Phase 1 Study of ROR1 Specific CAR T Cells in Advanced Hematopoietic and Epithelial Malignancies.

PURPOSE: The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is expressed in hematopoietic and epithelial cancers but has limited expression on normal adult tissues. This phase 1 study evaluated the safety of targeting ROR1 with autologous T-lymphocytes engineered to express a ROR1 chimeric antigen receptor (CAR). Secondary objectives evaluated persistence, trafficking, and antitumor activity of CAR T cells. PATIENTS & METHODS: Twenty-one patients with ROR1+ tumors received CAR T cells at one of four dose levels (DL): 3.3x105/1x106/3.3x106/1x107 cells/kg, administered after lymphodepletion with Cyclophosphamide/Fludarabine (Cy/Flu) or Oxaliplatin/Cyclophosphamide (Ox/Cy). Cohort A included patients with chronic lymphocytic leukemia (CLL, n=3); cohort B included patients with triple-negative breast cancer (TNBC, n=10) or non-small-cell lung cancer (NSCLC, n=8). A second infusion was administered to one patient in cohort A with residual CLL in the marrow and three patients in cohort B with stable disease after first infusion. RESULTS: Treatment was well tolerated apart from one dose limiting toxicity at DL4 in a patient with advanced NSCLC. Two of the three (67%) CLL patients showed robust CAR T expansion and a rapid antitumor response. In patients with NSCLC and TNBC, CAR T cells expanded to variable levels, infiltrated tumor poorly, and one of eighteen patients (5.5%) achieved partial response by RECIST 1.1. CONCLUSION: ROR1 CAR T cells were well tolerated in most patients. Antitumor activity was observed in CLL but was limited in TNBC and NSCLC. Immunogenicity of the CAR and lack of sustained tumor infiltration were identified as limitations.

Toll-Like Receptor 4 Agonist Injection With Concurrent Radiotherapy in Patients With Metastatic Soft Tissue Sarcoma: A Phase 1 Nonrandomized Controlled Trial.

Importance: Metastatic soft tissue sarcomas (STSs) have limited systemic therapy options, and immunomodulation has not yet meaningfully improved outcomes. Intratumoral (IT) injection of the toll-like receptor 4 (TLR4) agonist glycopyranosyl lipid A in stable-emulsion formulation (GLA-SE) has been studied as immunotherapy in other contexts. Objective: To evaluate the safety, efficacy, and immunomodulatory effects of IT GLA-SE with concurrent radiotherapy in patients with metastatic STS with injectable lesions. Design, Setting, and Participants: This phase 1 nonrandomized controlled trial of patients with STS was performed at a single academic sarcoma specialty center from November 17, 2014, to March 16, 2016. Data analysis was performed from August 2016 to September 2022. Interventions: Two doses of IT GLA-SE (5 µg and 10 µg for 8 weekly doses) were tested for safety in combination with concurrent radiotherapy of the injected lesion. Main Outcomes and Measures: Primary end points were safety and tolerability. Secondary and exploratory end points included local response rates as well as measurement of antitumor immunity with immunohistochemistry and T-cell receptor (TCR) sequencing of tumor-infiltrating and circulating lymphocytes. Results: Twelve patients (median [range] age, 65 [34-78] years; 8 [67%] female) were treated across the 2 dose cohorts. Intratumoral GLA-SE was well tolerated, with only 1 patient (8%) experiencing a grade 2 adverse event. All patients achieved local control of the injected lesion after 8 doses, with 1 patient having complete regression (mean regression, -25%; range, -100% to 4%). In patients with durable local response, there were detectable increases in tumor-infiltrating lymphocytes. In 1 patient (target lesion -39% at 259 days of follow-up), TCR sequencing revealed expansion of preexisting and de novo clonotypes, with convergence of numerous rearrangements coding for the same binding sequence (suggestive of clonal convergence to antitumor targets). Single-cell sequencing identified these same expanded TCR clones in peripheral blood after treatment; these T cells had markedly enhanced Tbet expression, suggesting TH1 phenotype. Conclusions and Relevance: In this nonrandomized controlled trial, IT GLA-SE with concurrent radiotherapy was well tolerated and provided more durable local control than radiotherapy alone. Patients with durable local response demonstrated enhanced IT T-cell clonal expansion, with matched expansion of these clonotypes in the circulation. Additional studies evaluating synergism of IT GLA-SE and radiotherapy with systemic immune modulation are warranted. Trial Registration: ClinicalTrials.gov Identifier: NCT02180698.

Conditionally Active, pH-Sensitive Immunoregulatory Antibodies Targeting VISTA and CTLA-4 Lead an Emerging Class of Cancer Therapeutics.

Immune checkpoints and other immunoregulatory targets can be difficult to precisely target due to expression on non-tumor immune cells critical to maintaining immune homeostasis in healthy tissues. On-target/off-tumor binding of therapeutics results in significant pharmacokinetic and pharmacodynamic problems. Target-mediated drug disposition (TMDD) significantly limits effective intratumoral drug levels and adversely affects anti-tumor efficacy. Target engagement outside the tumor environment may lead to severe immune-related adverse events (irAEs), resulting in a narrowing of the therapeutic window, sub-optimal dosing, or cessation of drug development altogether. Overcoming these challenges has become tractable through recent advances in antibody engineering and screening approaches. Here, we review the discovery and development of conditionally active antibodies with minimal binding to target at physiologic pH but high-affinity target binding at the low pH of the tumor microenvironment by focusing on the discovery and improved properties of pH-dependent mAbs targeting two T cell checkpoints, VISTA and CTLA-4.

Effects of HLA single chain trimer design on peptide presentation and stability.

MHC class I "single-chain trimer" molecules, coupling MHC heavy chain, ß2-microglobulin, and a specific peptide into a single polypeptide chain, are widely used in research. To more fully understand caveats associated with this design that may affect its use for basic and translational studies, we evaluated a set of engineered single-chain trimers with combinations of stabilizing mutations across eight different classical and non-classical human class I alleles with 44 different peptides, including a novel human/murine chimeric design. While, overall, single-chain trimers accurately recapitulate native molecules, care was needed in selecting designs for studying peptides longer or shorter than 9-mers, as single-chain trimer design could affect peptide conformation. In the process, we observed that predictions of peptide binding were often discordant with experiment and that yields and stabilities varied widely with construct design. We also developed novel reagents to improve the crystallizability of these proteins and confirmed novel modes of peptide presentation.

Transcriptional and functional analyses of neoantigen-specific CD4 T cells during a profound response to anti-PD-L1 in metastatic Merkel cell carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) often responds to PD-1 pathway blockade, regardless of tumor-viral status (~80% of cases driven by the Merkel cell polyomavirus (MCPyV)). Prior studies have characterized tumor-specific T cell responses to MCPyV, which have typically been CD8, but little is known about the T cell response to UV-induced neoantigens. METHODS: A patient in her mid-50s with virus-negative (VN) MCC developed large liver metastases after a brief initial response to chemotherapy. She received anti-PD-L1 (avelumab) and had a partial response within 4 weeks. Whole exome sequencing (WES) was performed to determine potential neoantigen peptides. Characterization of peripheral blood neoantigen T cell responses was evaluated via interferon-gamma (IFNγ) ELISpot, flow cytometry and single-cell RNA sequencing. Tumor-resident T cells were characterized by multiplexed immunohistochemistry. RESULTS: WES identified 1027 tumor-specific somatic mutations, similar to the published average of 1121 for VN-MCCs. Peptide prediction with a binding cut-off of ≤100 nM resulted in 77 peptides that were synthesized for T cell assays. Although peptides were predicted based on class I HLAs, we identified circulating CD4 T cells targeting 5 of 77 neoantigens. In contrast, no neoantigen-specific CD8 T cell responses were detected. Neoantigen-specific CD4 T cells were undetectable in blood before anti-PD-L1 therapy but became readily detectible shortly after starting therapy. T cells produced robust IFNγ when stimulated by neoantigen (mutant) peptides but not by the normal (wild-type) peptides. Single cell RNAseq showed neoantigen-reactive T cells expressed the Th1-associated transcription factor (T-bet) and associated cytokines. These CD4 T cells did not significantly exhibit cytotoxicity or non-Th1 markers. Within the pretreatment tumor, resident CD4 T cells were also Th1-skewed and expressed T-bet. CONCLUSIONS: We identified and characterized tumor-specific Th1-skewed CD4 T cells targeting multiple neoantigens in a patient who experienced a profound and durable partial response to anti-PD-L1 therapy. To our knowledge, this is the first report of neoantigen-specific T cell responses in MCC. Although CD4 and CD8 T cells recognizing viral tumor antigens are often detectible in virus-positive MCC, only CD4 T cells recognizing neoantigens were detected in this patient. These findings suggest that CD4 T cells can play an important role in the response to anti-PD-(L)1 therapy.

An In Vivo Model of Human Macrophages in Metastatic Melanoma.

Despite recent therapeutic progress, advanced melanoma remains lethal for many patients. The composition of the immune tumor microenvironment (TME) has decisive impacts on therapy response and disease outcome, and high-dimensional analyses of patient samples reveal the heterogeneity of the immune TME. Macrophages infiltrate TMEs and generally associate with tumor progression, but the underlying mechanisms are incompletely understood. Because experimental systems are needed to elucidate the functional properties of these cells, we developed a humanized mouse model reconstituted with human immune cells and human melanoma. We used two strains of recipient mice, supporting or not supporting the development of human myeloid cells. We found that human myeloid cells favored metastatic spread of the primary tumor, thereby recapitulating the cancer-supportive role of macrophages. We next analyzed the transcriptome of human immune cells infiltrating tumors versus other tissues. This analysis identified a cluster of myeloid cells present in the TME, but not in other tissues, which do not correspond to canonical M2 cells. The transcriptome of these cells is characterized by high expression of glycolytic enzymes and multiple chemokines and by low expression of gene sets associated with inflammation and adaptive immunity. Compared with humanized mouse results, we found transcriptionally similar myeloid cells in patient-derived samples of melanoma and other cancer types. The humanized mouse model described here thus complements patient sample analyses, enabling further elucidation of fundamental principles in melanoma biology beyond M1/M2 macrophage polarization. The model can also support the development and evaluation of candidate antitumor therapies.

Immunologic Gene Signature Analysis Correlates Myeloid Cells and M2 Macrophages with Time to Trabectedin Failure in Sarcoma Patients.

Patients with metastatic soft tissue sarcoma (STS) have a poor prognosis and few available systemic treatment options. Trabectedin is currently being investigated as a potential adjunct to immunotherapy as it has been previously shown to kill tumor-associated macrophages. In this retrospective study, we sought to identify biomarkers that would be relevant to trials combining trabectedin with immunotherapy. We performed a single-center retrospective study of sarcoma patients treated with trabectedin with long-term follow-up. Multiplex gene expression analysis using the NanoString platform was assessed, and an exploratory analysis using the lasso-penalized Cox regression and kernel association test for survival (MiRKAT-S) methods investigated tumor-associated immune cells and correlated their gene signatures to patient survival. In total, 147 sarcoma patients treated with trabectedin were analyzed, with a mean follow-up time of 5 years. Patients with fewer prior chemotherapy regimens were more likely to stay on trabectedin longer (pairwise correlation = -0.17, p = 0.04). At 5 years, increased PD-L1 expression corresponded to worse outcomes (HR = 1.87, p = 0.04, q = 0.199). Additionally, six immunologic gene signatures were associated with up to 7-year survival by MiRKAT-S, notably myeloid-derived suppressor cells (p = 0.023, q = 0.058) and M2 macrophages (p = 0.03, q = 0.058). We found that the number of chemotherapy regimens prior to trabectedin negatively correlated with the number of trabectedin cycles received, suggesting that patients may benefit from receiving trabectedin earlier in their therapy course. The correlation of trabectedin outcomes with immune cell infiltrates supports the hypothesis that trabectedin may function as an immune modulator and supports ongoing efforts to study trabectedin in combination with immunotherapy. Furthermore, tumors with an immunosuppressive microenvironment characterized by macrophage infiltration and high PD-L1 expression were less likely to benefit from trabectedin, which could guide clinicians in future treatment decisions.

Neoadjuvant Therapy Induces a Potent Immune Response to Sarcoma, Dominated by Myeloid and B Cells.

PURPOSE: To characterize changes in the soft-tissue sarcoma (STS) tumor immune microenvironment induced by standard neoadjuvant therapy with the goal of informing neoadjuvant immunotherapy trial design. EXPERIMENTAL DESIGN: Paired pre- and postneoadjuvant therapy specimens were retrospectively identified for 32 patients with STSs and analyzed by three modalities: multiplexed IHC, NanoString, and RNA sequencing with ImmunoPrism analysis. RESULTS: All 32 patients, representing a variety of STS histologic subtypes, received neoadjuvant radiotherapy and 21 (66%) received chemotherapy prior to radiotherapy. The most prevalent immune cells in the tumor before neoadjuvant therapy were myeloid cells (45% of all immune cells) and B cells (37%), with T (13%) and natural killer (NK) cells (5%) also present. Neoadjuvant therapy significantly increased the total immune cells infiltrating the tumors across all histologic subtypes for patients receiving neoadjuvant radiotherapy with or without chemotherapy. An increase in the percentage of monocytes and macrophages, particularly M2 macrophages, B cells, and CD4+ T cells was observed postneoadjuvant therapy. Upregulation of genes and cytokines associated with antigen presentation was also observed, and a favorable pathologic response (≥90% necrosis postneoadjuvant therapy) was associated with an increase in monocytic infiltrate. Upregulation of the T-cell checkpoint TIM3 and downregulation of OX40 were observed posttreatment. CONCLUSIONS: Standard neoadjuvant therapy induces both immunostimulatory and immunosuppressive effects within a complex sarcoma microenvironment dominated by myeloid and B cells. This work informs ongoing efforts to incorporate immune checkpoint inhibitors and novel immunotherapies into the neoadjuvant setting for STSs.

Impact of HPV status on immune responses in head and neck squamous cell carcinoma.

The main objective of our study was to understand the impact of immune cell composition and the tumor-reactivity of tumor infiltrating lymphocytes (TIL) in HPV-positive (HPV+) and HPV-negative (HPV-) head and neck squamous cell carcinoma (HNSCC). TIL cultures were established from primary HNSCC tumors, the T cell subsets were phenotypically characterized using flow cytometry, and Interferon (IFN)-γ ELISA assay was used to determine TIL function. NanoString Immune Profiler was used to determine an immune signature by HPV-status, and multiplex immunohistochemistry (MIHC) was used to quantify immune cell distributions and their spatial relationships. Results showed that HPV+ and HPV- HNSCC had similar capacity to expand IFN-γ reactive TIL populations, and these TIL populations had similar characteristics. NanoString analysis revealed increased differential expression of genes related to B cell functions in HPV+ HNSCC, which were significant at a Benjamini-Yekutieli adjusted p-value of < 0.001. MIHC also displayed increased CD8+ T cell and CD19/CD20+ B cell densities in the tumor region of HPV+ HNSCC as opposed to HPV- HNSCC (p < 0.01). Increases in a combined metric of tumor B cell content and stromal plasma cell content was associated with increased progression-free survival in HPV- HNSCC patients treated with immune checkpoint inhibitor therapy (p = 0.03). In summary, TIL populations expanded from HPV+ and HPV- HNSCC displayed similar IFN-γ reactivity. However, we identified a strong B-cell signature present within HPV+ HNSCC, and higher B and plasma cell content associated with improved PFS in HPV- HNSCC patients treated with immune checkpoint inhibitors.

Targeting an alternate Wilms' tumor antigen 1 peptide bypasses immunoproteasome dependency.

Designing effective antileukemic immunotherapy will require understanding mechanisms underlying tumor control or resistance. Here, we report a mechanism of escape from immunologic targeting in an acute myeloid leukemia (AML) patient, who relapsed 1 year after immunotherapy with engineered T cells expressing a human leukocyte antigen A*02 (HLA-A2)-restricted T cell receptor (TCR) specific for a Wilms' tumor antigen 1 epitope, WT1126-134 (TTCR-C4). Resistance occurred despite persistence of functional therapeutic T cells and continuous expression of WT1 and HLA-A2 by the patient's AML cells. Analysis of the recurrent AML revealed expression of the standard proteasome, but limited expression of the immunoproteasome, specifically the beta subunit 1i (ß1i), which is required for presentation of WT1126-134. An analysis of a second patient treated with TTCR-C4 demonstrated specific loss of AML cells coexpressing ß1i and WT1. To determine whether the WT1 protein continued to be processed and presented in the absence of immunoproteasome processing, we identified and tested a TCR targeting an alternative, HLA-A2-restricted WT137-45 epitope that was generated by immunoproteasome-deficient cells, including WT1-expressing solid tumor lines. T cells expressing this TCR (TTCR37-45) killed the first patients' relapsed AML resistant to WT1126-134 targeting, as well as other primary AML, in vitro. TTCR37-45 controlled solid tumor lines lacking immunoproteasome subunits both in vitro and in an NSG mouse model. As proteasome composition can vary in AML, defining and preferentially targeting these proteasome-independent epitopes may maximize therapeutic efficacy and potentially circumvent AML immune evasion by proteasome-related immunoediting.

Mavorixafor, an Orally Bioavailable CXCR4 Antagonist, Increases Immune Cell Infiltration and Inflammatory Status of Tumor Microenvironment in Patients with Melanoma.

Purpose: Mavorixafor is an oral, selective inhibitor of the CXCR4 chemokine receptor that modulates immune cell trafficking. A biomarker-driven phase Ib study (NCT02823405) was conducted in 16 patients with melanoma to investigate the hypothesis that mavorixafor favorably modulates immune cell profiles in the tumor microenvironment (TME) and to evaluate the safety of mavorixafor alone and in combination with pembrolizumab. Experimental Design: Serial biopsies of melanoma lesions were assessed after 3 weeks of mavorixafor monotherapy and after 6 weeks of combination treatment for immune cell markers by NanoString analysis for gene expression and by multiplexed immunofluorescent staining for in situ protein expression. Serum samples taken at biopsy timepoints were evaluated for key chemokine and cytokine alterations using the Myriad Rules Based Medicine multiplex immunoassays. Results: Within the TME, mavorixafor alone increased CD8+ T-cell infiltration, granzyme B signal, antigen presentation machinery, and both tumor inflammatory signature (TIS) and IFNγ gene expression signature scores. Increases in the key serum cytokines CXCL9 and CXCL10 were further enhanced when mavorixafor was combined with pembrolizumab. Adverse events (AE), as assessed by the investigator according to NCI Common Terminology Criteria for Adverse Events (v4.03), related to either mavorixafor or pembrolizumab (≥15%) were diarrhea, fatigue, maculopapular rash, and dry eye. Reported AEs were all ≤ grade 3. Conclusion/Discussion: Treatment with single-agent mavorixafor resulted in enhanced immune cell infiltration and activation in the TME, leading to increases in TIS and IFNγ gene signatures. Mavorixafor as a single agent, and in combination with pembrolizumab, has an acceptable safety profile. These data support further investigation of the use of mavorixafor for patients unresponsive to checkpoint inhibitors. Significance: Despite survival improvements in patients with melanoma treated with checkpoint inhibitor therapy, a significant unmet medical need exists for therapies that enhance effectiveness. We propose that mavorixafor sensitizes the melanoma tumor microenvironment and enhances the activity of checkpoint inhibitors, and thereby may translate to a promising treatment for broader patient populations.

The Liver-Immunity Nexus and Cancer Immunotherapy.

The impact of liver metastases on immune checkpoint-inhibitor effectiveness in patients with solid-tumor malignancies has been the focus of several recent clinical and translational studies. We review the literature describing the immune functions of the liver and particularly the mechanistic observations in these studies. The initial clinical observation was that pembrolizumab appeared to be much less effective in melanoma and non-small cell lung cancer (NSCLC) patients with liver metastasis. Subsequently other clinical studies have extended and reported similar findings with programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1) inhibitors in many cancers. Two recent translational studies in animal models have dissected the mechanism of this systemic immune suppression. In both studies CD11b+ suppressive macrophages generated by liver metastasis in a two-site MC38 model appear to delete CD8+ T cells in a FasL-dependent manner. In addition, regulatory T-cell (Treg) activation was observed and contributed to the distal immunosuppression. Finally, we discuss some of the interventions reported to address liver immune suppression, such as radiation therapy, combination checkpoint blockade, and Treg depletion.

Immune cell topography predicts response to PD-1 blockade in cutaneous T cell lymphoma.

Cutaneous T cell lymphomas (CTCL) are rare but aggressive cancers without effective treatments. While a subset of patients derive benefit from PD-1 blockade, there is a critically unmet need for predictive biomarkers of response. Herein, we perform CODEX multiplexed tissue imaging and RNA sequencing on 70 tumor regions from 14 advanced CTCL patients enrolled in a pembrolizumab clinical trial (NCT02243579). We find no differences in the frequencies of immune or tumor cells between responders and non-responders. Instead, we identify topographical differences between effector PD-1+ CD4+ T cells, tumor cells, and immunosuppressive Tregs, from which we derive a spatial biomarker, termed the SpatialScore, that correlates strongly with pembrolizumab response in CTCL. The SpatialScore coincides with differences in the functional immune state of the tumor microenvironment, T cell function, and tumor cell-specific chemokine recruitment and is validated using a simplified, clinically accessible tissue imaging platform. Collectively, these results provide a paradigm for investigating the spatial balance of effector and suppressive T cell activity and broadly leveraging this biomarker approach to inform the clinical use of immunotherapies.

CD4+ T cell and M2 macrophage infiltration predict dedifferentiated liposarcoma patient outcomes.

BACKGROUND: Dedifferentiated liposarcoma (DDLPS) is one of the most common soft tissue sarcoma subtypes and is devastating in the advanced/metastatic stage. Despite the observation of clinical responses to PD-1 inhibitors, little is known about the immune microenvironment in relation to patient prognosis. METHODS: We performed a retrospective study of 61 patients with DDLPS. We completed deep sequencing of the T-cell receptor (TCR) ß-chain and RNA sequencing for predictive modeling, evaluating both immune markers and tumor escape genes. Hierarchical clustering and recursive partitioning were employed to elucidate relationships of cellular infiltrates within the tumor microenvironment, while an immune score for single markers was created as a predictive tool. RESULTS: Although many DDLPS samples had low TCR clonality, high TCR clonality combined with low T-cell fraction predicted lower 3-year overall survival (p=0.05). Higher levels of CD14+ monocytes (p=0.02) inversely correlated with 3-year recurrence-free survival (RFS), while CD4+ T-cell infiltration (p=0.05) was associated with a higher RFS. Genes associated with longer RFS included PD-1 (p=0.003), ICOS (p=0.006), BTLA (p=0.033), and CTLA4 (p=0.02). In a composite immune score, CD4+ T cells had the strongest positive predictive value, while CD14+ monocytes and M2 macrophages had the strongest negative predictive values. CONCLUSIONS: Immune cell infiltration predicts clinical outcome in DDLPS, with CD4+ cells associated with better outcomes; CD14+ cells and M2 macrophages are associated with worse outcomes. Future checkpoint inhibitor studies in DDLPS should incorporate immunosequencing and gene expression profiling techniques that can generate immune landscape profiles.

IL-15 mediated expansion of rare durable memory T cells following adoptive cellular therapy.

BACKGROUND: Synovial sarcoma (SS) and myxoid/round cell liposarcoma (MRCL) are ideal solid tumors for the development of adoptive cellular therapy (ACT) targeting NY-ESO-1, as a high frequency of tumors homogeneously express this cancer-testes antigen. Data from early phase clinical trials have shown antitumor activity after the adoptive transfer of NY-ESO-1-specific T cells. In these studies, persistence of NY-ESO-1 specific T cells is highly correlated with response to ACT, but patients often continue to have detectable transferred cells in their peripheral blood following progression. METHOD: We performed a phase I clinical trial evaluating the safety of NY-ESO-1-specific endogenous T cells (ETC) following cyclophosphamide conditioning. Peripheral blood mononuclear cells (PBMCs) from treated patients were evaluated by flow cytometry and gene expression analysis as well as through ex vivo culture assays with and without IL-15. RESULTS: Four patients were treated in a cohort using ETC targeting NY-ESO-1 following cyclophosphamide conditioning. Treatment was well tolerated without significant toxicity, but all patients ultimately had disease progression. In two of four patients, we obtained post-treatment tumor tissue and in both, NY-ESO-1 antigen was retained despite clear detectable persisting NY-ESO-1-specific T cells in the peripheral blood. Despite a memory phenotype, these persisting cells lacked markers of proliferation or activation. However, in ex vivo culture assays, they could be induced to proliferate and kill tumor using IL-15. These results were also seen in PBMCs from two patients who received gene-engineered T-cell receptor-based products at other centers. CONCLUSIONS: ETC targeting NY-ESO-1 with single-agent cyclophosphamide alone conditioning was well tolerated in patients with SS and those with MRCL. IL-15 can induce proliferation and activity in persisting NY-ESO-1-specific T cells even in patients with disease progression following ACT. These results support future work evaluating whether IL-15 could be incorporated into ACT trials post-infusion or at the time of progression.

Intratumoral Plasmid IL12 Expands CD8+ T Cells and Induces a CXCR3 Gene Signature in Triple-negative Breast Tumors that Sensitizes Patients to Anti-PD-1 Therapy.

PURPOSE: Triple-negative breast cancer (TNBC) is an aggressive disease with limited therapeutic options. Antibodies targeting programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1) have entered the therapeutic landscape in TNBC, but only a minority of patients benefit. A way to reliably enhance immunogenicity, T-cell infiltration, and predict responsiveness is critically needed. PATIENTS AND METHODS: Using mouse models of TNBC, we evaluate immune activation and tumor targeting of intratumoral IL12 plasmid followed by electroporation (tavokinogene telseplasmid; Tavo). We further present a single-arm, prospective clinical trial of Tavo monotherapy in patients with treatment refractory, advanced TNBC (OMS-I140). Finally, we expand these findings using publicly available breast cancer and melanoma datasets. RESULTS: Single-cell RNA sequencing of murine tumors identified a CXCR3 gene signature (CXCR3-GS) following Tavo treatment associated with enhanced antigen presentation, T-cell infiltration and expansion, and PD-1/PD-L1 expression. Assessment of pretreatment and posttreatment tissue from patients confirms enrichment of this CXCR3-GS in tumors from patients that exhibited an enhancement of CD8+ T-cell infiltration following treatment. One patient, previously unresponsive to anti-PD-L1 therapy, but who exhibited an increased CXCR3-GS after Tavo treatment, went on to receive additional anti-PD-1 therapy as their immediate next treatment after OMS-I140, and demonstrated a significant clinical response. CONCLUSIONS: These data show a safe, effective intratumoral therapy that can enhance antigen presentation and recruit CD8 T cells, which are required for the antitumor efficacy. We identify a Tavo treatment-related gene signature associated with improved outcomes and conversion of nonresponsive tumors, potentially even beyond TNBC.

Detection of engineered T cells in FFPE tissue by multiplex in situ hybridization and immunohistochemistry.

Identifying engineered T cells in situ is important to understand the location, persistence, and phenotype of these cells in patients after adoptive T cell therapy. While engineered cells are routinely characterized in fresh tissue or blood from patients by flow cytometry, it is difficult to distinguish them from endogenous cells in formalin-fixed, paraffin-embedded (FFPE) tissue biopsies. To overcome this limitation, we have developed a method for characterizing engineered T cells in fixed tissue using in situ hybridization (ISH) to the woodchuck hepatitis post-transcriptional regulatory element (WPRE) common in many lentiviral vectors used to transduce chimeric antigen receptor T (CAR-T) and T cell receptor T (TCR-T) cells, coupled with alternative permeabilization conditions that allows subsequent multiplex immunohistochemical (mIHC) staining within the same image. This new method provides the ability to mark the cells by ISH, and simultaneously stain for cell-associated proteins to immunophenotype CAR/TCR modified T cells within tumors, as well as assess potential roles of these cells in on-target/off-tumor toxicity in other tissue.

Microdissected "cuboids" for microfluidic drug testing of intact tissues.

As preclinical animal tests often do not accurately predict drug effects later observed in humans, most drugs under development fail to reach the market. Thus there is a critical need for functional drug testing platforms that use human, intact tissues to complement animal studies. To enable future multiplexed delivery of many drugs to one small biopsy, we have developed a multi-well microfluidic platform that selectively treats cuboidal-shaped microdissected tissues or "cuboids" with well-preserved tissue microenvironments. We create large numbers of uniformly-sized cuboids by semi-automated sectioning of tissue with a commercially available tissue chopper. Here we demonstrate the microdissection method on normal mouse liver, which we characterize with quantitative 3D imaging, and on human glioma xenograft tumors, which we evaluate after time in culture for viability and preservation of the microenvironment. The benefits of size uniformity include lower heterogeneity in future biological assays as well as facilitation of their physical manipulation by automation. Our prototype platform consists of a microfluidic circuit whose hydrodynamic traps immobilize the live cuboids in arrays at the bottom of a multi-well plate. Fluid dynamics simulations enabled the rapid evaluation of design alternatives and operational parameters. We demonstrate the proof-of-concept application of model soluble compounds such as dyes (CellTracker, Hoechst) and the cancer drug cisplatin. Upscaling of the microfluidic platform and microdissection method to larger arrays and numbers of cuboids could lead to direct testing of human tissues at high throughput, and thus could have a significant impact on drug discovery and personalized medicine.

Immunogenic Chemotherapy Enhances Recruitment of CAR-T Cells to Lung Tumors and Improves Antitumor Efficacy when Combined with Checkpoint Blockade.

Adoptive therapy using chimeric antigen receptor-modified T cells (CAR-T cells) is effective in hematologic but not epithelial malignancies, which cause the greatest mortality. In breast and lung cancer patients, CAR-T cells targeting the tumor-associated antigen receptor tyrosine kinase-like orphan receptor 1 (ROR1) infiltrate tumors poorly and become dysfunctional. To test strategies for enhancing efficacy, we adapted the KrasLSL-G12D/+;p53f/f autochthonous model of lung adenocarcinoma to express the CAR target ROR1. Murine ROR1 CAR-T cells transferred after lymphodepletion with cyclophosphamide (Cy) transiently control tumor growth but infiltrate tumors poorly and lose function, similar to what is seen in patients. Adding oxaliplatin (Ox) to the lymphodepletion regimen activates tumor macrophages to express T-cell-recruiting chemokines, resulting in improved CAR-T cell infiltration, remodeling of the tumor microenvironment, and increased tumor sensitivity to anti-PD-L1. Combination therapy with Ox/Cy and anti-PD-L1 synergistically improves CAR-T cell-mediated tumor control and survival, providing a strategy to improve CAR-T cell efficacy in the clinic.