Chemokines and other mediators in the development and functional organization of lymph nodes
Secondary lymphoid organs like lymph nodes (LNs) are the main inductive sites for adaptive immune responses. Lymphocytes are constantly entering LNs, scanning the environment for their cognate antigen and get replenished by incoming cells after a certain period of time. As only a minor percentage of lymphocytes recognizes cognate antigen, this mechanism of permanent recirculation ensures fast and effective immune responses when necessary. Thus, homing, positioning, and activation as well as egress require precise regulation within LNs. In this review we discuss the mediators, including chemokines, cytokines, growth factors, and others that are involved in the formation of the LN anlage and subsequent functional organization of LNs. We highlight very recent findings in the fields of LN development, steady-state migration in LNs, and the intranodal processes during an adaptive immune response.
The Emergence of Universal Immune Receptor T Cell Therapy for Cancer
Chimeric antigen receptor (CAR) T cells have shown great success in the treatment of CD19+ hematological malignancies, leading to their recent approval by the FDA as a new cancer treatment modality. However, their broad use is limited since a CAR targets a single tumor associated antigen (TAA), which is not effective against tumors with heterogeneous TAA expression or emerging antigen loss variants. Further, stably engineered CAR T cells can continually and uncontrollably proliferate and activate in response to antigen, potentially causing fatal on-target off-tumor toxicity, cytokine release syndrome, or neurotoxicity without a method of control or elimination. To address these issues, our lab and others have developed various universal immune receptors (UIRs) that allow for targeting of multiple TAAs by T cells expressing a single receptor. UIRs function through the binding of an extracellular adapter domain which acts as a bridge between intracellular T cell signaling domains and a soluble tumor antigen targeting ligand (TL). The dissociation of TAA targeting and T cell signaling confers many advantages over standard CAR therapy, such as dose control of T cell effector function, the ability to simultaneously or sequentially target multiple TAAs, and control of immunologic synapse geometry. There are currently four unique UIR platform types: ADCC-mediating Fc-binding immune receptors, bispecific protein engaging immune receptors, natural binding partner immune receptors, and anti-tag CARs. These UIRs all allow for potential benefits over standard CARs, but also bring unique engineering challenges that will have to be addressed to achieve maximal efficacy and safety in the clinic. Still, UIRs present an exciting new avenue for adoptive T cell transfer therapies and could lead to their expanded use in areas which current CAR therapies have failed. Here we review the development of each UIR platform and their unique functional benefits, and detail the potential hurdles that may need to be overcome for continued clinical translation.
A Single-Cell Atlas of the Tumor and Immune Ecosystem of Human Breast Cancer
Breast cancer is a heterogeneous disease. Tumor cells and associated healthy cells form ecosystems that determine disease progression and response to therapy. To characterize features of breast cancer ecosystems and their associations with clinical data, we analyzed 144 human breast tumor and 50 non-tumor tissue samples using mass cytometry. The expression of 73 proteins in 26 million cells was evaluated using tumor and immune cell-centric antibody panels. Tumors displayed individuality in tumor cell composition, including phenotypic abnormalities and phenotype dominance. Relationship analyses between tumor and immune cells revealed characteristics of ecosystems related to immunosuppression and poor prognosis. High frequencies of PD-L1(+) tumor-associated macrophages and exhausted T cells were found in high-grade ER(+) and ER(-) tumors. This large-scale, single-cell atlas deepens our understanding of breast tumor ecosystems and suggests that ecosystem-based patient classification will facilitate identification of individuals for precision medicine approaches targeting the tumor and its immunoenvironment.
Exploratory trial of a biepitopic CAR T-targeting B cell maturation antigen in relapsed/refractory multiple myeloma
Relapsed and refractory (R/R) multiple myeloma (MM) patients have very poor prognosis. Chimeric antigen receptor modified T (CAR T) cells is an emerging approach in treating hematopoietic malignancies. Here we conducted the clinical trial of a biepitope-targeting CAR T against B cell maturation antigen (BCMA) (LCAR-B38M) in 17 R/R MM cases. CAR T cells were i.v. infused after lymphodepleting chemotherapy. Two delivery methods, three infusions versus one infusion of the total CAR T dose, were tested in, respectively, 8 and 9 cases. No response differences were noted among the two delivery subgroups. Together, after CAR T cell infusion, 10 cases experienced a mild cytokine release syndrome (CRS), 6 had severe but manageable CRS, and 1 died of a very severe toxic reaction. The abundance of BCMA and cytogenetic marker del(17p) and the elevation of IL-6 were the key indicators for severe CRS. Among 17 cases, the overall response rate was 88.2%, with 13 achieving stringent complete response (sCR) and 2 reaching very good partial response (VGPR), while 1 was a nonresponder. With a median follow-up of 417 days, 8 patients remained in sCR or VGPR, whereas 6 relapsed after sCR and 1 had progressive disease (PD) after VGPR. CAR T cells were high in most cases with stable response but low in 6 out of 7 relapse/PD cases. Notably, positive anti-CAR antibody constituted a high-risk factor for relapse/PD, and patients who received prior autologous hematopoietic stem cell transplantation had more durable response. Thus, biepitopic CAR T against BCMA represents a promising therapy for R/R MM, while most adverse effects are clinically manageable.
Transforming Growth Factor-beta Signaling in Immunity and Cancer
Transforming growth factor (TGF)-beta is a crucial enforcer of immune homeostasis and tolerance, inhibiting the expansion and function of many components of the immune system. Perturbations in TGF-beta signaling underlie inflammatory diseases and promote tumor emergence. TGF-beta is also central to immune suppression within the tumor microenvironment, and recent studies have revealed roles in tumor immune evasion and poor responses to cancer immunotherapy. Here, we present an overview of the complex biology of the TGF-beta family and its context-dependent nature. Then, focusing on cancer, we discuss the roles of TGF-beta signaling in distinct immune cell types and how this knowledge is being leveraged to unleash the immune system against the tumor.
The CTLA-4 x OX40 bispecific antibody ATOR-1015 induces anti-tumor effects through tumor-directed immune activation
BACKGROUND: The CTLA-4 blocking antibody ipilimumab has demonstrated substantial and durable effects in patients with melanoma. While CTLA-4 therapy, both as monotherapy and in combination with PD-1 targeting therapies, has great potential in many indications, the toxicities of the current treatment regimens may limit their use. Thus, there is a medical need for new CTLA-4 targeting therapies with improved benefit-risk profile. METHODS: ATOR-1015 is a human CTLA-4 x OX40 targeting IgG1 bispecific antibody generated by linking an optimized version of the Ig-like V-type domain of human CD86, a natural CTLA-4 ligand, to an agonistic OX40 antibody. In vitro evaluation of T-cell activation and T regulatory cell (Treg) depletion was performed using purified cells from healthy human donors or cell lines. In vivo anti-tumor responses were studied using human OX40 transgenic (knock-in) mice with established syngeneic tumors. Tumors and spleens from treated mice were analyzed for CD8(+) T cell and Treg frequencies, T-cell activation markers and tumor localization using flow cytometry. RESULTS: ATOR-1015 induces T-cell activation and Treg depletion in vitro. Treatment with ATOR-1015 reduces tumor growth and improves survival in several syngeneic tumor models, including bladder, colon and pancreas cancer models. It is further demonstrated that ATOR-1015 induces tumor-specific and long-term immunological memory and enhances the response to PD-1 inhibition. Moreover, ATOR-1015 localizes to the tumor area where it reduces the frequency of Tregs and increases the number and activation of CD8(+) T cells. CONCLUSIONS: By targeting CTLA-4 and OX40 simultaneously, ATOR-1015 is directed to the tumor area where it induces enhanced immune activation, and thus has the potential to be a next generation CTLA-4 targeting therapy with improved clinical efficacy and reduced toxicity. ATOR-1015 is also expected to act synergistically with anti-PD-1/PD-L1 therapy. The pre-clinical data support clinical development of ATOR-1015, and a first-in-human trial has started (NCT03782467).
Unleashing Type-2 Dendritic Cells to Drive Protective Antitumor CD4(+) T Cell Immunity
Differentiation of proinflammatory CD4(+) conventional T cells (Tconv) is critical for productive antitumor responses yet their elicitation remains poorly understood. We comprehensively characterized myeloid cells in tumor draining lymph nodes (tdLN) of mice and identified two subsets of conventional type-2 dendritic cells (cDC2) that traffic from tumor to tdLN and present tumor-derived antigens to CD4(+) Tconv, but then fail to support antitumor CD4(+) Tconv differentiation. Regulatory T cell (Treg) depletion enhanced their capacity to elicit strong CD4(+) Tconv responses and ensuing antitumor protection. Analogous cDC2 populations were identified in patients, and as in mice, their abundance relative to Treg predicts protective ICOS(+) PD-1(lo) CD4(+) Tconv phenotypes and survival. Further, in melanoma patients with low Treg abundance, intratumoral cDC2 density alone correlates with abundant CD4(+) Tconv and with responsiveness to anti-PD-1 therapy. Together, this highlights a pathway that restrains cDC2 and whose reversal enhances CD4(+) Tconv abundance and controls tumor growth.
Systemic clinical tumor regressions and potentiation of PD1 blockade with in situ vaccination
Indolent non-Hodgkin's lymphomas (iNHLs) are incurable with standard therapy and are poorly responsive to checkpoint blockade. Although lymphoma cells are efficiently killed by primed T cells, in vivo priming of anti-lymphoma T cells has been elusive. Here, we demonstrate that lymphoma cells can directly prime T cells, but in vivo immunity still requires cross-presentation. To address this, we developed an in situ vaccine (ISV), combining Flt3L, radiotherapy, and a TLR3 agonist, which recruited, antigen-loaded and activated intratumoral, cross-presenting dendritic cells (DCs). ISV induced anti-tumor CD8(+) T cell responses and systemic (abscopal) cancer remission in patients with advanced stage iNHL in an ongoing trial ( NCT01976585 ). Non-responding patients developed a population of PD1(+)CD8(+) T cells after ISV, and murine tumors became newly responsive to PD1 blockade, prompting a follow-up trial of the combined therapy. Our data substantiate that recruiting and activating intratumoral, cross-priming DCs is achievable and critical to anti-tumor T cell responses and PD1-blockade efficacy.
Targeted antibody and cytokine cancer immunotherapies through collagen affinity
Cancer immunotherapy with immune checkpoint inhibitors (CPIs) and interleukin-2 (IL-2) has demonstrated clinical efficacy but is frequently accompanied with severe adverse events caused by excessive and systemic immune system activation. Here, we addressed this need by targeting both the CPI antibodies anti-cytotoxic T lymphocyte antigen 4 antibody (alphaCTLA4) + anti-programmed death ligand 1 antibody (alphaPD-L1) and the cytokine IL-2 to tumors via conjugation (for the antibodies) or recombinant fusion (for the cytokine) to a collagen-binding domain (CBD) derived from the blood protein von Willebrand factor (VWF) A3 domain, harnessing the exposure of tumor stroma collagen to blood components due to the leakiness of the tumor vasculature. We show that intravenously administered CBD protein accumulated mainly in tumors. CBD conjugation or fusion decreases the systemic toxicity of both alphaCTLA4 + alphaPD-L1 combination therapy and IL-2, for example, eliminating hepatotoxicity with the CPI molecules and ameliorating pulmonary edema with IL-2. Both CBD-CPI and CBD-IL-2 suppressed tumor growth compared to their unmodified forms in multiple murine cancer models, and both CBD-CPI and CBD-IL-2 increased tumor-infiltrating CD8(+) T cells. In an orthotopic breast cancer model, combination treatment with CPI and IL-2 eradicated tumors in 9 of 13 animals with the CBD-modified drugs, whereas it did so in only 1 of 13 animals with the unmodified drugs. Thus, the A3 domain of VWF can be used to improve safety and efficacy of systemically administered tumor drugs with high translational promise.
Unlocking the therapeutic potential of primary tumor-draining lymph nodes
Lymph nodes draining the primary tumor are essential for the initiation of an effective anti-tumor T-cell immune response. However, cancer-derived immune suppressive factors render the tumor-draining lymph nodes (TDLN) immune compromised, enabling tumors to invade and metastasize. Unraveling the different mechanisms underlying this immune escape will inform therapeutic intervention strategies to halt tumor spread in early clinical stages. Here, we review our findings from translational studies in melanoma, breast, and cervical cancer and discuss clinical opportunities for local immune modulation of TDLN in each of these indications.