Cabozantinib Plus Nivolumab in Patients with Non-Clear Cell Renal Cell Carcinoma: Updated Results from a Phase 2 Trial.

Treatment options are limited for patients with non-clear cell renal cell carcinoma (nccRCC). Patients with nccRCC experienced a favorable objective response rate (ORR) in a phase 2 trial of cabozantinib plus nivolumab. We now report updated efficacy and safety results at median follow-up of 34 mo for patients with papillary, unclassified, or translocation-associated RCC. Cabozantinib and nivolumab were administered at standard doses to patients with metastatic nccRCC that had progressed on zero or one line of systemic therapy. The primary endpoint was the ORR according to Response Evaluation Criteria in Solid Tumors v1.1. Secondary endpoints included progression-free survival (PFS), overall survival (OS), and adverse events. Forty patients were treated. At median follow-up of 34 mo for survivors, the ORR was 48% (95% confidence interval [CI] 31.5-63.9%). Median PFS was 13 mo (95% CI 7-16); the 12-mo and 24-mo PFS rates were 51% (95% CI 34-65%) and 23% (95% CI 11-37%), respectively. Median OS was 28 mo (95% CI 23-43); the 18-mo and 36-mo OS rates were 70% (95% CI 53-82%) and 44% (95% CI 28-60%), respectively. No new safety signals were seen with cabozantinib and nivolumab. This extended follow-up analysis demonstrates promising efficacy, and highlights the potential for sustained responses with cabozantinib plus nivolumab in patients with metastatic nccRCC. PATIENT SUMMARY: We evaluated outcomes for patients with metastatic kidney cancer of the non-clear cell (NCC) type who were treated with cabozantinib + nivolumab. We found that 48% of the patients responded to the treatment, and there were no unexpected side effects. Among patients who responded to the treatment, the response lasted for a median of 17 months. We conclude that cabozantinib + nivolumab is a safe and effective treatment for NCC kidney cancer.

A Phase 2 Trial of Talazoparib and Avelumab in Genomically Defined Metastatic Kidney Cancer.

BACKGROUND: Although different kidney cancers represent a heterogeneous group of malignancies, multiple subtypes including Von Hippel-Lindau (VHL)-altered clear cell renal cell carcinoma (ccRCC), fumarate hydratase (FH)- and succinate dehydrogenase (SDH)-deficient renal cell carcinoma (RCC), and renal medullary carcinoma (RMC) are affected by genomic instability. Synthetic lethality with poly ADP-ribose polymerase inhibitors (PARPis) has been suggested in preclinical models of these subtypes, and paired PARPis with immune checkpoint blockade (ICB) may achieve additive and/or synergistic effects in patients with previously treated advanced kidney cancers. OBJECTIVE: To evaluate combined PARPi + ICB in treatment-refractory metastatic kidney cancer. DESIGN, SETTING, AND PARTICIPANTS: We conducted a single-center, investigator-initiated phase 2 trial in two genomically selected advanced kidney cancer cohorts: (1) VHL-altered RCC with at least one prior ICB agent and one vascular endothelial growth factor (VEGF) inhibitor, and (2) FH- or SDH-deficient RCC with at least one prior ICB agent or VEGF inhibitor and RMC with at least one prior line of chemotherapy. INTERVENTION: Patients received talazoparib 1 mg daily plus avelumab 800 mg intravenously every 14 d in 28-d cycles. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary endpoint was objective response rate (ORR) by Immune Response Evaluation Criteria in Solid Tumors at 4 mo, and the secondary endpoints included progression-free survival (PFS), overall survival, and safety. RESULTS AND LIMITATIONS: Cohort 1 consisted of ten patients with VHL-altered ccRCC. All patients had previously received ICB. The ORR was 0/9 patients; one patient was not evaluable due to missed doses. In this cohort, seven patients achieved stable disease (SD) as the best response. The median PFS was 3.5 mo (95% confidence interval [CI] 1.0, 3.9 mo). Cohort 2 consisted of eight patients; four had FH-deficient RCC, one had SDH-deficient RCC, and three had RMC. In this cohort, six patients had previously received ICB. The ORR was 0/8 patients; two patients achieved SD as the best response and the median PFS was 1.2 mo (95% CI 0.4, 2.9 mo). The most common treatment-related adverse events of all grades were fatigue (61%), anemia (28%), nausea (22%), and headache (22%). There were seven grade 3-4 and no grade 5 events. CONCLUSIONS: The first clinical study of combination PARPi and ICB therapy in advanced kidney cancer did not show clinical benefit in multiple genomically defined metastatic RCC cohorts or RMC. PATIENT SUMMARY: We conducted a study to look at the effect of two medications, talazoparib and avelumab, in patients with metastatic kidney cancer who had disease progression on standard treatment. Talazoparib blocks the normal activity of molecules called poly ADP-ribose polymerase, which then prevents tumor cells from repairing themselves and growing, while avelumab helps the immune system recognize and kill cancer cells. We found that the combination of these agents was safe but not effective in specific types of kidney cancer.

Clinical and Genomic Landscape of FGFR3-Altered Urothelial Carcinoma and Treatment Outcomes with Erdafitinib: A Real-World Experience.

PURPOSE: Erdafitinib is the only FDA-approved targeted therapy for FGFR2/3-altered metastatic urothelial cancer. We characterized the genetic landscape of FGFR-altered urothelial carcinoma and real-world clinical outcomes with erdafitinib, including on-treatment genomic evolution. EXPERIMENTAL DESIGN: Prospectively collected clinical data were integrated with institutional genomic data to define the landscape of FGFR2/3-altered urothelial carcinoma. To identify mechanisms of erdafitinib resistance, a subset of patients underwent prospective cell-free (cf) DNA assessment. RESULTS: FGFR3 alterations predictive of erdafitinib sensitivity were identified in 39% (199/504) of patients with non-muscle invasive, 14% (75/526) with muscle-invasive, 43% (81/187) with localized upper tract, and 26% (59/228) with metastatic specimens. One patient had a potentially sensitizing FGFR2 fusion. Among 27 FGFR3-altered cases with a primary tumor and metachronous metastasis, 7 paired specimens (26%) displayed discordant FGFR3 status. Erdafitinib achieved a response rate of 40% but median progression-free and overall survival of only 2.8 and 6.6 months, respectively (n = 32). Dose reductions (38%, 12/32) and interruptions (50%, 16/32) were common. Putative resistance mutations detected in cfDNA involved TP53 (n = 5), AKT1 (n = 1), and second-site FGFR3 mutations (n = 2). CONCLUSIONS: FGFR3 mutations are common in urothelial carcinoma, whereas FGFR2 alterations are rare. Discordance of FGFR3 mutational status between primary and metastatic tumors occurs frequently and raises concern over sequencing archival primary tumors to guide patient selection for erdafitinib therapy. Erdafitinib responses were typically brief and dosing was limited by toxicity. FGFR3, AKT1, and TP53 mutations detected in cfDNA represent putative mechanisms of acquired erdafitinib resistance.

Scratching the Surface: NECTIN-4 as a Surrogate for Enfortumab Vedotin Resistance.

Clinical data with enfortumab vedotin (EV) suggest that most bladder cancers overexpress NECTIN-4. A recent article shows that NECTIN-4 membranous expression changes with progression to metastatic disease and that low NECTIN-4 expression in metastatic biopsies is potentially associated with EV resistance. These data argue for incorporation of NECTIN-4 expression into future biomarker strategies. See related article by Klümper et al., p. 1496.

Progression-free Survival After Second Line of Therapy for Metastatic Clear Cell Renal Cell Carcinoma in Patients Treated with First-line Immunotherapy Combinations.

Immunotherapy (IO)-based combinations used to treat metastatic clear cell renal cell carcinoma (ccRCC) include dual immune checkpoint inhibition with ipilimumab and nivolumab (IO/IO) and several combinations of vascular endothelial growth factor receptor-targeting tyrosine kinase inhibitors (TKIs) with an immune checkpoint inhibitor (TKI/IO). IO/IO and TKI/IO approaches have not been compared directly, and it is unknown whether patients who do not respond to first-line IO/IO can salvage long-term survival by receiving a second-line TKI. Progression-free survival after second-line therapy (PFS-2) evaluates the ability to be salvaged by second-line therapy. We retrospectively evaluated 173 patients treated with first-line IO/IO or TKI/IO for metastatic ccRCC at Memorial Sloan Kettering Cancer Center and report PFS-2, overall survival, and response to second line of therapy (ORR2nd) for groups defined by first-line category. Although ORR2nd was significantly higher with IO/IO than with TKI/IO (47% vs 13%, p < 0.001), there was no significant difference in median PFS-2 for TKI/IO versus IO/IO (44 vs 23 mo, log-rank p = 0.1) or restricted mean survival time (RMST) for PFS-2 when adjusted for propensity score (33 vs 30 mo; difference 2.6 mo [95% confidence interval {CI}: -2.6, 7.9]; p = 0.3). There was also no significant difference in RMST for overall survival when adjusted for propensity score (38 vs 37 mo; group difference 1.0 mo [95% CI: -3.4, 5.5]; p = 0.7). These findings do not support a change in current utilization practices for IO/IO and TKI/IO treatment strategies for ccRCC. PATIENT SUMMARY: In cases of metastatic clear cell renal cell carcinoma, no significant difference was observed in progression-free survival after second line of therapy between patients receiving ipilimumab plus nivolumab and those receiving a combination of a tyrosine kinase inhibitor and an immune checkpoint inhibitor.

Single-cell protein activity analysis identifies recurrence-associated renal tumor macrophages.

Clear cell renal carcinoma (ccRCC) is a heterogeneous disease with a variable post-surgical course. To assemble a comprehensive ccRCC tumor microenvironment (TME) atlas, we performed single-cell RNA sequencing (scRNA-seq) of hematopoietic and non-hematopoietic subpopulations from tumor and tumor-adjacent tissue of treatment-naive ccRCC resections. We leveraged the VIPER algorithm to quantitate single-cell protein activity and validated this approach by comparison to flow cytometry. The analysis identified key TME subpopulations, as well as their master regulators and candidate cell-cell interactions, revealing clinically relevant populations, undetectable by gene-expression analysis. Specifically, we uncovered a tumor-specific macrophage subpopulation characterized by upregulation of TREM2/APOE/C1Q, validated by spatially resolved, quantitative multispectral immunofluorescence. In a large clinical validation cohort, these markers were significantly enriched in tumors from patients who recurred following surgery. The study thus identifies TREM2/APOE/C1Q-positive macrophage infiltration as a potential prognostic biomarker for ccRCC recurrence, as well as a candidate therapeutic target.

Considerations for treatment duration in responders to immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICIs) have improved overall survival for cancer patients, however, optimal duration of ICI therapy has yet to be defined. Given ICIs were first used to treat patients with metastatic melanoma, a condition that at the time was incurable, little attention was initially paid to how much therapy would be needed for a durable response. As the early immunotherapy trials have matured past 10 years, a significant per cent of patients have demonstrated durable responses; it is now time to determine whether patients have been overtreated, and if durable remissions can still be achieved with less therapy, limiting the physical and financial toxicity associated with years of treatment. Well-designed trials are needed to identify optimal duration of therapy, and to define biomarkers to predict who would benefit from shorter courses of immunotherapy. Here, we outline key questions related to health, financial and societal toxicities of over treating with ICI and present four unique clinical trials aimed at exposing criteria for early cessation of ICI. Taken together, there is a serious liability to overtreating patients with ICI and future work is warranted to determine when it is safe to stop ICI.

Blocking IL1 Beta Promotes Tumor Regression and Remodeling of the Myeloid Compartment in a Renal Cell Carcinoma Model: Multidimensional Analyses.

PURPOSE: Intratumoral immunosuppression mediated by myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) represents a potential mechanism of immune checkpoint inhibitor (ICI) resistance in solid tumors. By promoting TAM and MDSC infiltration, IL1ß may drive adaptive and innate immune resistance in renal cell carcinoma (RCC) and in other tumor types. EXPERIMENTAL DESIGN: Using the RENCA model of RCC, we evaluated clinically relevant combinations of anti-IL1ß plus either anti-PD-1 or the multitargeted tyrosine kinase inhibitor (TKI), cabozantinib. We performed comprehensive immune profiling of established RENCA tumors via multiparameter flow cytometry, tumor cytokine profiling, and single-cell RNA sequencing (RNA-seq). Similar analyses were extended to the MC38 tumor model. RESULTS: Analyses via multiparameter flow cytometry, tumor cytokine profiling, and single-cell RNA-seq showed that anti-IL1ß reduces infiltration of polymorphonuclear MDSCs and TAMs. Combination treatment with anti-IL1ß plus anti-PD-1 or cabozantinib showed increased antitumor activity that was associated with decreases in immunosuppressive MDSCs and increases in M1-like TAMs. CONCLUSIONS: Single-cell RNA-seq analyses show that IL1ß blockade and ICI or TKI remodel the myeloid compartment through nonredundant, relatively T-cell-independent mechanisms. IL1ß is an upstream mediator of adaptive myeloid resistance and represents a potential target for kidney cancer immunotherapy.

Targeting PD-1 or PD-L1 in Metastatic Kidney Cancer: Combination Therapy in the First-Line Setting.

Recent FDA approvals of regimens targeting programmed death 1 (PD-1) in combination with anti-CTLA-4 or with VEGF tyrosine kinase inhibitors are reshaping front-line therapy for metastatic kidney cancer. In parallel, therapeutics specific for programmed death ligand 1 (PD-L1), one of the two major ligands for PD-1, are under continued investigation. Surprisingly, not all PD-1 and PD-L1 agents lead to similar clinical outcomes, potentially due to biological differences in the cellular expression and regulation of these targets. Here, we review current clinical data on combination immune checkpoint inhibitor therapy in metastatic kidney cancer and discuss the relevant biology of PD-1 and PD-L1. The design of future rational combination therapy trials in metastatic renal cell carcinoma will rely upon an understanding of this biology, along with an evolving understanding of immune cell populations and their functional states in the tumor microenvironment.

Association between immunosuppressive cytokines and PSA progression in biochemically recurrent prostate cancer treated with intermittent hormonal therapy.

BACKGROUND: Immunosuppressive cytokines have the potential to promote prostate cancer progression. Assessing their longitudinal changes may implicate mechanisms of progression, treatment resistance, and suggest new therapeutic targets. METHODS: Thirty-seven men with biochemically recurrent (BCR) prostate cancer who received 6 months of androgen deprivation therapy (ADT) and were monitored until the time to prostate-specific antigen progression (TTPP) were identified from a completed phase III trial (NCT00020085). Serum samples were archived at baseline, 3 months after ADT, and at TTPP. Cytokine concentrations were quantified using a 36-parameter electrochemiluminescence assay. The Wilcoxon signed-rank sum test was used to compare observations between time points. Kaplan-Meier analysis was used to calculate TTPP dichotomized by cytokine values above or below the median. Pearson's rank correlation coefficient was used to compare continuous variables. RESULTS: Median TTPP was 399 days (range, 114-1641). Median prostate-specific antigen (PSA) at baseline and progression were 8.5 and 5.3 ng/mL, respectively. Twenty-three patients (62%) achieved undetectable PSA with ADT. Castrate levels of testosterone (<50 ng/dL) after 3 months of ADT occurred in 35 patients (95%). TNF-α (P = .002), IL-23 (P = .002), and CXCL10 (P = .001) significantly increased from baseline to post ADT. Certain cytokines correlated longitudinally: TNF-α correlated with IL-23 (r = .72; P < .001) and IL-8 (r = .59; P < .001) from baseline to post ADT and to PSA progression. Neutrophil-to-lymphocyte ratio correlated with IL-27 (r = .57; P < .001) and MIP-3α (r = .56; P < .001). Patients with a detectable PSA after ADT had elevated levels of IL-6 (P = .049) and IL-8 (P = .013) at PSA progression as compared with those with an undetectable PSA. There was a trend toward shorter TTPP in patients with TNF-α levels above the median (P = .042). CONCLUSIONS: Several innate cytokines were associated with biochemically recurrent prostate cancer.

A novel approach to assess real-world efficacy of cancer therapy in metastatic prostate cancer. Analysis of national data on Veterans treated with abiraterone and enzalutamide.

BACKGROUND: With 1.3 million new cases in 2018 worldwide, prostate cancer remains a challenge. Development of novel therapies targeting the androgen pathway followed recognition of the continued importance of androgens in castrate-resistant prostate cancer. To assess abiraterone and enzalutamide efficacy we analyzed data from US Veterans Administration Medical Centers (VAMCs). METHODS: We used a novel method independent of assessment intervals and ideal for real-world analysis to estimate rates of tumor growth (g) and regression (d). FINDINGS: Using the VA Informatics and Computing Infrastructure, we collected data from 5,116 Veterans with castrate-resistant prostate cancer prescribed abiraterone, enzalutamide or both. We estimated values for g and d and demonstrated a correlation of g with overall survival (P < .0001). Abiraterone and enzalutamide slowed growth rates across age groups and across the entire VAMC system, although less so in Veterans previously treated with a taxane and those with Gleason grade group 5 tumors. Abiraterone and enzalutamide efficacy in first-line were comparable although abiraterone in first-line slowed growth rates significantly more in African Americans than in Caucasians; enzalutamide was a better salvage therapy. When abiraterone was first-line and g was low, switching to enzalutamide was associated with a faster g in 67%. INTERPRETATION: In the real-world g can be estimated using a novel analysis method indifferent to assessment intervals that correlates highly with OS. While we show excellent real-world outcomes with abiraterone and enzalutamide, 2 effective and tolerable therapies, our results in VAMCs suggest enzalutamide should follow abiraterone. Changing therapies may be detrimental and consideration should be given to continue monitoring of growth rates over time. Funding Support from the Prostate Cancer Foundation and the Blavatnik Family Foundation.

Generation of higher affinity T cell receptors by antigen-driven differentiation of progenitor T cells in vitro.

Many promising targets for T-cell-based cancer immunotherapies are self-antigens. During thymic selection, T cells bearing T cell receptors (TCRs) with high affinity for self-antigen are eliminated. The affinity of the remaining low-avidity TCRs can be improved to increase their antitumor efficacy, but conventional saturation mutagenesis approaches are labor intensive, and the resulting TCRs may be cross-reactive. Here we describe the in vitro maturation and selection of mouse and human T cells on antigen-expressing feeder cells to develop higher-affinity TCRs. The approach takes advantage of natural Tcrb gene rearrangement to generate diversity in the length and composition of CDR3ß. In vitro differentiation of progenitors transduced with a known Tcra gene in the presence of antigen drives differentiation of cells with a distinct agonist-selected phenotype. We purified these cells to generate TCRß chain libraries pre-enriched for target antigen specificity. Several TCRß chains paired with a transgenic TCRα chain to produce a TCR with higher affinity than the parental TCR for target antigen, without evidence of cross-reactivity.

Biomarkers for immunotherapy in bladder cancer: a moving target.

Treatment options for metastatic urothelial carcinoma (mUC) remained relative unchanged over the last 30 years with combination chemotherapy as the mainstay of treatment. Within the last year the landscape for mUC has seismically shifted following the approval of five therapies targeting the programmed cell death protein (PD-1)/programmed cell death ligand 1 (PD-L1) axis. Notably, the anti-PD-1 antibody pembrolizumab demonstrated improved OS relative to chemotherapy in a randomized phase III study for second line treatment of mUC; this level 1 evidence led to approval from the U.S. Food and Drug Administration (FDA). The PD-1 antibody nivolumab also demonstrated an overall survival benefit, in this case in comparison to historical controls. Similarly, antibodies targeting PD-L1 including atezolizumab, durvalumab, and avelumab have now received accelerated approval from the FDA as second line treatments for mUC, with durable response lasting more than 1 year in some patients. Some of these agents are approved in the first line setting as well - based on single-arm phase II studies atezolizumab and pembrolizumab received accelerated approval for first-line treatment of cisplatin ineligible patients. Despite these multiple approvals, the development of clinically useful biomarkers to determine the optimal treatment for patients remains somewhat elusive. In this review, we examine key clinical trial results with anti-PD1/PD-L1 antibodies and discuss progress towards developing novel biomarkers beyond PD-L1 expression.

A novel T cell receptor single-chain signaling complex mediates antigen-specific T cell activity and tumor control.

Adoptive transfer of genetically modified T cells to treat cancer has shown promise in several clinical trials. Two main strategies have been applied to redirect T cells against cancer: (1) introduction of a full-length T cell receptor (TCR) specific for a tumor-associated peptide-MHC, or (2) introduction of a chimeric antigen receptor, including an antibody fragment specific for a tumor cell surface antigen, linked intracellularly to T cell signaling domains. Each strategy has advantages and disadvantages for clinical applications. Here, we present data on the in vitro and in vivo effectiveness of a single-chain signaling receptor incorporating a TCR variable fragment as the targeting element (referred to as TCR-SCS). This receptor contained a single-chain TCR (Vα-linker-Vß) from a high-affinity TCR called m33, linked to the intracellular signaling domains of CD28 and CD3ζ. This format avoided mispairing with endogenous TCR chains and mediated specific T cell activity when expressed in either CD4 or CD8 T cells. TCR-SCS-transduced CD8-negative cells showed an intriguing sensitivity, compared to full-length TCRs, to higher densities of less stable pepMHC targets. T cells that expressed this peptide-specific receptor persisted in vivo, and exhibited polyfunctional responses. Growth of metastatic antigen-positive tumors was significantly inhibited by T cells that expressed this receptor, and tumor cells that escaped were antigen-loss variants. TCR-SCS receptors represent an alternative targeting receptor strategy that combines the advantages of single-chain expression, avoidance of TCR chain mispairing, and targeting of intracellular antigens presented in complex with MHC proteins.

Enhanced-affinity murine T-cell receptors for tumor/self-antigens can be safe in gene therapy despite surpassing the threshold for thymic selection.

Many of the most promising tumor antigens for T-cell-based cancer immunotherapies are unmodified self-antigens. Unfortunately, the avidity of T cells specific for these antigens is limited by central tolerance during T-cell development in the thymus, resulting in decreased anti-tumor efficacy of these T cells. One approach to overcoming this obstacle is to mutate T-cell receptor (TCR) genes from naturally occurring T cells to enhance the affinity for the target antigen. These enhanced-affinity TCRs can then be developed for use in TCR gene therapy. Although TCRs with significantly enhanced affinity have been generated using this approach, it is not clear whether these TCRs, which bypass the affinity limits imposed by negative selection, remain unresponsive to the low levels of self-antigen generally expressed by some normal tissues. Here we show that 2 variants of a high-affinity WT1-specific TCR with enhanced affinity for WT1 are safe and do not mediate autoimmune tissue infiltration or damage when transduced into peripheral CD8 T cells and transferred in vivo. However, if expressed in developing T cells and subjected to thymic selection, the same enhanced-affinity TCRs signal tolerance mechanisms in the thymus, resulting in T cells with attenuated antigen sensitivity in the periphery.

A sensitivity scale for targeting T cells with chimeric antigen receptors (CARs) and bispecific T-cell Engagers (BiTEs).

Although T cells can mediate potent antitumor responses, immune tolerance mechanisms often result in the deletion or inactivation of T cells that express T-cell receptors (TCRs) against potentially effective target epitopes. Various approaches have been devised to circumvent this problem. In one approach, the gene encoding an antibody against a cancer-associated antigen is linked, in the form of a single-chain variable fragment (scFv), to genes that encode transmembrane and signaling domains. This chimeric antigen receptor (CAR) is then introduced into T cells for adoptive T-cell therapy. In another approach, the anti-cancer scFv is fused to a scFv that binds to the CD3ε subunit of the TCR/CD3 complex. This fusion protein serves as a soluble, injectable product that has recently been termed bispecific T-cell engager (BiTE). Both strategies have now been tested in clinical trials with promising results, but the comparative efficacies are not known. Here, we performed a direct comparison of the in vitro sensitivity of each strategy, using the same anti-cancer scFv fragments, directed against a tumor-specific glycopeptide epitope on the sialomucin-like transmembrane glycoprotein OTS8, which results form a cancer-specific mutation of Cosmc. While both approaches showed specific responses to the epitope as revealed by T cell-mediated cytokine release and target cell lysis, CAR-targeted T cells were more sensitive than BiTE-targeted T cells to low numbers of antigens per cell. The sensitivity scale described here provides a guide to the potential use of these two different approaches.

T cell receptor engineering.

T lymphocytes express on their surface a heterodimeric αß receptor, called the T cell receptor (TCR), which recognizes foreign antigens. Unlike antibodies, the recognition requires both an antigenic peptide epitope and a protein encoded by the major histocompatibility complex (MHC). In contrast to conventional antibody-directed target antigens, antigens recognized by the TCR can include the entire array of potential intracellular proteins, which are processed and delivered to the cell surface as a peptide/MHC complex. In the past 10 years, there have been significant efforts to engineer TCRs in various formats, which would allow improved recognition and destruction of virus-infected cells or cancer. The proposed therapeutic approaches involve either the use of engineered, high-affinity TCRs in soluble forms, analogous to antibody-directed therapies, or the use of engineered TCRs whose genes are reintroduced into autologous T cells and transferred back into patients (T cell adoptive therapies). This chapter describes three methods associated with the engineering of TCRs for these therapeutic purposes: (1) use of a yeast display system to engineer higher affinity single-chain VαVß TCRs, called scTv; (2) use of a T cell display system to engineer higher affinity full-length TCRs; and (3) expression, purification, and characterization of soluble TCRs in an Escherichia coli system.

Opposite effects of endogenous peptide-MHC class I on T cell activity in the presence and absence of CD8.

Nonstimulatory or endogenous peptide-MHC (pepMHC) presented on the surfaces of APCs, either alone or alongside agonist pepMHC, plays various roles in T cell selection and activation. To examine these properties in more detail, we explored several model systems of TCR and pepMHC ligands with sufficient affinity to be activated in the absence of CD8. The TCRs had a range of affinities for agonist and nonstimulatory ligands and were restricted by MHC class I alleles with different properties. We observed CD8-independent antagonism from TCR-pepMHC interactions with very low affinities (e.g., K(D) = 300 µM). In addition, endogenous peptide-L(d) complexes on APCs antagonized activation of coreceptor (CD8)-negative 2C T cells even by the strong agonist QL9-L(d). In contrast, TCRs m33 and 3D-PYY, restricted by K(b) and D(b), respectively, did not show signs of antagonism by endogenous pepMHC in the absence of CD8. This did not appear to be an inherent difference in the ability of the TCRs to be antagonized, as altered peptide ligands could antagonize each TCR. In the presence of CD8, endogenous pepMHC ligands acted in some cases as coagonists. These results show that endogenous pepMHC molecules exhibit complex behavior in T cells, leading to either reduced activity (e.g., in cases of low coreceptor levels) or enhanced activity (e.g., in presence of coreceptor). The behavior may be influenced by the ability of different TCRs to recognize endogenous pepMHC but also perhaps by the inherent properties of the presenting MHC allele.

Identification and engineering of human variable regions that allow expression of stable single-chain T cell receptors.

Single-chain antibody fragments (scFv), consisting of two linked variable regions (V(H) and V(L)), are a versatile format for engineering and as potential antigen-specific therapeutics. Although the analogous format for T cell receptors (TCRs), consisting of two linked V regions (Vα and Vß; referred to here as scTv), could provide similar opportunities, all wild-type scTv proteins examined to date are unstable. This obstacle has prevented scTv fragments from being widely used for engineering or therapeutics. To further explore whether some stable human scTv fragments could be expressed, we used a yeast system in which display of properly folded domains correlates with ability to express the folded scTv in soluble form. We discovered that, unexpectedly, scTv fragments that contained the human Vα2 region (IMGT: TRAV12 family) were displayed and properly associated with different Vß regions. Furthermore, a single polymorphic residue (Ser(α49)) in the framework region conferred additional thermal stability. These stabilized Vα2-containing scTv fragments could be expressed at high levels in Escherichia coli, and used to stain target cells that expressed the specific pep-HLA-A2 complexes. Thus, the scTv fragments can serve as a platform for engineering TCRs with diverse specificities, and possibly for therapeutic or diagnostic applications.