-targeting CD3 T-cell engager, in metastatic melanoma: active drug monitoring to assess the impact of immune response on drug exposure.

Introduction: Although checkpoint inhibitors (CPIs) have improved outcomes for patients with metastatic melanoma, those progressing on CPIs have limited therapeutic options. To address this unmet need and overcome CPI resistance mechanisms, novel immunotherapies, such as T-cell engaging agents, are being developed. The use of these agents has sometimes been limited by the immune response mounted against them in the form of anti-drug antibodies (ADAs), which is challenging to predict preclinically and can lead to neutralization of the drug and loss of efficacy. Methods: TYRP1-TCB (RO7293583; RG6232) is a T-cell engaging bispecific (TCB) antibody that targets tyrosinase-related protein 1 (TYRP1), which is expressed in many melanomas, thereby directing T cells to kill TYRP1-expressing tumor cells. Preclinical studies show TYRP1-TCB to have potent anti-tumor activity. This first-in-human (FIH) phase 1 dose-escalation study characterized the safety, tolerability, maximum tolerated dose/optimal biological dose, and pharmacokinetics (PK) of TYRP1-TCB in patients with metastatic melanoma (NCT04551352). Results: Twenty participants with cutaneous, uveal, or mucosal TYRP1-positive melanoma received TYRP1-TCB in escalating doses (0.045 to 0.4 mg). All participants experienced ≥1 treatment-related adverse event (TRAE); two participants experienced grade 3 TRAEs. The most common toxicities were grade 1-2 cytokine release syndrome (CRS) and rash. Fractionated dosing mitigated CRS and was associated with lower levels of interleukin-6 and tumor necrosis factor-alpha. Measurement of active drug (dual TYPR1- and CD3-binding) PK rapidly identified loss of active drug exposure in all participants treated with 0.4 mg in a flat dosing schedule for ≥3 cycles. Loss of exposure was associated with development of ADAs towards both the TYRP1 and CD3 domains. A total drug PK assay, measuring free and ADA-bound forms, demonstrated that TYRP1-TCB-ADA immune complexes were present in participant samples, but showed no drug activity in vitro. Discussion: This study provides important insights into how the use of active drug PK assays, coupled with mechanistic follow-up, can inform and enable ongoing benefit/risk assessment for individuals participating in FIH dose-escalation trials. Translational studies that lead to a better understanding of the underlying biology of cognate T- and B-cell interactions, ultimately resulting in ADA development to novel biotherapeutics, are needed.

Circulating tumor DNA: Opportunities and challenges for pharmacometric approaches.

To support further development of model-informed drug development approaches leveraging circulating tumor DNA (ctDNA), we performed an exploratory analysis of the relationships between treatment-induced changes to ctDNA levels, clinical response and tumor size dynamics in patients with cancer treated with checkpoint inhibitors and targeted therapies. This analysis highlights opportunities for pharmacometrics approaches such as for optimizing sampling design strategies. It also highlights challenges related to the nature of the data and associated variability overall emphasizing the importance of mechanistic modeling studies of the underlying biology of ctDNA processes such as shedding, release and clearance and their relationships with tumor size dynamic and treatment effects.

Phase I/II Trial of StemRegenin-1 Expanded Umbilical Cord Blood Hematopoietic Stem Cells Supports Testing as a Stand-Alone Graft.

Clinical application of umbilical cord blood (UCB) as a source of hematopoietic stem cells for transplantation is limited by low CD34+ cell dose, increased risk of graft failure, and slow hematopoietic recovery. While the cell dose limitation is partially mitigated by using two UCB units, larger-dosed single units would be preferable. We have evaluated the feasibility and safety of StemRegenin-1 (SR-1), an aryl hydrocarbon receptor antagonist that expands CD34+ cells, by placing one of the two units in expansion culture. SR-1 produced a 330-fold increase in CD34+ cells and led to engraftment in 17/17 patients at a median of 15 days for neutrophils and 49 days for platelets, significantly faster than in patients treated with unmanipulated UCB. Taken together, the marked expansion, absence of graft failure, and enhanced hematopoietic recovery support testing of SR-1 expansion as a stand-alone graft and suggest it may ameliorate a limitation of UCB transplant.

Evidence that a neutrophil-keratinocyte crosstalk is an early target of IL-17A inhibition in psoriasis.

The response of psoriasis to antibodies targeting the interleukin (IL)-23/IL-17A pathway suggests a prominent role of T-helper type-17 (Th17) cells in this disease. We examined the clinical and immunological response patterns of 100 subjects with moderate-to-severe psoriasis receiving 3 different intravenous dosing regimens of the anti-IL-17A antibody secukinumab (1 × 3 mg/kg or 1 × 10 mg/kg on Day 1, or 3 × 10 mg/kg on Days 1, 15 and 29) or placebo in a phase 2 trial. Baseline biopsies revealed typical features of active psoriasis, including epidermal accumulation of neutrophils and formation of microabscesses in >60% of cases. Neutrophils were the numerically largest fraction of infiltrating cells containing IL-17 and may store the cytokine preformed, as IL-17A mRNA was not detectable in neutrophils isolated from active plaques. Significant clinical responses to secukinumab were observed 2 weeks after a single infusion, associated with extensive clearance of cutaneous neutrophils parallel to the normalization of keratinocyte abnormalities and reduction of IL-17-inducible neutrophil chemoattractants (e.g. CXCL1, CXCL8); effects on numbers of T cells and CD11c-positive dendritic cells were more delayed. Histological and immunological improvements were generally dose dependent and not observed in the placebo group. In the lowest-dose group, a recurrence of neutrophils was seen in some subjects at Week 12; these subjects relapsed faster than those without microabscesses. Our findings are indicative of a neutrophil-keratinocyte axis in psoriasis that may involve neutrophil-derived IL-17 and is an early target of IL-17A-directed therapies such as secukinumab.

Conversion of the thymus into a bipotent lymphoid organ by replacement of FOXN1 with its paralog, FOXN4.

The thymus is a lymphoid organ unique to vertebrates, and it provides a unique microenvironment that facilitates the differentiation of immature hematopoietic precursors into mature T cells. We subjected the evolutionary trajectory of the thymic microenvironment to experimental analysis. A hypothetical primordial form of the thymus was established in mice by replacing FOXN1, the vertebrate-specific master regulator of thymic epithelial cell function, with its metazoan ancestor, FOXN4, thereby resetting the regulatory and coding changes that have occurred since the divergence of these two paralogs. FOXN4 exhibited substantial thymopoietic activity. Unexpectedly, histological changes and a functional imbalance between the lymphopoietic cytokine IL7 and the T cell specification factor DLL4 within the reconstructed thymus resulted in coincident but spatially segregated T and B cell development. Our results identify an evolutionary mechanism underlying the conversion of a general lymphopoietic organ to a site of exclusive T cell generation.

Maintenance of thymic epithelial phenotype requires extrinsic signals in mouse and zebrafish.

Thymopoiesis strictly depends on proper differentiation of the thymic epithelial anlage. Differentiation of thymic epithelial cells (TECs) is controlled by the Foxn1 transcription factor. The in vivo signals initiating and maintaining Foxn1 expression in the future thymus anlage are unknown. In the mouse, bone morphogenetic protein (BMP) signaling is required for the maintenance of Foxn1 expression in TECs, as shown here by lineage tracing using a Foxn1-driven Cre transgene. Loss of Foxn1 expression after BMP inhibition reverts TECs to a basal state of pharyngeal epithelium unable to support T cell development; it does not divert them into a parathyroid fate. In zebrafish larvae, BMP inhibition likewise causes loss of foxn1 expression in the thymic anlage and subsequent impairment of thymopoiesis. These results indicate an evolutionarily conserved role of BMP signaling in the maintenance of Foxn1 expression.

Ltbetar signaling does not regulate Aire-dependent transcripts in medullary thymic epithelial cells.

Thymic medullary epithelial cells (mTECs) play a major role in central tolerance induction by expressing tissue-specific Ags (TSAs). The expression of a subset of TSAs in mTECs is under the control of Aire (autoimmune regulator). Humans defective for AIRE develop a syndrome characterized by autoimmune disease in several endocrine glands. Aire has been proposed to be regulated by lymphotoxin beta receptor (Ltbetar) signaling and there is evidence that, additionally, Aire-independent transcripts may be regulated by this pathway. Given the potential clinical importance of Aire regulation in mTECs for the control of autoimmunity, we investigated the relation between Ltbetar signaling and TSA expression by whole genome transcriptome analysis. In this study, we show that the absence of Ltbetar has no effect on the expression of Aire and Aire-dependent TSAs. Also, the lack of Ltbetar signaling does not disturb regulatory T cells or the distribution of dendritic cells in the thymus. However, mTECs in Ltbetar-deficient mice show an aberrant distribution within the thymic medulla with disruption of their three-dimensional architecture. This is predicted to impair the interaction between mTECs and thymocytes as shown by the reduced surface uptake of MHCII by mature thymocytes in Ltbetar-deficient mice. We propose that the physiological medullary architecture ensures negative-selection by supporting lympho-epithelial interaction through a large epithelial cell surface distributed evenly across the medulla.

The stream of precursors that colonizes the thymus proceeds selectively through the early T lineage precursor stage of T cell development.

T cell development in the thymus depends on continuous colonization by hematopoietic precursors. Several distinct T cell precursors have been identified, but whether one or several independent precursor cell types maintain thymopoiesis is unclear. We have used thymus transplantation and an inducible lineage-tracing system to identify the intrathymic precursor cells among previously described thymus-homing progenitors that give rise to the T cell lineage in the thymus. Extrathymic precursors were not investigated in these studies. Both approaches show that the stream of T cell lineage precursor cells, when entering the thymus, selectively passes through the early T lineage precursor (ETP) stage. Immigrating precursor cells do not exhibit characteristics of double-negative (DN) 1c, DN1d, or DN1e stages, or of populations containing the common lymphoid precursor 2 (CLP-2) or the thymic equivalent of circulating T cell progenitors (CTPs). It remains possible that an unknown hematopoietic precursor cell or previously described extrathymic precursors with a CLP, CLP-2, or CTP phenotype feed into T cell development by circumventing known intrathymic T cell lineage progenitor cells. However, it is clear that of the known intrathymic precursors, only the ETP population contributes significant numbers of T lineage precursors to T cell development.

The Foxn1-dependent transcripts PCOLCE2 and mPPP1R16B are not required for normal thymopoiesis.

The adaptive immune system relies on the thymic microenvironment for the production of a diverse, self-tolerant T cell receptor repertoire. The central cellular organizer of the thymic microenvironment is the thymic epithelial cell (TEC). The development of TEC from endodermal precursor cells is under the control of the forkhead/winged helix transcription factor Foxn1 but the transcriptional program that leads to this unique epithelial differentiation has not been investigated functionally. Here, we show that expression of procollagen C-proteinase enhancer 2 (PCOLCE2) is absent in Foxn1-deficient TEC. In order to study the functional role of this gene in TEC differentiation, we have genetically inactivated PCOLCE2 and the gene encoding phosphatase 1 regulatory inhibitory subunit 16B (mPPP1R16B), another transcript lacking in Foxn1-deficient TEC. Mice deficient for either one or both of these transcripts presented a normal thymic microenvironment and undisturbed thymopoiesis. While there is no evidence for a functional role of PCOLCE2 and mPPP1R16B in thymus development, our results suggest that the lack of thymopoiesis in Foxn1-deficient mice is caused by multiple functional defects.

A silent chemokine receptor regulates steady-state leukocyte homing in vivo.

The location of leukocytes in different microenvironments is intimately connected to their function and, in the case of leukocyte precursors, to the executed differentiation and maturation program. Leukocyte migration within lymphoid organs has been shown to be mediated by constitutively expressed chemokines, but how the bioavailability of these homeostatic chemokines is regulated remains unknown. Here, we report in vivo evidence for the role of a nonsignaling chemokine receptor in the migration of leukocytes under physiological, i.e., noninflammatory, conditions. We have studied the in vivo role of the silent chemokine receptor CCX-CKR1 by both loss- and gain-of-function approaches. CCX-CKR1 binds the constitutively expressed chemokines CC chemokine ligand (CCL)19, CCL21, and CCL25. We find that CCX-CKR1 is involved in the steady-state homing of CD11c(+)MHCII(high) dendritic cells to skin-draining lymph nodes, and it affects the homing of embryonic thymic precursors to the thymic anlage. These observations indicate that the silent chemokine receptor CCX-CKR1, which is exclusively expressed by stroma cells, but not hematopoietic cells themselves, regulates homeostatic leukocyte migration by controlling the availability of chemokines in the extracellular space. This finding adds another level of complexity to our understanding of leukocyte homeostatic migration.

Bone marrow-derived hemopoietic precursors commit to the T cell lineage only after arrival in the thymic microenvironment.

T lymphocytes develop in the thymus from hemopoietic precursors that commit to the T cell lineage under the influence of Notch signals. In this study, we show by single cell analyses that the most immature hemopoietic precursors in the adult mouse thymus are uncommitted and specify to the T cell lineage only after their arrival in the thymus. These precursors express high levels of surface Notch receptors and rapidly lose B cell potential upon the provision of Notch signals. Using a novel culture system with complexed, soluble Notch ligands that allows the titration of T cell lineage commitment, we find that these precursors are highly sensitive to both Delta and Jagged ligands. In contrast, their phenotypical and functional counterparts in the bone marrow are resistant to Notch signals that efficiently induce T cell lineage commitment in thymic precursors. Mechanistically, this is not due to differences in receptor expression, because early T lineage precursors, bone marrow lineage marker-negative, Sca-1-positive, c-Kit-positive and common lymphoid progenitor cells, express comparable amounts of surface Notch receptors. Our data demonstrate that the sensitivity to Notch-mediated T lineage commitment is stage-dependent and argue against the bone marrow as the site of T cell lineage commitment.

The evolutionary history of lymphoid organs.

Lymphoid organs are important regulators of lymphocyte development and immune responses. During vertebrate evolution, primary lymphoid organs appeared earlier than secondary lymphoid organs. Among the sites of primary lymphopoiesis during evolution and ontogeny, those for B cell differentiation have differed considerably, although they often have had myelolymphatic characteristics. In contrast, only a single site for T cell differentiation has occurred, exclusively the thymus. Based on those observations and the known features of variable-diversity-joining gene recombination, we propose a model for the successive specification of different lymphocyte lineages during vertebrate evolution. According to our model, T cells were the first lymphocytes to acquire variable-diversity-joining-type receptors, and the thymus was the first lymphoid organ to evolve in vertebrates to deal with potentially autoreactive, somatically diversified T cell receptors.

Thymus-homing precursors and the thymic microenvironment.

T cells develop in the thymus from precursors that are generated in the bone marrow and continuously seed the thymus through the blood. During evolution, 'outsourcing' the development of one blood lineage, namely the T-cell lineage, to an anatomically distinct hematopoietic organ required the generation of migratory precursors in the bone marrow, their homing to specialized, precursor-retaining thymic niches and their subsequent differentiation. Niche building and precursor homing are therefore intricately linked and should be viewed in context. In this review, we discuss recent findings on the developmental and genetic events that prepare the thymic epithelial microenvironment for its complex tasks, and highlight recent progress in the definition of the thymus-settling cells and the homing process that leads them into the thymus.

Formation of a functional thymus initiated by a postnatal epithelial progenitor cell.

The thymus is essential for the generation of self-tolerant effector and regulatory T cells. Intrathymic T-cell development requires an intact stromal microenvironment, of which thymic epithelial cells (TECs) constitute a major part. For instance, cell-autonomous genetic defects of forkhead box N1 (Foxn1) and autoimmune regulator (Aire) in thymic epithelial cells cause primary immunodeficiency and autoimmunity, respectively. During development, the thymic epithelial rudiment gives rise to two major compartments, the cortex and medulla. Cortical TECs positively select T cells, whereas medullary TECs are involved in negative selection of potentially autoreactive T cells. It has long been unclear whether these two morphologically and functionally distinct types of epithelial cells arise from a common bi-potent progenitor cell and whether such progenitors are still present in the postnatal period. Here, using in vivo cell lineage analysis in mice, we demonstrate the presence of a common progenitor of cortical and medullary TECs after birth. To probe the function of postnatal progenitors, a conditional mutant allele of Foxn1 was reverted to wild-type function in single epithelial cells in vivo. This led to the formation of small thymic lobules containing both cortical and medullary areas that supported normal thymopoiesis. Thus, single epithelial progenitor cells can give rise to a complete and functional thymic microenvironment, suggesting that cell-based therapies could be developed for thymus disorders.

Evidence for a functional second thymus in mice.

The thymus organ supports the development of T cells and is located in the thorax. Here, we report the existence of a second thymus in the mouse neck, which develops after birth and grows to the size of a small lymph node. The cervical thymus had a typical medulla-cortex structure, was found to support T cell development, and could correct T cell deficiency in athymic nude mice upon transplantation. The identification of a regular second thymus in the mouse may provide evolutionary links to thymus organogenesis in other vertebrates and suggests a need to reconsider the effect of thoracic thymectomy on de novo T cell production.

BMP signaling is required for normal thymus development.

The microenvironment of the thymus fosters the generation of a diverse and self-tolerant T cell repertoire from a pool of essentially random specificities. Epithelial as well as mesenchymal cells contribute to the thymic stroma, but little is known about the factors that allow for communication between the two cells types that shape the thymic microenvironment. In this study, we investigated the role of bone morphogenetic protein (BMP) signaling in thymus development. Transgenic expression of the BMP antagonist Noggin in thymic epithelial cells under the control of a Foxn1 promoter in the mouse leads to dysplastic thymic lobes of drastically reduced size that are ectopically located in the neck at the level of the hyoid bone. Interestingly, the small number of thymocytes in these thymic lobes develops with normal kinetics and shows a wild-type phenotype. Organ initiation of the embryonic thymic anlage in these Noggin transgenic mice occurs as in wild-type mice, but the tight temporal and spatial regulation of BMP4 expression is abrogated in subsequent differentiation stages. We show that transgenic Noggin blocks BMP signaling in epithelial as well as mesenchymal cells of the thymic anlage. Our data demonstrate that BMP signaling is crucial for thymus development and that it is the thymic stroma rather than developing thymocytes that depends on BMP signals.

A multipotent precursor in the thymus maps to the branching point of the T versus B lineage decision.

Hematopoietic precursors continuously colonize the thymus where they give rise mainly to T cells, but also to B and dendritic cells. The lineage relationship between these three cell types is unclear, and it remains to be determined if precursors in the thymus are multipotent, oligopotent, or lineage restricted. Resolution of this question necessitates the determination of the clonal differentiation potential of the most immature precursors in the thymus. Using a CC chemokine receptor 9-enhanced green fluorescent protein knock-in allele like a surface marker of unknown function, we identify a multipotent precursor present in bone marrow, blood, and thymus. Single cells of this precursor give rise to T, B, and dendritic cells. A more differentiated stage of this multipotent precursor in the thymus has lost the capacity to generate B but not T, dendritic, and myeloid cells. Thus, the newly identified precursor maps to the branching point of the T versus B lineage decision in the hematopoietic lineage hierarchy.

Homing of immature thymocytes to the subcapsular microenvironment within the thymus is not an absolute requirement for T cell development.

T cell development is thought to occur in distinct microenvironments within the thymus. Namely, the subcapsular zone, the cortex and the medulla have been described to support expansion of the immature thymocyte pool, positive selection of useful specificities and elimination of potentially self-reactive specificities, respectively. Consistent with this model, thymocytes show a highly ordered migration pattern and move into these niches in the expected sequence. Here we show that the chemokine receptor CCR9 plays a nonredundant role in the homing of immature thymocytes to the subcapsular zone. In CCR9-deficient mice, T cells in early stages of development do not accumulate in their physiological microenvironment underneath the thymic capsule and are instead homogeneously distributed across the thymic cortex. Remarkably, this abnormality does not result in a detectable defect in T cell development in CCR9-deficient mice, suggesting that the transit of immature thymocytes through the subcapsular microenvironment is not an absolute requirement for proper T cell development.

Genetic dissection of thymus development in mouse and zebrafish.

Lymphoid organs represent a specialized microenvironment for interaction of stromal and lymphoid cells. In primary lymphoid organs, these interactions are required to establish a self-tolerant repertoire of lymphocytes. While detailed information is available about the genes that control lymphocyte differentiation, little is known about the genes that direct the establishment and differentiation of principal components of such microenvironments. Here, we discuss genetic studies addressing the role of thymic epithelial cells (TECs) during thymopoiesis. We have identified an evolutionarily conserved key regulator of TEC differentiation, Foxn1, that is required for the immigration of prothymocytes into the thymic primordium. Because Foxn1 specifies the prospective endodermal domain that gives rise to thymic epithelial cells, it can be used to identify the evolutionary origins of this specialized cell type. In the course of these studies, we have found that early steps of thymus development in zebrafish are very similar to those in mice. Subsequently, we have used chemical mutagenesis to derive zebrafish lines with aberrant thymus development. Strengths and weaknesses of mouse and zebrafish models are largely complementary such that genetic analysis of mouse and zebrafish mutants may lead to a better understanding of thymus development.

Thymic medullary epithelial cell differentiation, thymocyte emigration, and the control of autoimmunity require lympho-epithelial cross talk via LTbetaR.

Thymocytes depend on the interaction with thymic epithelial cells for the generation of a diverse, nonautoreactive T cell repertoire. In turn, thymic epithelial cells acquire their three-dimensional cellular organization via instructive signals from developing thymocytes. The nature of these signals has been elusive so far. We show that thymocytes and medullary epithelial cells (MECs) communicate via the lymphotoxin beta receptor (LTbetaR) signaling axis. Normal differentiation of thymic MECs requires LTbetaR ligand on thymocytes and LTbetaR together with nuclear factor-kappaB-inducing kinase (Nik) in thymic epithelial cells. Impaired lympho-epithelial cross talk in the absence of the LTbetaR causes aberrant differentiation and reduced numbers of thymic MECs, leads to the retention of mature T lymphocytes, and is associated with autoimmune phenomena, suggesting an unexpected role for LTbetaR signaling in central tolerance induction.