Overall survival of patients with cHL who progress after autologous stem cell transplant: results in the novel agent era.

In the pre-novel agent era, the median postprogression overall survival (PPS) of patients with classic Hodgkin lymphoma (cHL) who progress after autologous stem cell transplant (ASCT) was 2 to 3 years. Recently, checkpoint inhibitors (CPI) and brentuximab vedotin (BV) have improved the depth and durability of response in this population. Here, we report the estimate of PPS in patients with relapsed cHL after ASCT in the era of CPI and BV. In this multicenter retrospective study of 15 participating institutions, adult patients with relapsed cHL after ASCT were included. Study objective was postprogression overall survival (PPS), defined as the time from posttransplant progression to death or last follow-up. Of 1158 patients who underwent ASCT, 367 had progressive disease. Median age was 34 years (range, 27-46) and 192 were male. Median PPS was 114.57 months (95% confidence interval [CI], 91-not achieved) or 9.5 years. In multivariate analysis, increasing age, progression within 6 months, and pre-ASCT positive positron emission tomography scan were associated with inferior PPS. When adjusted for these features, patients who received CPI, but not BV, as first treatment for post-ASCT progression had significantly higher PPS than the no CPI/no BV group (hazard ratio, 3.5; 95% CI, 1.6-7.8; P = .001). Receipt of allogeneic SCT (Allo-SCT) did not improve PPS. In the era of novel agents, progressive cHL after ASCT had long survival that compares favorably with previous reports. Patients who receive CPI as first treatment for progression had higher PPS. Receipt to Allo-SCT was not associated with PPS in this population.

Improved outcomes for relapsed/refractory Hodgkin lymphoma after autologous transplantation in the era of novel agents.

The treatment landscape of relapsed/refractory (R/R) classic Hodgkin lymphoma (cHL) has evolved significantly over the past decade after the approval of brentuximab vedotin (BV) and the programmed death-1 (PD-1) inhibitors. We evaluated how outcomes and practice patterns have changed for patients with R/R cHL who underwent autologous hematopoietic cell transplantation (AHCT) at our institution from 2011 to 2020 (N = 183) compared with those from 2001 to 2010 (N = 159) and evaluated prognostic factors for progression-free survival (PFS) and overall survival (OS) in both eras. OS was superior in the modern era with a trend toward lower nonrelapse mortality beyond 2 years after transplant. Among patients who progressed after AHCT, 4-year postprogression survival increased from 43.3% to 71.4% in the modern era, reflecting increasing use of BV and the PD-1 inhibitors. In multivariable analysis for patients that underwent transplant in the modern era, age ≥45 years, primary refractory disease, and lack of complete remission pre-AHCT were associated with inferior PFS, whereas receipt of a PD-1 inhibitor-based regimen pre-AHCT was associated with superior PFS. Extranodal disease at relapse was associated with inferior OS. Our study demonstrates improved survival for R/R cHL after AHCT in the modern era attributed to more effective salvage regimens allowing for better disease control pre-AHCT and improved outcomes for patients who progressed after AHCT. Excellent outcomes were observed with PD-1 inhibitor-based salvage regimens pre-AHCT and support a randomized trial evaluating immunotherapy in the second line setting.

Checkpoint inhibitor-based salvage regimens prior to autologous stem cell transplant improve event-free survival in relapsed/refractory classic Hodgkin lymphoma.

Clinical trials of novel salvage therapies have encouraging outcomes for relapsed/refractory transplant-eligible classic Hodgkin lymphoma (R/R cHL) but comparison with conventional chemotherapy is lacking. Herein, we report the final analysis of a multicenter retrospective cohort of R/R cHL assessing outcomes by type of salvage therapy before autologous stem cell transplant (ASCT). R/R cHL patients who underwent ASCT at 14 institutions across the United States were included. Outcomes were compared among patients receiving conventional chemotherapy, brentuximab vedotin (BV) + chemotherapy, BV alone, and a checkpoint inhibitor (CPI)-based regimens before ASCT. Study endpoints included event-free survival (EFS), progression-free survival (PFS), and overall survival (OS). All endpoints are defined from relapse. Of 936 patients, 728 received conventional chemotherapy, 73 received BV + chemotherapy, 70 received BV alone, and 65 received CPI-based regimens prior to ASCT. When adjusted for time to relapse, pre-ASCT response and use of BV maintenance, patients receiving CPI-based regimens had superior 2-year EFS compared to conventional chemotherapy, BV + chemotherapy, and BV alone (79.7, 49.6, 62.3, and 36.9%, respectively, p < .0001). Among 649 patients transplanted after 1 line of salvage therapy, CPI-based regimens were associated with superior 2-year PFS compared to conventional chemotherapy (98% vs. 68.8%, hazard ratio: 0.1, 95% confidence interval: 0.03-0.5, p < .0001). OS did not differ by pre-ASCT salvage regimen. In this large multicenter retrospective study, CPI-based regimens improved EFS and PFS compared to other salvage regimens independent of pre-ASCT response. These data support earlier sequencing of CPI-based regimens in R/R cHL in the pre-ASCT setting.

Targeted Mutational Profiling Reveals Clonal Relationships in Metachronous Occurrence of Classic Hodgkin and Mediastinal Large B-Cell Lymphomas.

Classic Hodgkin lymphoma (CHL) patients may infrequently present with a prior or recurrent disease with discordant histology resembling non-Hodgkin lymphomas. These include primary mediastinal large B-cell lymphoma (PMBL), diffuse large B-cell lymphoma (DLBCL), or mediastinal gray-zone lymphoma (MGZL). Such patients are often refractory to standard therapy and their diagnosis is hampered by significant morphologic and immunophenotypic overlap and insufficient molecular data. Among 509 CHL patients seen at an academic medical center, 6 patients had a prior or subsequent diagnosis different from CHL. Paired tissue samples were evaluated by targeted mutational analysis using a 164-gene panel. Our findings show multiple shared variants indicative of a clonal relationship between the CHL and the PMBL, DLBCL, or MGZL diagnoses. Most frequent mutated genes included TNFAIP3 (4 of 6, 66.7%), STAT6 (3 or 6, 50%), ARID1A (3 of 6, 50%), and XPO1 (3 of 5, 60%). Three patients showed the same oncogenic variant within the XPO1 gene (E571K), and mutations in TNFAIP3 and B2M were observed in 2 of the 5 patients with shared variants. In addition, differences in the mutation profile between the lymphoma pairs were also observed, which could represent clonal evolution. Mutational profiling could be of benefit in patients with recurrent/refractory disease with discordant histology, where the clonal relationship could be helpful to inform and guide therapeutic decisions. These findings provide further evidence of a true biological continuum surrounding CHL, PMBL, DLBCL, and MGZL and shed light on underlying genetic events and their clinical impact.

Engineering a natural ligand-based CAR: directed evolution of the stress-receptor NKp30.

B7H6, a stress-induced ligand which binds to the NK cell receptor NKp30, has recently emerged as a promising candidate for immunotherapy due to its tumor-specific expression on a broad array of human tumors. NKp30 can function as a chimeric antigen receptor (CAR) extracellular domain but exhibits weak binding with a fast on and off rate to B7H6 compared to the TZ47 anti-B7H6 single-chain variable fragment (scFv). Here, directed evolution using yeast display was employed to isolate novel NKp30 variants that bind to B7H6 with higher affinity compared to the native receptor but retain its fast association and dissociation profile. Two variants, CC3 and CC5, were selected for further characterization and were expressed as soluble Fc-fusion proteins and CARs containing CD28 and CD3ς intracellular domains. We observed that Fc-fusion protein forms of NKp30 and its variants were better able to bind tumor cells expressing low levels of B7H6 than TZ47, and that the novel variants generally exhibited improved in vitro tumor cell killing relative to NKp30. Interestingly, CAR T cells expressing the engineered variants produced unique cytokine signatures in response to multiple tumor types expressing B7H6 compared to both NKp30 and TZ47. These findings suggest that natural CAR receptors can be fine-tuned to produce more desirable signaling outputs while maintaining evolutionary advantages in ligand recognition relative to scFvs.

Engineering a potent inhibitor of matriptase from the natural hepatocyte growth factor activator inhibitor type-1 (HAI-1) protein.

Dysregulated matriptase activity has been established as a key contributor to cancer progression through its activation of growth factors, including the hepatocyte growth factor (HGF). Despite its critical role and prevalence in many human cancers, limitations to developing an effective matriptase inhibitor include weak binding affinity, poor selectivity, and short circulating half-life. We applied rational and combinatorial approaches to engineer a potent inhibitor based on the hepatocyte growth factor activator inhibitor type-1 (HAI-1), a natural matriptase inhibitor. The first Kunitz domain (KD1) of HAI-1 has been well established as a minimal matriptase-binding and inhibition domain, whereas the second Kunitz domain (KD2) is inactive and involved in negative regulation. Here, we replaced the inactive KD2 domain of HAI-1 with an engineered chimeric variant of KD2/KD1 domains and fused the resulting construct to an antibody Fc domain to increase valency and circulating serum half-life. The final protein variant contains four stoichiometric binding sites that we showed were needed to effectively inhibit matriptase with a Ki of 70 ± 5 pm, an increase of 120-fold compared with the natural HAI-1 inhibitor, to our knowledge making it one of the most potent matriptase inhibitors identified to date. Furthermore, the engineered inhibitor demonstrates a protease selectivity profile similar to that of wildtype KD1 but distinct from that of HAI-1. It also inhibits activation of the natural pro-HGF substrate and matriptase expressed on cancer cells with at least an order of magnitude greater efficacy than KD1.

Development of a Protease Biosensor Based on a Dimerization-Dependent Red Fluorescent Protein.

Dysregulated activity of the protease matriptase is a key contributor to aggressive tumor growth, cancer metastasis, and osteoarthritis. Methods for the detection and quantification of matriptase activity and inhibition would be useful tools. To address this need, we developed a matriptase-sensitive protein biosensor based on a dimerization-dependent red fluorescent protein (ddRFP) reporter system. In this platform, two adjoining protein domains, connected by a protease-labile linker, produce fluorescence when assembled and are nonfluorescent when the linker is cleaved by matriptase. A panel of ddRFP-based matriptase biosensor designs was created that contained different linker lengths between the protein domains. These constructs were characterized for linker-specific cleavage, matriptase activity, and matriptase selectivity; a biosensor containing a RSKLRVGGH linker (termed B4) was expressed at high yields and displayed both high catalytic efficiency and matriptase specificity. This biosensor detects matriptase inhibition by soluble and yeast cell surface expressed inhibitor domains with up to a 5-fold dynamic range and also detects matriptase activity expressed by human cancer cell lines. In addition to matriptase, we highlight a strategy that can be used to create effective biosensors for quantifying activity and inhibition of other proteases of interest.

A critical role for the mTORC2 pathway in lung fibrosis.

A characteristic of dysregulated wound healing in IPF is fibroblastic-mediated damage to lung epithelial cells within fibroblastic foci. In these foci, TGF-ß and other growth factors activate fibroblasts that secrete growth factors and matrix regulatory proteins, which activate a fibrotic cascade. Our studies and those of others have revealed that Akt is activated in IPF fibroblasts and it mediates the activation by TGF-ß of pro-fibrotic pathways. Recent studies show that mTORC2, a component of the mTOR pathway, mediates the activation of Akt. In this study we set out to determine if blocking mTORC2 with MLN0128, an active site dual mTOR inhibitor, which blocks both mTORC1 and mTORC2, inhibits lung fibrosis. We examined the effect of MLN0128 on TGF-ß-mediated induction of stromal proteins in IPF lung fibroblasts; also, we looked at its effect on TGF-ß-mediated epithelial injury using a Transwell co-culture system. Additionally, we assessed MLN0128 in the murine bleomycin lung model. We found that TGF-ß induces the Rictor component of mTORC2 in IPF lung fibroblasts, which led to Akt activation, and that MLN0128 exhibited potent anti-fibrotic activity in vitro and in vivo. Also, we observed that Rictor induction is Akt-mediated. MLN0128 displays multiple anti-fibrotic and lung epithelial-protective activities; it (1) inhibited the expression of pro-fibrotic matrix-regulatory proteins in TGF-ß-stimulated IPF fibroblasts; (2) inhibited fibrosis in a murine bleomycin lung model; and (3) protected lung epithelial cells from injury caused by TGF-ß-stimulated IPF fibroblasts. Our findings support a role for mTORC2 in the pathogenesis of lung fibrosis and for the potential of active site mTOR inhibitors in the treatment of IPF and other fibrotic lung diseases.