Exploration of Tumor Biopsy Gene Signatures to Understand the Role of the Tumor Microenvironment in Outcomes to Lisocabtagene Maraleucel.

In the TRANSCEND NHL 001 study, 53% of patients with relapsed/refractory large B-cell lymphoma (LBCL) treated with lisocabtagene maraleucel (liso-cel) achieved a complete response (CR). To determine characteristics of patients who did and did not achieve a CR, we examined the tumor biology and microenvironment from lymph node tumor biopsies. LBCL biopsies from liso-cel-treated patients were taken pretreatment and ∼11 days posttreatment for RNA sequencing (RNA-seq) and multiplex immunofluorescence (mIF). We analyzed gene expression data from pretreatment biopsies (N = 78) to identify gene sets enriched in patients who achieved a CR to those with progressive disease. Pretreatment biopsies from month-3 CR patients displayed higher expression levels of T-cell and stroma-associated genes, and lower expression of cell-cycle genes. To interpret whether LBCL samples were "follicular lymphoma (FL)-like," we constructed an independent gene expression signature and found that patients with a higher "FL-like" gene expression score had longer progression-free survival (PFS). Cell of origin was not associated with response or PFS, but double-hit gene expression was associated with shorter PFS. The day 11 posttreatment samples (RNA-seq, N = 73; mIF, N = 53) had higher levels of chimeric antigen receptor (CAR) T-cell densities and CAR gene expression, general immune infiltration, and immune activation in patients with CR. Further, the majority of T cells in the day 11 samples were endogenous. Gene expression signatures in liso-cel-treated patients with LBCL can inform the development of combination therapies and next-generation CAR T-cell therapies.

Antitumor Potency of an Anti-CD19 Chimeric Antigen Receptor T-Cell Therapy, Lisocabtagene Maraleucel in Combination With Ibrutinib or Acalabrutinib.

Chimeric antigen receptor (CAR) T-cell therapy is a promising treatment for patients with CD19 B-cell malignancies. Combination strategies that improve CAR T-cell potency, limit tumor environment-mediated immune dysfunction, and directly reduce tumor burden may increase the potential for durable clinical benefit of CAR T-cell therapy. Lisocabtagene maraleucel (liso-cel) is a product therapy candidate being tested in patients with relapsed/refractory non-Hodgkin lymphoma or chronic lymphocytic leukemia. This study assessed the in vitro and in vivo functionality of CAR T cells transduced to express the anti-CD19 CAR of liso-cel in combination with ibrutinib or acalabrutinib. In prolonged stimulation assays, the presence of ibrutinib or acalabrutinib improved the CAR T-cell effector function. RNA-Seq analysis and surface marker profiling of these CAR T cells treated with ibrutinib but not acalabrutinib revealed gene expression changes consistent with skewing toward a memory-like, type 1 T-helper, Bruton tyrosine kinase phenotype. Ibrutinib or acalabrutinib improved CD19 tumor clearance and prolonged survival of tumor-bearing mice when used in combination with CAR T cells. A combination of the defined cell product therapy candidate, liso-cel, with ibrutinib or acalabrutinib is an attractive approach that may potentiate the promising clinical responses already achieved in CD19 B-cell malignancies with each of these single agents.

Expamers: a new technology to control T cell activation.

T cell activation is a cornerstone in manufacturing of T cell-based therapies, and precise control over T cell activation is important in the development of the next generation T-cell based therapeutics. This need cannot be fulfilled by currently available methods for T cell stimulation, in particular not in a time dependent manner. Here, we describe a modular activation reagent called Expamers, which addresses these limitations. Expamers are versatile stimuli that are intended for research and clinical use. They are readily soluble and can be rapidly bound and removed from the cell surface, allowing nearly instantaneous initiation and termination of activation signal, respectively. Hence, Expamers enable precise regulation of T cell stimulation duration and provide promise of control over T cell profiles in future products. Expamers can be easily adopted to different T cell production formats and have the potential to increase efficacy of T cell immunotherapeutics.

SLC46A3 Is Required to Transport Catabolites of Noncleavable Antibody Maytansine Conjugates from the Lysosome to the Cytoplasm.

Antibody-drug conjugates (ADC) target cytotoxic drugs to antigen-positive cells for treating cancer. After internalization, ADCs with noncleavable linkers are catabolized to amino acid-linker-warheads within the lysosome, which then enter the cytoplasm by an unknown mechanism. We hypothesized that a lysosomal transporter was responsible for delivering noncleavable ADC catabolites into the cytoplasm. To identify candidate transporters, we performed a phenotypic shRNA screen with an anti-CD70 maytansine-based ADC. This screen revealed the lysosomal membrane protein SLC46A3, the genetic attenuation of which inhibited the potency of multiple noncleavable antibody-maytansine ADCs, including ado-trastuzumab emtansine. In contrast, the potencies of noncleavable ADCs carrying the structurally distinct monomethyl auristatin F were unaffected by SLC46A3 attenuation. Structure-activity experiments suggested that maytansine is a substrate for SLC46A3. Notably, SLC46A3 silencing led to relative increases in catabolite concentrations in the lysosome. Taken together, our results establish SLC46A3 as a direct transporter of maytansine-based catabolites from the lysosome to the cytoplasm, prompting further investigation of SLC46A3 as a predictive response marker in breast cancer specimens.

Measurement of Cancer Cell Growth Heterogeneity through Lentiviral Barcoding Identifies Clonal Dominance as a Characteristic of In Vivo Tumor Engraftment.

Advances in the fields of cancer initiating cells and high-throughput in vivo shRNA screens have highlighted a need to observe the growth of tumor cells in cancer models at the clonal level. While in vivo cancer cell growth heterogeneity in xenografts has been described, it has yet to be measured. Here, we tested an approach to quantify the clonal growth heterogeneity of cancer cells in subcutaneous xenograft mouse models. Using a high-throughput sequencing method, we followed the fate in vitro and in vivo of ten thousand HCT-116 cells individually tagged with a unique barcode delivered by lentiviral transduction. While growth in vitro was less homogeneous than anticipated, we still find that 95% of the final cells derived from 80% of the original cells. In xenografts, however, 95% of the retrieved barcoded cells originated from only 6% of the initially injected cells, an effect we term "clonal dominance". We observed this clonal dominance in two additional xenograft models (MDA-MB-468 and A2780(cis)) and in two different host strains (NSG and Nude). By precisely and reproducibly quantifying clonal cancer cell growth in vivo, we find that a small subset of clones accounts for the vast majority of the descendant cells, even with HCT-116, a cell line reported to lack a tumor-initiating compartment. The stochastic in vivo selection process we describe has important implications for the fields of in vivo shRNA screening and tumor initiating cells.