Chimeric antigen receptor (CAR)-engineered T cells are efficacious in controlling advanced leukemia and lymphoma, however, they fail in the treatment of solid cancer, which is thought to be due to insufficient T cell activation. We revealed that the immune response of CAR T cells with specificity for carcinoembryonic antigen (CEA) was more efficacious against CEA+ cancer cells when simultaneously incubated with an anti-CD30 immunotoxin or anti-CD30 CAR T cells, although the targeted cancer cells lack CD30. The same effect was achieved when the anti-CD30 single-chain variable fragment (scFv) was integrated into the extracellular domain of the anti-CEA CAR. Improvement in T cell activation was due to interfering with the T cell CD30-CD30L interaction by the antagonistic anti-CD30 scFv HRS3; an agonistic anti-CD30 scFv or targeting the high-affinity interleukin-2 (IL-2) receptor was not effective. T cells with the anti-CD30/CEA CAR showed superior immunity against established CEA+ CD30- tumors in a mouse model. The concept is broadly applicable since anti-CD30/TAG72 CAR T cells also showed improved elimination of TAG72+ CD30- cancer cells. Taken together, targeting CD30 on CAR T cells by the HRS3 scFv within the anti-tumor CAR improves the redirected immune response against solid tumors.

CD30 is expressed on Hodgkin lymphomas (HL), many non-Hodgkin lymphomas (NHLs), and non-lymphoid malignancies in children and adults. Tumor expression, combined with restricted expression in healthy tissues, identifies CD30 as a promising immunotherapy target. An anti-CD30 antibody-drug conjugate (ADC) has been approved by the FDA for HL. While anti-CD30 ADCs and chimeric antigen receptors (CARs) have shown promise, their shortcomings and toxicities suggest that alternative treatments are needed. We developed novel anti-CD30 x anti-CD3 bispecific antibodies (biAbs) to coat activated patient T cells (ATCs) ex vivo prior to autologous re-infusions. Our goal is to harness the dual specificity of the biAb, the power of cellular therapy, and the safety of non-genetically modified autologous T cell infusions. We present a comprehensive characterization of the CD30 binding and tumor cell killing properties of these biAbs. Five unique murine monoclonal antibodies (mAbs) were generated against the extracellular domain of human CD30. Resultant anti-CD30 mAbs were purified and screened for binding specificity, affinity, and epitope recognition. Two lead mAb candidates with unique sequences and CD30 binding clusters that differ from the ADC in clinical use were identified. These mAbs were chemically conjugated with OKT3 (an anti-CD3 mAb). ATCs were armed and evaluated in vitro for binding, cytokine production, and cytotoxicity against tumor lines and then in vivo for tumor cell killing. Our lead mAb was subcloned to make a Master Cell Bank (MCB) and screened for binding against a library of human cell surface proteins. Only huCD30 was bound. These studies support a clinical trial in development employing ex vivo-loading of autologous T cells with this novel biAb.

Hodgkin's lymphoma and anaplastic large cell lymphoma, especially relapsed or refractory diseases, could recently be cured by CD30-targeted immunotherapy. However, the CD30 antigen releases the soluble ectodomain of CD30, which might obscure the targeted therapy. Therefore, the membrane epitope of CD30 (mCD30), left on the cancer cells, might be a prospective target for lymphoma treatment. The discovery of novel mCD30 monoclonal antibodies (mAbs) using phage technology yielded 59 potential human single-chain variable fragments (HuscFvs). Ten candidate HuscFv clones have been selected based on various methods, i.e., direct PCR, ELISA and western blot assays, and nucleotide sequencing techniques. Fortunately, only one potential HuscFv clone, clone #A4, was determined by the prediction of HuscFv-peptide molecular docking and the binding affinity test using isothermal titration calorimetry. Finally, we proved that the HuscFv #A4, which had a binding affinity (Kd) of 421e-9 ± 2.76e-6 M, might be the novel mCD30 mAb. We generated chimeric antigen receptor-modified T lymphocytes using HuscFv #A4 as an antigen detection part (anti-mCD30-H4CART). The cytotoxicity assay of anti-mCD30-H4CART cells showed significant eradication of the CD30-expressing cell line, K562 (p = 0.0378). We found a novel mCD30 HuscFv using human phage technology. We systematically examined and proved that our HuscFv #A4 could specifically eradicate CD30-expressing cancers.

Patients with classical Hodgkin lymphoma (cHL) have an impaired cellular immune response as indicated by an anergic reaction against standard recall antigens and a diminished rejection reaction of allogeneic skin transplant. This clinical observation can be linked to the histopathological feature of cHL since the typical pattern of a cHL manifestation is characterized by sparse large CD30+ tumor-infiltrating Hodgkin-Reed-Sternberg (HRS) cells that are surrounded by a dense inflammatory immune microenvironment with mixed cellularity. Despite this extensive polymorphous inflammatory infiltrate, there is only a poor antitumor immune response seen to the neoplastic HRS cells. This is primarily mediated by a high expression of PD-L1 and PD-L2 ligands on the HRS cell surface which in turn antagonizes the activity of programmed death-1 (PD-1) antigen-positive T cells. PD-L1/L2 overexpression is caused by gene amplification at the 9p24.1 locus and/or latent Epstein-Barr virus infection present in around 40% of cHL cases. The blockade of the PD-L1/L2-PD-1 pathway by monoclonal antibodies can restore local T cell activity and leads to impressive tumor responses, some of which are long lasting and eventually curative. Another feature of HRS cells is the high CD30 antigen expression. Monoclonal antibody technology allowed for the successful development of CD30-specific immunotoxins, bispecific antibodies, and reprogrammed autologous T cells with the first one already approved for the treatment of high risk or relapsed cHL. Altogether, the discovery of the described pathomechanism of immune suppression and the identification of preferential target antigens has rendered cHL to be a prime subject for the successful development of new immunotherapeutic approaches.

Dendritic cells (DCs) are the most potent antigen-presenting cells that link innate and adaptive immune responses, playing a pivotal role in triggering antigen-specific immunity. Antigen uptake by DCs induces maturational changes that include increased surface expression of major histocompatibility complex (MHC) and costimulatory molecules. In addition, DCs actively migrate to regional lymph nodes and activate antigen-specific naive T cells after capturing antigens. We characterize the functional changes of DCs infected with Orientia tsutsugamushi, the causative agent of scrub typhus, since there is limited knowledge of the role played by DCs in O. tsutsugamushi infection.

Over the last few years, treatment principles have been changed towards more targeted therapy for many B-cell lymphoma subtypes and in chronic lymphocytic leukemia (CLL). Immunotherapeutic modalities, namely monoclonal antibodies (mAbs), bispecific antibodies (bsAbs), antibody-drug conjugates (ADCs), and chimeric antigen receptor T (CAR-T) cell therapy, commonly use B-cell-associated antigens (CD19, CD20, CD22, and CD79b) as one of their targets. T-cell engagers (TCEs), a subclass of bsAbs, work on a similar mechanism as CAR-T cell therapy without the need of previous T-cell manipulation. Currently, several anti-CD20xCD3 TCEs have demonstrated promising efficacy across different lymphoma subtypes with slightly better outcomes in the indolent subset. Anti-CD19xCD3 TCEs are being developed as well but only blinatumomab has been evaluated in clinical trials yet. The results are not so impressive as those with anti-CD19 CAR-T cell therapy. Antibody-drug conjugates targeting different B-cell antigens (CD30, CD79b, CD19) seem to be effective in combination with mAbs, standard chemoimmunotherapy, or immune checkpoint inhibitors. Further investigation will show whether immunotherapy alone or in combinatory regimens has potential to replace chemotherapeutic agents from the first line treatment.

Antibodies that target cell-surface molecules on T cells can enhance anti-tumor immune responses, resulting in sustained immune-mediated control of cancer. We set out to find new cancer immunotherapy targets by phenotypic screening on human regulatory T (Treg) cells and report the discovery of novel activators of tumor necrosis factor receptor 2 (TNFR2) and a potential role for this target in immunotherapy. A diverse phage display library was screened to find antibody mimetics with preferential binding to Treg cells, the most Treg-selective of which were all, without exception, found to bind specifically to TNFR2. A subset of these TNFR2 binders were found to agonise the receptor, inducing iκ-B degradation and NF-κB pathway signalling in vitro. TNFR2 was found to be expressed by tumor-infiltrating Treg cells, and to a lesser extent Teff cells, from three lung cancer patients, and a similar pattern was also observed in mice implanted with CT26 syngeneic tumors. In such animals, TNFR2-specific agonists inhibited tumor growth, enhanced tumor infiltration by CD8+ T cells and increased CD8+ T cell IFN-γ synthesis. Together, these data indicate a novel mechanism for TNF-α-independent TNFR2 agonism in cancer immunotherapy, and demonstrate the utility of target-agnostic screening in highlighting important targets during drug discovery.

Flow cytometry allows the visualization of physical, functional, and/or biological properties of cells including antigens, cytokines, size, and complexity. With increasingly large flow cytometry panels able to analyze up to 50 parameters, there is a need to standardize flow cytometry protocols to achieve high-quality data that can be input into analysis algorithms. Without this clean data, algorithms may incorrectly categorize the cell populations present in the samples. In this protocol, we outline a comprehensive methodology to prepare samples for polychromatic flow cytometry. The use of multiple washing steps and rigorous controls creates high-quality data with good separation between cell populations. Experimental data acquired using this protocol can be analyzed via computational algorithms that perform end-to-end analysis. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of single-cell suspension for flow cytometry Support Protocol 1: Lung preparation Support Protocol 2: Counting cells on a flow cytometer Basic Protocol 2: Surface and intracellular flow cytometry staining Support Protocol 3: Single-color bead controls.

Mast cell neoplasms are characterized by abnormal growth and focal accumulation of mast cells (MC) in one or more organs. Although several cytokines, including stem cell factor (SCF) and interleukin-9 (IL-9) have been implicated in growth of normal MC, little is known about pro-oncogenic molecules and conditions triggering differentiation and growth of MC far enough to lead to the histopathological picture of overt mastocytosis. The anaplastic lymphoma kinase (ALK) has recently been implicated in growth of neoplastic cells in malignant lymphomas. Here, we describe that transplantation of NPM-ALK-transplanted mouse bone marrow progenitors into lethally irradiated IL-9 transgenic mice not only results in lymphoma-formation, but also in the development of a neoplastic disease exhibiting histopathological features of systemic mastocytosis, including multifocal dense MC-infiltrates, occasionally with devastating growth in visceral organs. Transplantation of NPM-ALK-transduced progenitors into normal mice or maintenance of IL-9-transgenic mice without NPM-ALK each resulted in MC hyperplasia, but not in mastocytosis. Neoplastic MC in mice not only displayed IL-9, but also the IL-9 receptor, and the same was found to hold true for human neoplastic MC. Together, our data show that neoplastic MC express IL-9 receptors, that IL-9 and NPM-ALK upregulate MC-production in vivo, and that both'hits' act in concert to induce a mastocytosis-like disease in mice. These data may have pathogenetic and clinical implications and fit well with the observation that neoplastic MC in advanced SM strongly express NPM and multiple "lymphoid" antigens including CD25 and CD30.

Antibody-drug conjugates (ADCs) are composed of an antibody linked to cytotoxic anticancer payloads. ADCs recognize tumor-specific cell surface antigens and are internalized into lysosomes where proteolytic enzymes release the cytotoxic payloads. Efflux transporters on plasma membrane that play a significant role on multi-drug resistance in chemotherapy can be internalized on lysosomal membrane and sequester the cytotoxic payloads. In the present study, ATP binding cassette (ABC) efflux transporters including breast cancer resistance protein (BCRP), P-glycoprotein (P-gp-MDR1), multidrug resistance protein (MRP) 2, MRP3 and MRP4 in lysosomal, and plasma membrane of tumor cells were quantified by targeted quantitative proteomics. The cytotoxicity of brentuximab vedotin and its cytotoxic payload monomethyl auristatin E (MMAE) to the tumor cell lines in the presence and absence of elacridar (P-gp-MDR1 inhibitor) or chloroquine (lysosomotropic agent) were evaluated. MMAE is a substrate for P-gp-MDR1, as the apparent efflux ratio in MDR1 transfected MDCK cell monolayers was 44.5, and elacridar abolished the MMAE efflux. Cell lines that highly express P-gp-MDR1 show higher EC50s toward the cell killing effects of MMAE. Co-incubation with chloroquine or elacridar resulted in left shift of MMAE EC50 by 2.9-16-fold and 4.2-22-fold, respectively. Similarly co-incubation with chloroquine or elacridar or in combination of chloroquine and elacridar increased cytotoxic effects of brentuximab vedotin by 2.8- to 21.4-fold on KM-H2 cells that express a specific tumor antigen CD30 and P-gp-MDR1. These findings demonstrate important roles of P-gp-MDR1 on cytotoxic effects of brentuximab vedotin and its payload MMAE. Collectively, ABC transporter-mediated drug extrusion and/or sequestration needs to be early assessed for selection of optimal payloads and linkers when developing ADCs.

Technological advances now allow us to rapidly produce CARs and other antibody-derived therapeutics targeting cell surface receptors. To maximize the potential of these new technologies, relevant extracellular targets must be identified. The Pediatric Oncology Branch of the NCI curates a freely accessible database of gene expression data for both pediatric cancers and normal tissues, through which we have defined discrete sets of over-expressed transcripts in 12 pediatric cancer subtypes as compared to normal tissues. We coupled gene expression profiles to current annotation databases (i.e., Affymetrix, Gene Ontology, Entrez Gene), in order to categorize transcripts by their sub-cellular location. In this manner we generated a list of potential immune targets expressed on the cell surface, ranked by their difference from normal tissue. Global differences from normal between each of the pediatric tumor types studied varied, indicating that some malignancies expressed transcript sets that were more highly diverged from normal tissues than others. The validity of our approach is seen by our findings for pre-B cell ALL, where targets currently in clinical trials were top-ranked hits (CD19, CD22). For some cancers, reagents already in development could potentially be applied to a new disease class, as exemplified by CD30 expression on sarcomas. Moreover, several potential new targets shared among several pediatric solid tumors are herein identified, such as MCAM (MUC18), metadherin (MTDH), and glypican-2 (GPC2). These targets have been identified at the mRNA level and are yet to be validated at the protein level. The safety of targeting these antigens has yet to be demonstrated and therefore the identified transcripts should be considered preliminary candidates for new CAR and therapeutic antibody targets. Prospective candidate targets will be evaluated by proteomic analysis including Westerns and immunohistochemistry of normal and tumor tissues.

Nodal mature T-cell lymphomas (nMTCL) comprises a heterogeneous group of rare malignancies with aggressive biological behavior and poor prognosis. Epigenetic phenomena, including mutations in genes that control DNA methylation and histone deacetylation, in addition to inactivating mutations in the RhoA GTPase, play a central role in its pathogenesis and constitute potential new targets for therapeutic intervention. Tumor mutational burden (TMB) reflects the process of clonal evolution, predicts response to anti-cancer therapies and has emerged as a prognostic biomarker in several solid neoplasms; however, its potential prognostic impact remains unknown in nMTCL. In this study, we conducted Sanger sequencing of formalin-fixed paraffin-embedded (FFPE) diagnostic tumor samples using a target-panel to search for recurrent mutations involving the IDH-1/IDH-2, TET-2, DNMT3A and RhoA genes in 59 cases of nMTCL. For the first time, we demonstrated that high-TMB, defined by the presence of ≥ two mutations involving the aforementioned genes, was associated with decreased overall survival in nMTCL patients treated with CHOP-like regimens. Additionally, high-TMB was correlated with bulky disease, lower overall response rate, and higher mortality. Future studies using larger cohorts may validate our preliminary results that indicate TMB as a potential molecular biomarker associated with adverse prognosis in nMTCL.

Early-life establishment of tolerance to commensal bacteria at barrier surfaces carries enduring implications for immune health but remains poorly understood. Here, we showed that tolerance in skin was controlled by microbial interaction with a specialized subset of antigen-presenting cells. More particularly, CD301b+ type 2 conventional dendritic cells (DCs) in neonatal skin were specifically capable of uptake and presentation of commensal antigens for the generation of regulatory T (Treg) cells. CD301b+ DC2 were enriched for phagocytosis and maturation programs, while also expressing tolerogenic markers. In both human and murine skin, these signatures were reinforced by microbial uptake. In contrast to their adult counterparts or other early-life DC subsets, neonatal CD301b+ DC2 highly expressed the retinoic-acid-producing enzyme, RALDH2, the deletion of which limited commensal-specific Treg cell generation. Thus, synergistic interactions between bacteria and a specialized DC subset critically support early-life tolerance at the cutaneous interface.