A blood-based immune marker for resistance to pembrolizumab in patients with metastatic urothelial cancer.

PD1 inhibition is effective in patients with metastatic urothelial cancer (mUC), yet a large fraction of patients does not respond. In this study, we aimed to identify a blood-based immune marker associated with non-response to facilitate patient selection for anti-PD1. To this end, we quantified 18 immune cell populations using multiplex flow cytometry in blood samples from 71 patients with mUC (as part of a biomarker discovery trial; NCT03263039, registration date 28-08-2017). Patients were classified as responder (ongoing complete or partial response, or stable disease; n = 25) or non-responder (progressive disease; n = 46) according to RECIST v1.1 at 6 months of treatment with pembrolizumab. We observed no differences in numbers of lymphocytes, T-cells, granulocytes, monocytes or their subsets between responders and non-responders at baseline. In contrast, analysis of ratios of immune cell populations revealed that a high mature neutrophil-to-T-cell ratio (MNTR) exclusively identified non-responders. In addition, the survival of patients with high versus low MNTR was poor: median overall survival (OS) 2.2 vs 8.9 months (hazard ratio (HR) 6.6; p < 0.00001), and median progression-free survival (PFS) 1.5 vs 5.2 months (HR 5.6; p < 0.0001). The associations with therapy response, OS, and PFS for the MNTR were stronger than for the classical neutrophil-to-lymphocyte ratio (HR for OS 3.5, and PFS 3) and the PD-L1 combined positivity score (HR for OS 1.9, and PFS 2.1). In conclusion, the MNTR distinctly and uniquely identified non-responders to treatment and may represent a novel pre-treatment blood-based immune metric to select patients with mUC for treatment with pembrolizumab.

Lack of B and T cell reactivity towards IDH1R132H in blood and tumor tissue from LGG patients.

PURPOSE: Mutations in the isocitrate dehydrogenase-1 gene (IDH1) occur at high frequency in grade II-III gliomas (LGGs). IDH1 mutations are somatic, missense and heterozygous affecting codon 132 in the catalytic pocket of the enzyme. In LGG, most mutations (90%) result in an arginine to histidine substitution (IDH1R132H) providing a neo-epitope that is expressed in all tumor cells. To assess the immunogenic nature of this epitope, and its potential use to develop T cell treatments, we measured IDH1R132H-specific B and T cell reactivity in blood and tumor tissue of LGG patients. METHODS: Sera from IDH1R132H-mutated LGG patients (n = 27) were assayed for the presence of a neo-specific antibody response using ELISA. In addition, PBMCs (n = 36) and tumor-infiltrating lymphocytes (TILs, n = 10) were measured for T cell activation markers and IFN-γ production by flow cytometry and ELISA. In some assays, frequencies of CD4 T cells specific for mutated peptide presented by HLA-DR were enriched prior to T cell monitoring assays. RESULTS: Despite high sensitivity of our assay, we failed to detect IDH1R132H-specific IgG in sera of LGG patients. Similarly, we did not observe CD4 T cell reactivity towards IDH1R132H in blood, neither did we observe such reactivity following pre-enrichment of frequencies of IDH1R132H-specific CD4 T cells. Finally, we did not detect IDH1R132H-specific CD4 T cells among TILs. CONCLUSIONS: The absence of both humoral and cellular responses in blood and tumors of LGG patients indicates that IDH1R132H is not sufficiently immunogenic and devaluates its further therapeutic exploitation, at least in the majority of LGG patients.

MAGE-C2-Specific TCRs Combined with Epigenetic Drug-Enhanced Antigenicity Yield Robust and Tumor-Selective T Cell Responses.

Adoptive T cell therapy has shown significant clinical success for patients with advanced melanoma and other tumors. Further development of T cell therapy requires improved strategies to select effective, yet nonself-reactive, TCRs. In this study, we isolated 10 TCR sequences against four MAGE-C2 (MC2) epitopes from melanoma patients who showed clinical responses following vaccination that were accompanied by significant frequencies of anti-MC2 CD8 T cells in blood and tumor without apparent side effects. We introduced these TCRs into T cells, pretreated tumor cells of different histological origins with the epigenetic drugs azacytidine and valproate, and tested tumor and self-reactivities of these TCRs. Pretreatment of tumor cells upregulated MC2 gene expression and enhanced recognition by T cells. In contrast, a panel of normal cell types did not express MC2 mRNA, and similar pretreatment did not result in recognition by MC2-directed T cells. Interestingly, the expression levels of MC2, but not those of CD80, CD86, or programmed death-ligand 1 or 2, correlated with T cell responsiveness. One of the tested TCRs consistently recognized pretreated MC2(+) cell lines from melanoma, head and neck, bladder, and triple-negative breast cancers but showed no response to MHC-eluted peptides or peptides highly similar to MC2. We conclude that targeting MC2 Ag, combined with epigenetic drug-enhanced antigenicity, allows for significant and tumor-selective T cell responses.

TCRs genetically linked to CD28 and CD3ε do not mispair with endogenous TCR chains and mediate enhanced T cell persistence and anti-melanoma activity.

Adoptive transfer of T cells that are gene engineered to express a defined TCR represents a feasible and promising therapy for patients with tumors. However, TCR gene therapy is hindered by the transient presence and effectiveness of transferred T cells, which are anticipated to be improved by adequate T cell costimulation. In this article, we report the identification and characterization of a novel two-chain TCR linked to CD28 and CD3ε (i.e., TCR:28ε). This modified TCR demonstrates enhanced binding of peptide-MHC and mediates enhanced T cell function following stimulation with peptide compared with wild-type TCR. Surface expression of TCR:28ε depends on the transmembrane domain of CD28, whereas T cell functions depend on the intracellular domains of both CD28 and CD3ε, with IL-2 production showing dependency on CD28:LCK binding. TCR:28ε, but not wild-type TCR, induces detectable immune synapses in primary human T cells, and such immune synapses show significantly enhanced accumulation of TCR transgenes and markers of early TCR signaling, such as phosphorylated LCK and ERK. Importantly, TCR:28ε does not show signs of off-target recognition, as evidenced by lack of TCR mispairing, as well as preserved specificity. Notably, when testing TCR:28ε in immune-competent mice, we observed a drastic increase in T cell survival, which was accompanied by regression of large melanomas with limited recurrence. Our data argue that TCR transgenes that contain CD28, and, thereby, may provide T cell costimulation in an immune-suppressive environment, represent candidate receptors to treat patients with tumors.

TCR gene transfer: MAGE-C2/HLA-A2 and MAGE-A3/HLA-DP4 epitopes as melanoma-specific immune targets.

Adoptive therapy with TCR gene-engineered T cells provides an attractive and feasible treatment option for cancer patients. Further development of TCR gene therapy requires the implementation of T-cell target epitopes that prevent "on-target" reactivity towards healthy tissues and at the same time direct a clinically effective response towards tumor tissues. Candidate epitopes that meet these criteria are MAGE-C2(336-344)/HLA-A2 (MC2/A2) and MAGE-A3(243-258)/HLA-DP4 (MA3/DP4). We molecularly characterized TCRαß genes of an MC2/A2-specific CD8 and MA3/DP4-specific CD4 T-cell clone derived from melanoma patients who responded clinically to MAGE vaccination. We identified MC2/A2 and MA3/DP4-specific TCR-Vα3/Vß28 and TCR-Vα38/Vß2 chains and validated these TCRs in vitro upon gene transfer into primary human T cells. The MC2 and MA3 TCR were surface-expressed and mediated CD8 T-cell functions towards melanoma cell lines and CD4 T-cell functions towards dendritic cells, respectively. We intend to start testing these MAGE-specific TCRs in phase I clinical trial.

Gene Engineering T Cells with T-Cell Receptor for Adoptive Therapy.

Prior to clinical testing of adoptive T-cell therapy with T-cell receptor (TCR)-engineered T cells, TCRs need to be retrieved, annotated, gene-transferred, and extensively tested in vitro to accurately assess specificity and sensitivity of target recognition. Here, we present a fundamental series of protocols that cover critical preclinical parameters, thereby enabling the selection of candidate TCRs for clinical testing.

Germline Variation in PDCD1 Is Associated with Overall Survival in Patients with Metastatic Melanoma Treated with Anti-PD-1 Monotherapy.

A substantial number of melanoma patients do not benefit from therapy with anti-PD-1. Therefore, we investigated the predictive value of single nucleotide polymorphisms (SNPs) in genes related to the PD-1 axis in patients with metastatic melanoma. From 119 consecutive melanoma patients who were treated with pembrolizumab or nivolumab monotherapy, blood samples were genotyped for 11 SNPs in nine genes. Associations between SNPs and OS were tested using Cox regression analysis and internally validated by bootstrapping. For SNPs with a statistical significance, an expression quantitative trait loci (eQTL) analysis was performed. In a subset of patients, immunophenotyping was performed. Patients with a SNP in PDCD1 (804C > T; rs2227981) had a significantly poorer OS with a 3-year OS rate of 51.8%, as compared to 71% in wild type patients (hazard ratio [HR] 2.37; 95% CI: 1.11-5.04; p = 0.026). eQTL analysis showed that this SNP was associated with decreased gene expression. In addition, PDCD1 804C > T carriers had a reduced fraction of peripheral PD-1+CD4+ T cells. No other associations between SNPs and OS were found. PDCD1 804C > T is associated with poorer OS after anti-PD-1 monotherapy in patients with metastatic melanoma. This SNP may affect clinical benefit from ICIs by decreasing transcription initiation and expression of PD-1 in T cells.

T Cells Expressing a TCR-Like Antibody Selected Against the Heteroclitic Variant of a Shared MAGE-A Epitope Do Not Recognise the Cognate Epitope.

Antibodies-recognising peptides bound to the major histocompatibility complex (pMHC) represent potentially valuable and promising targets for chimeric antigen receptor (CAR) T cells to treat patients with cancer. Here, a human phage-Fab library has been selected using HLA-A2 complexed with a heteroclitic peptide variant from an epitope shared among multiple melanoma-associated antigens (MAGEs). DNA restriction analyses and phage ELISAs confirmed selection of unique antibody clones that specifically bind to HLA-A2 complexes or HLA-A2-positive target cells loaded with native or heteroclitic peptide. Antibodies selected against heteroclitic peptide, in contrast to native peptide, demonstrated significantly lower to even negligible binding towards native peptide or tumour cells that naturally expressed peptides. The binding to native peptide was not rescued by phage panning with antigen-positive tumour cells. Importantly, when antibodies directed against heteroclitic peptides were engineered into CARs and expressed by T cells, binding to native peptides and tumour cells was minimal to absent. In short, TCR-like antibodies, when isolated from a human Fab phage library using heteroclitic peptide, fail to recognise its native peptide. We therefore argue that peptide modifications to improve antibody selections should be performed with caution as resulting antibodies, either used directly or as CARs, may lose activity towards endogenously presented tumour epitopes.

Blood-based kinase activity profiling: a potential predictor of response to immune checkpoint inhibition in metastatic cancer.

BACKGROUND: Many cancer patients do not obtain clinical benefit from immune checkpoint inhibition. Checkpoint blockade targets T cells, suggesting that tyrosine kinase activity profiling of baseline peripheral blood mononuclear cells may predict clinical outcome. METHODS: Here a total of 160 patients with advanced melanoma or non-small-cell lung cancer (NSCLC), treated with anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4) or anti-programmed cell death 1 (anti-PD-1), were divided into five discovery and cross-validation cohorts. The kinase activity profile was generated by analyzing phosphorylation of peripheral blood mononuclear cell lysates in a microarray comprising of 144 peptides derived from sites that are substrates for protein tyrosine kinases. Binary grouping into patients with or without clinical benefit was based on Response Evaluation Criteria in Solid Tumors V.1.1. Predictive models were trained using partial least square discriminant analysis (PLS-DA), performance of the models was evaluated by estimating the correct classification rate (CCR) using cross-validation. RESULTS: The kinase phosphorylation signatures segregated responders from non-responders by differences in canonical pathways governing T-cell migration, infiltration and co-stimulation. PLS-DA resulted in a CCR of 100% and 93% in the anti-CTLA-4 and anti-PD1 melanoma discovery cohorts, respectively. Cross-validation cohorts to estimate the accuracy of the predictive models showed CCRs of 83% for anti-CTLA-4 and 78% or 68% for anti-PD-1 in melanoma or NSCLC, respectively. CONCLUSION: Blood-based kinase activity profiling for response prediction to immune checkpoint inhibitors in melanoma and NSCLC revealed increased kinase activity in pathways associated with T-cell function and led to a classification model with a highly accurate classification rate in cross-validation groups. The predictive value of kinase activity profiling is prospectively verified in an ongoing trial.

CD45RA+CCR7- CD8 T cells lacking co-stimulatory receptors demonstrate enhanced frequency in peripheral blood of NSCLC patients responding to nivolumab.

BACKGROUND: Checkpoint inhibitors have become standard care of treatment for non-small cell lung cancer (NSCLC), yet only a limited fraction of patients experiences durable clinical benefit, highlighting the need for markers to stratify patient populations. METHODS: To prospectively identify patients showing response to therapy, we have stained peripheral blood samples of NSCLC patients treated with 2nd line nivolumab (n = 71), as well as healthy controls, with multiplex flow cytometry. By doing so, we enumerated 18 immune cell subsets and assessed expression for 28 T cell markers, which was followed by dimensionality reduction as well as rationale-based analyses. RESULTS: In patients with a partial response (PR), representing best overall response (BOR) according to RECIST v1.1, the number of CD8 T cells at baseline and during treatment is similar to those of healthy controls, but 2-fold higher than in patients with progressive and stable disease (PD and SD). CD8 T cell populations in PR patients show enhanced frequencies of T effector memory re-expressing CD45RA (TEMRA) cells, as well as T cells that express markers of terminal differentiation (CD95+) and egression from tumor tissue (CD69-). In PR patients, the fraction of CD8 T cells that lacks co-stimulatory receptors (CD28, ICOS, CD40L, 4-1BB, OX40) correlates significantly with the total numbers and differentiated phenotype of CD8 T cells. CONCLUSIONS: This study demonstrates that high numbers of peripheral CD8 T cells expressing differentiation markers and lacking co-stimulatory receptors at baseline are associated with response to nivolumab in NSCLC patients.

Enhanced recruitment of genetically modified CX3CR1-positive human T cells into Fractalkine/CX3CL1 expressing tumors: importance of the chemokine gradient.

BACKGROUND: Adoptive T-cell based immunotherapies constitute a promising approach to treat cancer, however, a major problem is to obtain effective and long-lasting anti-tumor responses. Lack of response may be due to insufficient trafficking of specific T cells to tumors. A key requirement for efficient migration of cytotoxic T cells is that they express chemokine receptors that match the chemokines produced by tumor or tumor-associated cells. METHODS: In this study, we investigated whether the in vivo tumor trafficking of activated T cells could be enhanced by the expression of the chemokine receptor CX3CR1. Two human colorectal cancer cell lines were used to set up a xenograft tumor model in immunodeficient mice; the NCI-H630, constitutively expressing the chemokine ligand CX3CL1 (Fractalkine), and the RKO cell line, transduced to express CX3CL1. RESULTS: Human primary T cells were transduced with the receptor CX3CR1-eGFP. Upon in vivo adoptive transfer of genetically modified CX3CR1-T cells in mice bearing NCI-H630 tumors, enhanced lymphocyte migration and tumor trafficking were observed, compared to mice receiving Mock-T cells, indicating improved homing ability towards ligand-expressing tumor cells. Furthermore, significant inhibition of tumor growth was found in mice receiving modified CX3CR1-T cells. In contrast, tumors formed by RKO cells transduced with the ligand (RKO-CX3CL1) were not affected, nor more infiltrated upon transfer of CX3CR1-T lymphocytes, likely because high levels of the chemokine were shed by tumor cells in the systemic circulation, thus nullifying the blood-tissue chemokine gradient. CONCLUSIONS: This study demonstrates that ectopic expression of CX3CR1 enhanced the homing of adoptively transferred T cells towards CX3CL1-producing tumors, resulting in increased T cell infiltration in tumor tissues and decreased tumor growth. Our results also establish that a correct chemokine gradient between the systemic circulation and the tumor is an essential requirement in adoptive T-cell based immunotherapy to efficiently recruit T cell effectors at the correct sites.

Targeting melanoma with immunoliposomes coupled to anti-MAGE A1 TCR-like single-chain antibody.

Therapy of melanoma using T-cells with genetically introduced T-cell receptors (TCRs) directed against a tumor-selective cancer testis antigen (CTA) NY-ESO1 demonstrated clear antitumor responses in patients without side effects. Here, we exploited the concept of TCR-mediated targeting through introduction of single-chain variable fragment (scFv) antibodies that mimic TCRs in binding major histocompatibility complex-restricted CTA. We produced scFv antibodies directed against Melanoma AntiGEn A1 (MAGE A1) presented by human leukocyte antigen A1 (HLA-A1), in short M1/A1, and coupled these TCR-like antibodies to liposomes to achieve specific melanoma targeting. Two anti-M1/A1 antibodies with different ligand-binding affinities were derived from a phage-display library and reformatted into scFvs with an added cysteine at their carboxyl termini. Protein production conditions, ie, bacterial strain, temperature, time, and compartments, were optimized, and following production, scFv proteins were purified by immobilized metal ion affinity chromatography. Batches of pure scFvs were validated for specific binding to M1/A1-positive B-cells by flow cytometry. Coupling of scFvs to liposomes was conducted by employing different conditions, and an optimized procedure was achieved. In vitro experiments with immunoliposomes demonstrated binding of M1/A1-positive B-cells as well as M1/A1-positive melanoma cells and internalization by these cells using flow cytometry and confocal microscopy. Notably, the scFv with nonenhanced affinity of M1/A1, but not the one with enhanced affinity, was exclusively bound to and internalized by melanoma tumor cells expressing M1/A1. Taken together, antigen-mediated targeting of tumor cells as well as promoting internalization of nanoparticles by these tumor cells is mediated by TCR-like scFv and can contribute to melanoma-specific targeting.

T cell receptor-engineered T cells to treat solid tumors: T cell processing toward optimal T cell fitness.

Therapy with autologous T cells that have been gene-engineered to express chimeric antigen receptors (CAR) or T cell receptors (TCR) provides a feasible and broadly applicable treatment for cancer patients. In a clinical study in advanced renal cell carcinoma (RCC) patients with CAR T cells specific for carbonic anhydrase IX (CAIX), we observed toxicities that (most likely) indicated in vivo function of CAR T cells as well as low T cell persistence and clinical response rates. The latter observations were confirmed by later clinical trials in other solid tumor types and other gene-modified T cells. To improve the efficacy of T cell therapy, we have redefined in vitro conditions to generate T cells with young phenotype, a key correlate with clinical outcome. For their impact on gene-modified T cell phenotype and function, we have tested various anti-CD3/CD28 mAb-based T cell activation and expansion conditions as well as several cytokines prior to and/or after gene transfer using two different receptors: CAIX CAR and MAGE-C2(ALK)/HLA-A2 TCR. In a total set of 16 healthy donors, we observed that T cell activation with soluble anti-CD3/CD28 mAbs in the presence of both IL15 and IL21 prior to TCR gene transfer resulted in enhanced proportions of gene-modified T cells with a preferred in vitro phenotype and better function. T cells generated according to these processing methods demonstrated enhanced binding of pMHC, and an enhanced proportion of CD8+, CD27+, CD62L+, CD45RA+T cells. These new conditions will be translated into a GMP protocol in preparation of a clinical adoptive therapy trial to treat patients with MAGE-C2-positive tumors.