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Supplementary Fig. 4: (A-D) BCR convergent groups converge to shared amino acid sequences. (A-B) Phylogenetic trees across all repertories of a representative naive patient (A) and of a representative treated patient (B). For each patient, social network analysis was performed using R packages: Ape (24) and igraph (25). A convergent group was defined as a cluster that includes the clones with the distance less than or equal to 1 (allowing maximum of 1 basepair mutation among clone sequences sharing the same V-gene, J-gene and CDR3 length). The phylogenetic tree was plotted by R package: ggtree (26) with the edge color was coded by amino acid sequences. (C) Social network of a selected V family (V01) across all repertories from the 6 time points of the representative treated patient. Each node represents a single full-length of BCR clonotype sequence colored by its corresponding CDR3 amino acid sequences; node size was characterized based on the corresponding abundance. The nodes connected by lines were within the same convergent group. (D) Social network figures of top 6 largest groups in the V01 family across all repertories from the 6 time points of the representative treated patient in (C). One convergent group was selected for illustration purpose with presenting the actual converged two CDR3 amino acid sequences. (E) Public clusters shared by treated patients at baseline and naïve patients post baseline. Across all repertories from treated patients at baseline and naïve patients post baseline, 5 public clones were identified first. Then social network analysis was performed to find the clones which were at most 1 basepair mismatch with those 5 public clones.

Supplementary Fig. 2A: Changes in rank of the top 100 BCR clones at week 2 for a representative naïve patient are shown. The top 100 clonotypes at week 2 were identified based on their abundances at week 2. The rank of those clones at other timepoints were also obtained, and plotted across all 6 time points. Supplementary Fig. 2B: Pairwise Morisita's distance for both treated and naïve. Morisita's distance of 0 and 1 means maximally and minimally dissimilar of the repertoires from two different time points of the same patient, respectively (27). Each panel illustrates the Morisita's distance of two time points. Within each panel left and right boxplots represent sipuleucel-T treated patients and sipuleucel-T naive patients, respectively. The asterisk shows significant higher in treated vs naïve (Wilcoxon sum rank test p

Sipuleucel-T is an autologous cellular immunotherapy, administered as three infusions, for metastatic castration-resistant prostate cancer (mCRPC). Sipuleucel-T induces T- and B-cell responses to prostatic acid phosphatase (PAP), correlating to improved survival. The long-term impact of sipuleucel-T on tumor antigen–specific immunologic memory remains unknown, in particular, B-cell responses, as measured by antigen-specific antibody responses and B-cell receptor (BCR) sequences. To evaluate whether sipuleucel-T could induce long-term immunologic memory, we examined circulating B-cell responses before and after sipuleucel-T treatment in two groups of patients with mCRPC: those who had previously received sipuleucel-T (treated; median, 8.9 years since the previous treatment) versus those who had not (naïve). Before re-treatment, previously treated patients exhibited persistent antibody responses as well as more focused and convergent BCR repertoires with distinct V(D)J gene usage compared with naïve patients. After re-treatment, previously treated patients maintained high-frequency clones and developed more convergent BCRs at earlier time points unlike naïve patients. With the first sipuleucel-T infusion specifically, previously treated patients had less shuffling within the 100 most abundant baseline clones. In contrast, naïve patients exhibited great BCR turnover with a continued influx of new B-cell clones. Social network analysis showed that previously treated patients had more highly organized B-cell repertoires, consistent with greater clonal maturation. Higher treatment-induced BCR clonality correlated with longer survival for naïve patients. These results demonstrated the capacity of sipuleucel-T to induce long-term immune memory and lasting changes to the B-cell repertoire.

Supplementary Fig. 5: Sensitivity analysis of social network analysis of the representative sipuleucel-T treated patient. (A) Density plot of the clone frequency for all clones of the treated patient (red) and density plot of the clone frequency for subsampled clones of the treated patient (green). Subsampling was done by weighted clone frequency. (B) Social network (V04 family gene) of the all clones of the treated patient. (C) Social network (V04 family gene) of the all clones of the naïve patient. (D) Social network (V04 family gene) of the subsampled clones of the treated patient. Subsampling was done by weighted clone frequency. For each patient, social network analysis was first performed using R packages: Ape (24) and igraph (25). A convergent group was defined as the cluster that included the clones with the distance less than or equal to 1 (allowing maximum of 1 basepair mutation among clone sequences sharing the same V-gene, J-gene and CDR3 length). For all social network figures, each node represents a single clone colored by its first presenting time point, and the node size was attributed based on the corresponding abundance. The nodes connected by lines were within the same convergent group.

Selected convergent groups. (A) Illustration of the groups that are persistently present across all time points for the treated patient. (B) Illustration of the groups induced in the treated patient. (C) Illustration of the induced groups in the naive patient. Each figure includes the actual nucleotide sequences corresponding to Fig 3E-3G. The colors were coded by ACTG (only first 60 base-pairs were retained due to space limitations). The yellow highlighting shows the hypermutation in the nucleotide sequence, while the bottom row shows the motif analysis. The tables present the clonal abundance for corresponding clones each time point.

Supplementary Table 2. The public clones shared between treated patients at baseline and naïve patients at post-baseline time points. The table is separated by the 5 public clones, for which, each was shared by two treated patients and two naïve patients. For each clone, the corresponding amino acid sequence, gene information, as well as their abundance at each timepoint for each shared patient were listed. NA = the corresponding blood sample was not available at that time point. 0 = the corresponding clonotype was not detected in the available blood sample.

Study Schema. The top panel is the study schema for P101 study (treated patients) and the bottom panel is for the STRIDE study schema (naïve patients). As shown in the figure, the treated patients had received sipuleucel-T treatment on average of 8.9 years ago before joined P10-1 study. For both studies, the patients had 3 infusions every 2 weeks (week 0, week 2 and week 4). The baseline was indicated as in the figure, which was right before the 1st infusion. The blood samples were collected at week 0, week 2 and week 4 right before each infusion, as well as at week 6, week 26 and week 52. Both studies have long term follow-up after one year.

Comparison of the proportions of pairwise overlapping BCR clones present at both earlier time point (T1) and later time point (T2) between treated and naïve patients. The proportion of overlapping BCR clones was calculated by the number of the overlapping clones divided by the total number of clones presented at either time point, i.e., the Jaccard index. Bold text highlights significant differences between treated and naïve patients (Wilcoxon sum rank test p < 0.05).

Purpose:Autoantibody responses in cancer are of great interest, as they may be concordant with T-cell responses to cancer antigens or predictive of response to cancer immunotherapies. Thus, we sought to characterize the antibody landscape of metastatic castration-resistant prostate cancer (mCRPC).Experimental Design:Serum antibody epitope repertoire analysis (SERA) was performed on patient serum to identify tumor-specific neoepitopes. Somatic mutation–specific neoepitopes were investigated by associating serum epitope enrichment scores with whole-genome sequencing results from paired solid tumor metastasis biopsies and germline blood samples. A protein-based immunome-wide association study (PIWAS) was performed to identify significantly enriched epitopes, and candidate serum antibodies enriched in select patients were validated by ELISA profiling. A distinct cohort of patients with melanoma was evaluated to validate the top cancer-specific epitopes.Results:SERA was performed on 1,229 serum samples obtained from 72 men with mCRPC and 1,157 healthy control patients. Twenty-nine of 6,636 somatic mutations (0.44%) were associated with an antibody response specific to the mutated peptide. PIWAS analyses identified motifs in 11 proteins, including NY-ESO-1 and HERVK-113, as immunogenic in mCRPC, and ELISA confirmed serum antibody enrichment in candidate patients. Confirmatory PIWAS, Identifying Motifs Using Next-generation sequencing Experiments (IMUNE), and ELISA analyses performed on serum samples from 106 patients with melanoma similarly revealed enriched cancer-specific antibody responses to NY-ESO-1.Conclusions:We present the first large-scale profiling of autoantibodies in advanced prostate cancer, utilizing a new antibody profiling approach to reveal novel cancer-specific antigens and epitopes. Our study recovers antigens of known importance and identifies novel tumor-specific epitopes of translational interest.

The supplementary file contains 3 supplemental figures. Supplementary Figure S1 shows the kinetics of Ki67 expression by CD4 and CD8 T cells following treatment with ipilimumab. Supplementary Figure S2 shows the change in TCR clonality over time following treatment with ipilimumab. Supplementary Figure S3 shows the changes in clonotype frequencies of sorted CD4+ and CD8+ T cells with ipilimumab.

Supplementary Figures S1-S9

While immune checkpoint blockade elicits efficacious responses in many patients with cancer, it also produces a diverse and unpredictable number of immune-related adverse events (IRAE). Mechanisms driving IRAEs are generally unknown. Because CTLA-4 blockade leads to proliferation of circulating T cells, we examined in this study whether ipilimumab treatment leads to clonal expansion of tissue-reactive T cells. Rather than narrowing the T-cell repertoire to a limited number of clones, ipilimumab induced greater diversification in the T-cell repertoire in IRAE patients compared with patients without IRAEs. Specifically, ipilimumab triggered increases in the numbers of clonotypes, including newly detected clones and a decline in overall T-cell clonality. Initial broadening in the repertoire occurred within 2 weeks of treatment, preceding IRAE onset. IRAE patients exhibited greater diversity of CD4+ and CD8+ T cells, but showed no differences in regulatory T-cell numbers relative to patients without IRAEs. Prostate-specific antigen responses to ipilimumab were also associated with increased T-cell diversity. Our results show how rapid diversification in the immune repertoire immediately after checkpoint blockade can be both detrimental and beneficial for patients with cancer. Cancer Res; 77(6); 1322–30. ©2016 AACR.

Supplementary Tables S1-S4

T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells1–3. Here we developed a clinical-grade approach based on CRISPR–Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRβ). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen–HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial (NCT03970382). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.

Ulcerative colitis (UC) is an inflammatory intestinal disorder driven by mucosal immune and stromal subsets, culminating in epithelial injury. Vedolizumab (VDZ) is an anti-integrin monoclonal antibody that is effective for treating UC. VDZ is thought to primarily inhibit lymphocyte trafficking to the intestine, but its effect on other cell subsets is less well characterized. To identify the inflammatory cells that contribute to colitis and respond to VDZ, we performed a single-cell transcriptomic and proteomic analysis of peripheral blood and colonic biopsies in healthy controls (HC) and patients with UC on either aminosalicylates or VDZ. We identified mononuclear phagocytes (MNPs) as a primary target of VDZ, with comparatively modest effects on lymphocytes. Spatial proteomics and transcriptomics demonstrated increased density and proximity of MNP and fibroblast subsets in UC biopsies when compared to HC, with inhibition by VDZ. VDZ non-responders were enriched for activated fibroblast and MNP signatures in a validation cohort.

The biology of human granzymes remains enigmatic in part due to our inability to probe their functions outside of in vitro assays or animal models with divergent granzyme species. We hypothesize that the biology of human granzymes could be better elaborated with a translational imaging technology to reveal the contexts in which granzymes are secreted and biochemically active in vivo. Here, we advance toward this goal by engineering a Granzyme targeting Restricted Interaction Peptide specific to family member B (GRIP B) to measure secreted granzyme B (GZMB) biochemistry with positron emission tomography. A proteolytic cleavage of 64Cu-labeled GRIP B liberates a radiolabeled form of Temporin L, which sequesters the radioisotope by binding to adjacent phospholipid bilayers. Thus, at extended time points postinjection (i.e., hours, not seconds), tissue biodistribution of the radioisotope in vivo reflects relative units of the GZMB activity. As a proof of concept, we show in three syngeneic mouse cancer models that 64Cu-GRIP B detects GZMB from T cells activated with immune checkpoint inhibitors (CPI). Remarkably, the radiotracer detects the proteolysis within tumors but also in lymphoid tissue, where immune cells are activated by a systemic CPI. Control experiments with an uncleavable analogue of 64Cu-GRIP B and tumor imaging studies in germline GZMB knockout mice were applied to show that 64Cu-GRIP B is specific for GZMB proteolysis. Furthermore, we explored a potential noncytotoxic function for GZMB by applying 64Cu-GRIP B to a model of pulmonary inflammation. In summary, we demonstrate that granzyme biochemistry can be assessed in vivo using an imaging modality that can be scaled vertically into human subjects., A protease-activated chemosensor for in vivo molecular imaging enables the detection of activated immune cells by specifically harnessing granzyme B catalysis.

Sex bias exists in the development and progression of nonreproductive organ cancers, but the underlying mechanisms are enigmatic. Studies so far have focused largely on sexual dimorphisms in cancer biology and socioeconomic factors. Here, we establish a role for CD8 + T cell–dependent antitumor immunity in mediating sex differences in tumor aggressiveness, which is driven by the gonadal androgen but not sex chromosomes. A male bias exists in the frequency of intratumoral antigen-experienced Tcf7 /TCF1 + progenitor exhausted CD8 + T cells that are devoid of effector activity as a consequence of intrinsic androgen receptor (AR) function. Mechanistically, we identify a novel sex-specific regulon in progenitor exhausted CD8 + T cells and a pertinent contribution from AR as a direct transcriptional transactivator of Tcf7 /TCF1. The T cell–intrinsic function of AR in promoting CD8 + T cell exhaustion in vivo was established using multiple approaches including loss-of-function studies with CD8-specific Ar knockout mice. Moreover, ablation of the androgen-AR axis rewires the tumor microenvironment to favor effector T cell differentiation and potentiates the efficacy of anti–PD-1 immune checkpoint blockade. Collectively, our findings highlight androgen-mediated promotion of CD8 + T cell dysfunction in cancer and imply broader opportunities for therapeutic development from understanding sex disparities in health and disease.

Immunotherapies have greatly expanded the armamentarium of cancer-directed therapies in the past decade, allowing the immune system to recognize and fight cancer. Immune checkpoint inhibitors (ICIs), in particular, have revolutionized cancer treatment and have demonstrated survival benefit in numerous types of cancer. These monoclonal antibodies increase anti-cancer immunity by blocking down-regulators of adaptive immunity, including cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), and its ligand (PD-L1), resulting in anti-tumor activity. As ICIs increase immune system activation, they can cause a wide range of inflammatory side effects, termed immune-released adverse events. Though these toxicities can affect nearly any organ, the most fatal toxicity is myocarditis. Here, we discuss the diverse spectrum of cardiovascular toxicities associated with ICI use. In addition, we provide insight and future directions on mechanisms and treatments for immune-related adverse events (irAEs) involving the myocardium, pericardium, vasculature, and conduction system.

T cells mediate anti-tumor immune responses and are the key target of immune checkpoint therapy, but they can also promote immune tolerance. A clear understanding of the specific contributions and biology of different T cell subsets is required to fully harness the curative potential of immunotherapies. Experts discuss the state of the field and key challenges for moving forward.

The mixed patient responses to antibodies targeting immune checkpoint proteins (e.g., CTLA-4, PD-1, PD-L1) have generated tremendous interest in discovering biomarkers that predict which patients will best respond to these treatments. To complement molecular biomarkers obtained from biopsies, the nuclear medicine community has begun developing radiopharmaceuticals that may provide a more holistic assessment of the biological character of all disease sites in patients. On the leading edge of clinical translation are a spectrum of radiolabeled antibodies targeting immune checkpoint proteins or T cell-specific antigens. The adoption of these reagents requires development of efficient and versatile methods for antibody bioconjugation and radiochemistry. We report herein protocols for the preparation of an anti-PD-L1 IgG1 (termed C4) labeled with zirconium-89. The approach is time and cost economical, high yielding, and adaptable to numerous antibody clones and platforms of interest to the immune-oncology community. Included also are representative methods for characterizing the pharmacology of the antibody post bioconjugation, and conducting an in vivo assessment of radiotracer biodistribution in tumor bearing mouse models.

Immune checkpoint inhibitors (ICI) are designed to activate exhausted tumor-reactive T cells thereby leading to tumor regression. Durvalumab, an ICI that binds to the programmed death ligand-1 (PD-L1) molecule, is approved as a consolidation therapy for treatment of patients with stage III, unresectable, non-small cell lung cancer (NSCLC). Immunophenotypic analysis of circulating immune cells revealed increases in circulating proliferating CD4 + and CD8 + T cells earlier after durvalumab treatment. To examine durvalumab’s mechanism of action and identify potential predictive biomarkers, we assessed the circulating T cells phenotypes and TCR genes of 71 NSCLC patients receiving durvalumab enrolled in a Phase I trial (NCT01693562, September 14, 2012). Next-generation sequencing of TCR repertoire was performed on these NSCLC patients’ peripheral blood samples at baseline and day 15. Though patients’ TCR repertoire diversity showed mixed responses to the treatment, patients exhibiting increased diversity on day 15 attained significantly longer overall survival (OS) (median OS was not reached vs 17.2 months for those with decreased diversity, p = 0.015). We applied network analysis to assess convergent T cell clonotypes indicative of an antigen-driven immune response. Patients with larger TCR clusters had improved OS (median OS was not reached vs 13.1 months for patients with smaller TCR clusters, p = 0.013). Early TCR repertoire diversification after durvalumab therapy for NSCLC may be predictive of increased survival and provides a mechanistic basis for durvalumab pharmacodynamic activity. Supplementary Information The online version contains supplementary material available at 10.1007/s00262-020-02833-z.

BackgroundImmune checkpoint inhibitors (ICI) can achieve durable responses in a subset of patients with locally advanced or metastatic urothelial carcinoma (aUC). The use of tumor genomic profiling in clinical practice may help suggest biomarkers to identify patients most likely to benefit from ICI.MethodsWe undertook a retrospective analysis of patients treated with an ICI for aUC at a large academic medical center. Patient clinical and histopathological variables were collected. Responses to treatment were assessed for all patients with at least one post-baseline scan or clear evidence of clinical progression following treatment start. Genomic profiling information was also collected for patients when available. Associations between patient clinical/genomic characteristics and objective response were assessed by logistic regression; associations between the characteristics and progression-free survival (PFS) and overall survival (OS) were examined by Cox regression. Multivariable analyses were performed to identify independent prognostic factors.ResultsWe identified 119 aUC patients treated with an ICI from December 2014 to January 2020. Genomic profiling was available for 78 patients. Overall response rate to ICI was 29%, and median OS (mOS) was 13.4 months. Favorable performance status at the start of therapy was associated with improved OS (HR 0.46, p=0.025) after accounting for other covariates. Similarly, the presence of a TERT promoter mutation was an independent predictor of improved PFS (HR 0.38, p=0.012) and OS (HR 0.32, p=0.037) among patients who had genomic profiling available. Patients with both a favorable performance status and a TERT promoter mutation had a particularly good prognosis with mOS of 21.1 months as compared with 7.5 months in all other patients (p=0.03).ConclusionsThe presence of a TERT promoter mutation was an independent predictor of improved OS in a cohort of aUC patients treated with an ICI who had genomic data available. Most of the clinical and laboratory variables previously shown to be prognostic in aUC patients treated with chemotherapy did not have prognostic value among patients treated with an ICI. Genomic profiling may provide important prognostic information and affect clinical decision making in this patient population. Validation of these findings in prospective patient cohorts is needed.

Background/aim The majority of colorectal cancer (CRC) cases, which are microsatellite stable (MSS) and do not harbor mismatch repair deficiency/microsatellite instability, are resistant to immunotherapy. Identification of patients with exceptional responses in MSS CRC and predictive biomarkers is an unmet need that needs to be addressed. Case report We report three cases of MSS CRC with durable clinical benefit from immunotherapy with anti-PD-1 checkpoint inhibitors. Two cases bear a POLE P286R mutation, which has been associated with lack of immunotherapy response in MSS CRC. Two cases bear alterations in Ataxia-Telangiectasia Mutated (ATM) which may contribute to observed responses, including interaction with a co-administered intratumoral stimulator of interferon genes (STING) pathway agonist in one patient. Conclusion Novel DNA damage repair alterations, including mutations in ATM, can provide insight into additional mechanisms by which genomic alterations can sensitize MSS CRC to diverse immunotherapies.

SUMMARY Cytotoxic T cells are important effectors of anti-tumor immunity. While tumor killing is ascribed to CD8+ T cell function, pre-clinical and clinical studies have identified intra-tumoral CD4+ T cells that possess cytotoxic programs and can directly kill cancer cells. Cytotoxic CD4+ T cells are found in other disease settings including infection and autoimmunity. Here, we review the phenotypic and functional characteristics of cytotoxic CD4+ T cells in non-cancer and cancer contexts. We conduct a comparative examination of cytolytic mechanisms of cytotoxic CD4+ T cells across disease states and synthesize features that define these cells independent of context. We discuss regulatory mechanisms driving ontogeny and effector function and evidence for the clinical relevance of cytotoxic CD4+ T cells in cancer. In this context, we highlight important gaps in understanding in the biology of cytotoxic CD4+ T cells as well as the potential use of these cells in immunotherapies for specific cancers.

Background CD38, a druggable ectoenzyme, is involved in the generation of adenosine, which is implicated in tumour immune evasion. Its expression and role in prostate tumour-infiltrating immune cells (TIICs) have not been elucidated. Objective To characterise CD38 expression on prostate cancer (PC) epithelial cells and TIICs, and to associate this expression with clinical outcomes. Design, setting, and participants RNAseq from 159 patients with metastatic castration-resistant prostate cancer (mCRPC) in the International Stand Up To Cancer/Prostate Cancer Foundation (SU2C/PCF) cohort and 171 mCRPC samples taken from 63 patients in the Fred Hutchinson Cancer Research Centre cohort were analysed. CD38 expression was immunohistochemically scored by a validated assay on 51 castration-resistant PC (CRPC) and matching, same-patient castration-sensitive PC (CSPC) biopsies obtained between 2016 and 2018, and was associated with retrospectively collected clinical data. Outcome measurements and statistical analysis mCRPC transcriptomes were analysed for associations between CD38 expression and gene expression signatures. Multiplex immunofluorescence determined CD38 expression in PC biopsies. Differences in CD38+ TIIC densities between CSPC and CRPC biopsies were analysed using a negative binomial mixed model. Differences in the proportions of CD38+ epithelial cells between non-matched benign prostatic epithelium and PC were compared using Fisher’s exact test. Differences in the proportions of biopsies containing CD38+ tumour epithelial cells between matched CSPC and CRPC biopsies were compared by McNemar’s test. Univariable and multivariable survival analyses were performed using Cox regression models. Results and limitations CD38 mRNA expression in mCRPC was most significantly associated with upregulated immune signalling pathways. CD38 mRNA expression was associated with interleukin (IL)-12, IL-23, and IL-27 signalling signatures as well as immunosuppressive adenosine signalling and T cell exhaustion signatures. CD38 protein was frequently expressed on phenotypically diverse TIICs including B cells and myeloid cells, but largely absent from tumour epithelial cells. CD38+ TIIC density increased with progression to CRPC and was independently associated with worse overall survival. Future studies are required to dissect TIIC CD38 function. Conclusions CD38+ prostate TIICs associate with worse survival and immunosuppressive mechanisms. The role of CD38 in PC progression warrants investigation as insights into its functions may provide rationale for CD38 targeting in lethal PC. Patient summary CD38 is expressed on the surface of white blood cells surrounding PC cells. These cells may impact PC growth and treatment resistance. Patients with PC with more CD38-expressing white blood cells are more likely to die earlier., Take Home Message CD38 is expressed by phenotypically diverse prostate tumour-infiltrating immune cells (TIICs). CD38+ TIICs are associated with a worse prognosis and may be implicated in prostate cancer immune tolerance. Existing CD38-directed therapies merit evaluation in metastatic prostate cancer clinical trials.

Sipuleucel-T is an autologous cellular immunotherapy, administered as three infusions, for metastatic castration-resistant prostate cancer (mCRPC). Sipuleucel-T induces T- and B-cell responses to prostatic acid phosphatase (PAP), correlating to improved survival. The long-term impact of sipuleucel-T on tumor antigen–specific immunologic memory remains unknown, in particular, B-cell responses, as measured by antigen-specific antibody responses and B-cell receptor (BCR) sequences. To evaluate whether sipuleucel-T could induce long-term immunologic memory, we examined circulating B-cell responses before and after sipuleucel-T treatment in two groups of patients with mCRPC: those who had previously received sipuleucel-T (treated; median, 8.9 years since the previous treatment) versus those who had not (naïve). Before re-treatment, previously treated patients exhibited persistent antibody responses as well as more focused and convergent BCR repertoires with distinct V(D)J gene usage compared with naïve patients. After re-treatment, previously treated patients maintained high-frequency clones and developed more convergent BCRs at earlier time points unlike naïve patients. With the first sipuleucel-T infusion specifically, previously treated patients had less shuffling within the 100 most abundant baseline clones. In contrast, naïve patients exhibited great BCR turnover with a continued influx of new B-cell clones. Social network analysis showed that previously treated patients had more highly organized B-cell repertoires, consistent with greater clonal maturation. Higher treatment-induced BCR clonality correlated with longer survival for naïve patients. These results demonstrated the capacity of sipuleucel-T to induce long-term immune memory and lasting changes to the B-cell repertoire.

PURPOSE: The swell of new and diverse radiotracers to predict or monitor tumor response to cancer immunotherapies invites the opportunity for comparative studies to identify optimal platforms. To probe the significance of antibody format on image quality for PD-L1 imaging, we developed and studied the biodistribution of a library of antibodies based on the anti-PD-L1 IgG1 clone C4. PROCEDURE: A C4 minibody and scFv were cloned, expressed and characterized. The antibodies were functionalized with desferrioxamine and radiolabeled with Zr-89 to enable rigorous comparison to prior data collected using (89)Zr-labeled C4 IgG1. The biodistribution of the radiotracers was evaluated in C57Bl6/J or nu/nu mice bearing B16F10 or H1975 tumors, respectively, which are models that represent high and low tumor autonomous PD-L1 expression. RESULTS: The tumor uptake of the (89)Zr-C4 minibody was higher than (89)Zr-C4 scFv, and equivalent to previous data collected using (89)Zr-C4 IgG1. However, the peak tumor to normal tissue ratios were generally higher for (89)Zr-C4 scFv compared to (89)Zr-C4 minibody and (89)Zr-IgG1. Moreover, an exploratory study showed that the rapid clearance of (89)Zr-C4 scFv enabled detection of endogenous PD-L1 on a genetically engineered and orthotopic model of hepatocellular carcinoma. CONCLUSION: In summary, these data support the use of low molecular weight constructs for PD-L1 imaging, especially for tumor types that manifest in abdominal organs that are obstructed by the clearance of high molecular weight radioligands.

Purpose:Autoantibody responses in cancer are of great interest, as they may be concordant with T-cell responses to cancer antigens or predictive of response to cancer immunotherapies. Thus, we sought to characterize the antibody landscape of metastatic castration-resistant prostate cancer (mCRPC).Experimental Design:Serum antibody epitope repertoire analysis (SERA) was performed on patient serum to identify tumor-specific neoepitopes. Somatic mutation–specific neoepitopes were investigated by associating serum epitope enrichment scores with whole-genome sequencing results from paired solid tumor metastasis biopsies and germline blood samples. A protein-based immunome-wide association study (PIWAS) was performed to identify significantly enriched epitopes, and candidate serum antibodies enriched in select patients were validated by ELISA profiling. A distinct cohort of patients with melanoma was evaluated to validate the top cancer-specific epitopes.Results:SERA was performed on 1,229 serum samples obtained from 72 men with mCRPC and 1,157 healthy control patients. Twenty-nine of 6,636 somatic mutations (0.44%) were associated with an antibody response specific to the mutated peptide. PIWAS analyses identified motifs in 11 proteins, including NY-ESO-1 and HERVK-113, as immunogenic in mCRPC, and ELISA confirmed serum antibody enrichment in candidate patients. Confirmatory PIWAS, Identifying Motifs Using Next-generation sequencing Experiments (IMUNE), and ELISA analyses performed on serum samples from 106 patients with melanoma similarly revealed enriched cancer-specific antibody responses to NY-ESO-1.Conclusions:We present the first large-scale profiling of autoantibodies in advanced prostate cancer, utilizing a new antibody profiling approach to reveal novel cancer-specific antigens and epitopes. Our study recovers antigens of known importance and identifies novel tumor-specific epitopes of translational interest.

Although the importance of T-cell immune responses is well appreciated in cancer, autoantibody responses are less well-characterized. Nevertheless, autoantibody responses are of great interest, as they may be concordant with T-cell responses to cancer antigens or predictive of response to cancer immunotherapies. We performed serum epitope repertoire analysis (SERA) on a total of 1,229 serum samples obtained from a cohort of 72 men with metastatic castration-resistant prostate cancer (mCRPC) and 1,157 healthy control patients to characterize the autoantibody landscape of mCRPC. Using whole-genome sequencing results from paired solid-tumor metastasis biopsies and germline specimens, we identified tumor-specific epitopes in 29 mutant and 11 non-mutant proteins. Autoantibody enrichments for the top candidate autoantigen (NY-ESO-1) were validated using ELISA performed on the prostate cancer cohort and an independent cohort of 106 patients with melanoma. Our study recovers antigens of known importance and identifies novel tumor-specific epitopes of translational interest in advanced prostate cancer.Statement of significanceAutoantibodies have been shown to inform treatment response and candidate drug targets in various cancers. We present the first large-scale profiling of autoantibodies in advanced prostate cancer, utilizing a new next-generation sequencing-based approach to antibody profiling to reveal novel cancer-specific antigens and epitopes.Disclosure of Potential Conflicts of InterestJJA reports receiving consulting income from Janssen Biotech and Merck and honoraria from Astellas for speaker’s fees. MR reports receiving commercial research support from Novartis, Johnson & Johnson, Merck, Astellas, and Medivation, and is a consultant/advisory board member for Constellation Pharmaceuticals, Amgen, Ambrx, Johnson & Johnson, and Bayer. A.R. has received honoraria from consulting with Amgen, Bristol-Myers Squibb, Chugai, Dynavax, Genentech, Merck, Nektar, Novartis, Roche and Sanofi, is or has been a member of the scientific advisory board and holds stock in Advaxis, Arcus Biosciences, Bioncotech Therapeutics, Compugen, CytomX, Five Prime, RAPT, ImaginAb, Isoplexis, Kite-Gilead, Lutris Pharma, Merus, PACT Pharma, Rgenix and Tango Therapeutics. FYF serves on the advisory board for Dendreon, EMD Serono, Janssen Oncology, Ferring, Sanofi, Blue Earth Diagnostics, Celgene, consults for Bayer, Medivation/Astellas, Genetech, and Nutcracker Therapeutics, has honoraria from Clovis Oncology, and is a founder and has an ownership stake in PFS Genomics. SGZ and FYF have patent applications with Decipher Biosciences. SGZ and FYF have a patent application licensed to PFS Genomics. SGZ and FYF have patent applications with Celgene. WAH, RW, KK, PSD, and JCS have ownership of stocks or shares at Serimmune, paid employment at Serimmune, board membership at Serimmune, and patent applications on behalf of Serimmune.

Introduction: Broad application of neoTCR-T cell therapy involves isolation of mutation-targeted CD8 T cells from the blood of patients with different tumor types. An ultra-sensitive process streamlined for high-throughput capture of neoE-specific T cells from blood is leveraged for producing a fully personalized adoptive T cell therapy for trial participants with BC or OC (NCT03970382). Methods: Expressed mutations were identified by comparative sequencing of blood cells and tumor tissue. HLA typing was performed with OpiType. Proprietary algorithms were used to predict and prioritize tumor-exclusive neoE-HLA candidates. A library of barcoded HLA-mutational neoantigen complexes was used to interrogate PBMCs from each patient using imPACT Isolation Technology® (Dalmas et al., 2020). NeoTCRs cloned from captured antigen-experienced CD8 T cells were precision genome engineered into healthy donor T cells for functional characterization, including secretion of cytokines and effector molecules (Jacoby et al., 2019; Sennino et al., 2019). Based on pre-specified prioritization criteria, up to 3 actionable neoTCRs were selected for manufacturing the products for each individual. Results: Six patients (2 BC/4 OC) successfully underwent neoTCR product selection. The median age was 59.5 years (range 38-69). Patients received a median of 5.5 prior regimens for metastatic disease (range 4-9). Median tumor mutational burden (TMB) was 2.7 (range 0.9-13.4). HLA-neoantigen libraries contained a median of 91 snares (range 29-262) and represented 6/6 HLA alleles in 4 patients and 5/6 in the remaining 2. A median of 8.5 distinct neoE-specific T cells (range 5-15) were isolated per patient. Following functional characterization 13 neoTCRs met selection criteria: three patients with 3 TCRs, one with 2 TCRs, and two had a single TCR. The median predicted neoE:HLA affinity of the selected TCRs was 77 nM (range 6.3 - 5866) and the median IFN-g EC50 was 4 ng/mL (range 1-431). Conclusion: Relevant antigen-experienced, tumor mutation-targeted CD8 T cells were isolated from the blood of heavily pre-treated BC and OC patients with low TMB ( Citation Format: Mihaela Cristea, Bartosz Chmielowski, David Y. Oh, Roel P. Funke, Daniela A. Bota, Mehrdad Abedi. Characterization of neoepitope (neoE)-specific T cells from peripheral blood for adoptive neoTCR-T cell therapy for patients with breast cancer (bc) or ovarian cancer (oc) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 331.

BackgroundManagement of pregnancy in patients with Kell-null phenotype can be challenging. The immune systems of these patients form an antibody that is universally reactive against the Kell Blood Group System and can cause hemolytic disease of the fetus and newborn.MethodsA 29-year-old woman, pregnant for the first time, developed anti-D and anti-Ku. The mother had to have labor induced when her fetus showed signs of severe anemia, but no compatible blood was available for transfusion. The induction was delayed so that a unit of blood could be collected from the mother.ResultsDue to delayed cord clamping at delivery, the newborn did not have anemia and did not require a transfusion. The remaining blood was frozen for future needs.ConclusionBlood donation by a pregnant woman for potential transfusion to a newborn with anemia is safe for the mother and fetus, and is possibly the only option in hemolytic disease of the newborn due to a rare antibody.

2573 Background: Immune checkpoint inhibitors (ICIs) have demonstrated durable clinical responses and improved survival in patients (pts) across numerous indications. Despite this progress, the benefit of ICIs is limited to a minority of overall metastatic cancer patients. There is a critical need for biomarkers agnostic of tumor type to inform which pts will benefit from nivo alone versus ipi/nivo combination treatment. Both pre-treatment tumoral CD8 + cells and recruitment of CD8+ T cells in response to ICIs are associated with improved clinical outcomes in patients treated with anti-PD-1 therapy. 1,2,3,4 Here we report the final results of a prospective clinical study in which pts with varying advanced solid tumors were assigned to nivo, with or without ipi, based on the percentage of tumoral CD8 cells at the time of treatment. Methods: We performed a prospective, non-randomized, open-label, multicenter study in which pts with tumoral CD8+ cells ≥ 15% (CD8+ high) received nivo 360mg IV Q3W, followed by nivo maintenance 480mg Q4W. Pts with tumoral CD8+ cells < 15% (CD8+ low) received nivo 360 mg IV Q3W, and ipi at 1 mg/kg IV Q3W for 2 doses and then Q6W for 2 doses, followed by nivo maintenance 480 mg IV Q4W until PD or intolerable toxicity. Primary endpoints were Disease Control Rate (DCR: CR, PR, or SD ≥ 6 months) and CD8 low to high conversion (< 15% to ≥ 15%). Baseline and on-treatment tumor, blood and stool samples were collected for multiomic biomarker analyses. This study was not powered for formal statistical analysis. Up to 200 pts could be enrolled to allow for adaptive exploration of response and CD8 changes. Results: N = 79 pts were enrolled:7 in CD8+ high arm (nivo) and 72 in CD8+ low arm (ipi/nivo). The study enrolled a wide variety of primary solid tumors; the most common were gynecological (n = 15), prostate (12), and head and neck (7). DCR was 14% (1/7; 95% CI 1 - 44) and 24% (17/72; 95% CI 15 - 34) in the CD8 high and CD8 low arms, respectively. Of 39 pts in CD8 low arm with an on-treatment biopsy, 14 (36%; 95% CI 22 - 51) had CD8 conversion; 7/14 pts (50%) who converted had DCR. Immune-related AEs (irAEs) were consistent with known safety profile of both drugs. Conclusions: Ipi/nivo demonstrated clinical responses and increased CD8% in a range of “cold” tumors with low tumoral CD8 cells. There may be an association between increasing CD8% and response. Baseline high CD8% alone does not appear to be sufficient as a pan-cancer predictive biomarker of response to nivo monotherapy. CD8 conversion, response, and irAEs associated with circulating and stool-based biomarkers are under evaluation as composite biomarkers may improve their predictive value. Clinical trial information: 03651271.

e16510 Background: Patients (pts) with muscle-invasive bladder cancer (MIBC) ineligible for cisplatin-based chemotherapy have no standard systemic therapy options and are prioritized for radical cystectomy (RC) alone. This prospective clinical trial investigated the safety and efficacy of escalating doses of neoadjuvant atezolizumab (N-ATZ) prior to RC in MIBC pts. Methods: This is a single-institution, phase II trial of escalating doses of N-ATZ (1200 mg IV every 3 weeks) in pts with MIBC. Key inclusion criteria were resectable urothelial carcinoma of the bladder (T2-T4a,N0-1,M0) and inability to receive cisplatin-based treatment (eGFR < 60 mL/min, G≥2 neuropathy/hearing loss, pt decision). Pts with high-risk disease at RC were eligible to receive adjuvant ATZ for up to 16 total doses. Pts were followed for up to 2 years following RC. Primary efficacy endpoint was pathologic complete response (pCR; pT0N0). Secondary endpoints were safety of treatment, rate of pathologic downstaging (≤pT1N0), response based on PD-L1 status, and overall survival (OS) and recurrence-free survival (RFS) at 1 and 2 years from RC. Results: A total of 20 pts were enrolled and sequentially treated with one (n=6), two (n=5), and three (n=9) cycles of N-ATZ prior to RC. Median age was 69 (range 61-81), 75% were male and 85% Caucasian. Pts were cisplatin-ineligible due to low GFR (35%), hearing loss (25%) or neuropathy (10%); the rest refused cisplatin (30%). Most pts had pT2 disease (80%); the remainder, pT3/pT4 (15%/5%), and 10% had cN1. Among 17 pts with available tumor PD-L1 status, 76% had PD-L1 positive (CPS≥10) tumors. pCR was observed in 2 pts (10%) with 1 and 2 ATZ doses, whereas pathologic downstaging was observed in 5 pts (25%) across all 3 doses (Table). All pts completed intended treatment and RC within the trial-defined timeframe. Perioperative TRAEs of any grade occurred in 75%, but only 10% had G3 TRAEs (diarrhea, fecal incontinence). There were no G4/G5 events. Median follow-up from RC was 23.6 months and 75% were still followed at the time of data cutoff in 2/2021. Among evaluable pts, 1-year RFS and OS were 72% and 94% while 2-year RFS and OS were 64% and 69%. PD-L1 positive pts had superior OS (logrank p=0.06) and RFS (p=0.10) relative to PD-L1 negative pts. Conclusions: N-ATZ was well tolerated at all three dose levels and did not delay or prevent surgery. As few as 1 to 2 ATZ doses resulted in pathologic downstaging, including pCR. Although pCR rate in this trial was lower than expected, most pts had a durable recurrence-free period and all evaluable pts with tumor downstaging were alive and recurrence-free at 2 years following RC. Increased tumor PD-L1 expression was suggestive of improved outcomes and further biomarker analyses are ongoing. Clinical trial information: NCT02451423. [Table: see text]

476 Background: Reliable predictive markers are lacking in patients (pts) with locally advanced or metastatic urothelial carcinoma (aUC) treated with immune checkpoint inhibitors (ICI). We sought to determine whether specific genomic alterations could be used to predict overall survival (OS) in this patient population. Methods: We undertook a retrospective cohort study of pts with aUC who received ICI and underwent genomic profiling by next-generation sequencing (NGS). All patients underwent NGS using commercially available platforms (e.g. Foundation Medicine, Strata, Invitae), or testing on the CLIA-certified institutional panel UCSF500. Associations between the 20 most frequently altered genes and OS were first examined by Cox regression. Genes with a p

442 Background: For patients (pts) with muscle-invasive bladder cancer (MIBC) who are ineligible for cisplatin-based chemotherapy (cisplatin), the standard of care option is radical cystectomy (RC) alone. This prospective clinical trial investigated the safety and efficacy of escalating doses of atezolizumab (ATZ) as neoadjuvant therapy prior to RC in pts with non-metastatic urothelial cancer. Methods: This single-arm, single institution, phase II trial investigated the administration of one (n = 6), two (n = 5) or three (n = 9) cycles of ATZ (1200 mg IV given every 3 weeks) in pts with MIBC who are either ineligible for or refused cisplatin prior to RC. Key inclusion criteria were urothelial carcinoma of the bladder (T2-T4a,N0-1,M0) and inability to receive cisplatin-based treatment (eGFR < 60 mL/min, G≥2 neuropathy/hearing loss, pt decision). Pts with high-risk disease ( > pT2 or LN+) at the time of RC were eligible to receive adjuvant ATZ for up to 16 total cycles. Primary efficacy endpoint was pathologic complete response (pCR; pT0N0). Important secondary endpoints were safety of treatment, rates of pathologic downstaging and biomarker assessments in serial tissue samples. Pts were followed for up to 2 years following RC. Results: Among 20 pts with MIBC, median age was 69 (range 61-81) and 75% were male. Most commonly pts were cisplatin-ineligible due to low GFR (35%), hearing loss (25%) or neuropathy (10%); remainder refused cisplatin (30%). At trial enrollment, pT2, pT3, and pT4 was present in 80%, 15%, and 5% of pts and 10% had enlarged pelvic lymph nodes ( > 10 mm) on scans. All pts completed intended treatment cycles and all had RC within the defined timeframe ( > 3 weeks from last and < 12 weeks from first treatment). pCR at RC was 10% (2/20 pts), and was observed in pts receiving 1 and 2 cycles of ATZ. Pathologic downstaging (≤pT1N0) was achieved in 25% (5/20 pts) and observed across all three dose levels. Adjuvant ATZ was given to 8 pts. TRAEs of any grade during perioperative period occurred in 75% and G3 TRAEs in 10% (diarrhea, fecal incontinence). There were no G4 or G5 events. Median follow-up from the time of RC was 21.4 months at the time of data cutoff in 10/2020. Among evaluable pts, 1-year RFS and OS were 71% and 94% while 2-year RFS and OS were 64% and 75%. Conclusions: This prospective trial supports the safety and efficacy of ATZ as neoadjuvant therapy in MIBC. Although pCR and rates of downstaging were lower than what was previously reported in comparable neoadjuvant trials of checkpoint inhibitors in MIBC, pCRs in this trial were seen even in pts receiving only 1-2 doses of ATZ. Many pts had a durable recurrence-free period and all 4 evaluable pts who had pathologic downstaging were alive and disease free at 2 years post RC. Translational and biomarker work from this study is also being pursued. Clinical trial information: NCT02451423.

Summary Responses to anti-PD-1 immunotherapy occur but are infrequent in bladder cancer. The specific T cells that mediate tumor rejection are unknown. T cells from human bladder tumors and non-malignant tissue were assessed with single-cell RNA and paired T cell receptor (TCR) sequencing of 30,604 T cells from 7 patients. We find that the states and repertoires of CD8+ T cells are not distinct in tumors compared with non-malignant tissues. In contrast, single-cell analysis of CD4+ T cells demonstrates several tumor-specific states, including multiple distinct states of regulatory T cells. Surprisingly, we also find multiple cytotoxic CD4+ T cell states that are clonally expanded. These CD4+ T cells can kill autologous tumors in an MHC class II-dependent fashion and are suppressed by regulatory T cells. Further, a gene signature of cytotoxic CD4+ T cells in tumors predicts a clinical response in 244 metastatic bladder cancer patients treated with anti-PD-L1., Graphical Abstract, Highlights • Human bladder tumors contain multiple clonally expanded cytotoxic CD4+ T cell states • Cytotoxic CD4+ T cells can kill autologous tumors in an MHC class II-dependent fashion • Autologous regulatory T cells can inhibit the activity of cytotoxic CD4+ T cells • A cytotoxic CD4+ gene signature predicts response to anti-PD-L1 in bladder cancer, Single-cell RNA and paired T cell receptor sequencing highlights enrichment of cytotoxic CD4+ T cells rather than CD8+ T cells in human bladder cancer. These CD4+ T cells are capable of killing autologous tumor cells and are subjected to inhibition by Tregs.

BackgroundCTLA-4 blockade with ipilimumab is Food and Drug Administration-approved for melanoma as a monotherapy and has been shown to modulate the circulating T-cell repertoire. We have previously reported clinical trials combining CTLA-4 blockade with granulocyte-macrophage colony-stimulating factor (GM-CSF) in metastatic melanoma patients and in metastatic castration resistant prostate cancer (mCRPC) patients. Here, we investigate the effect that cancer type has on circulating T cells in metastatic melanoma and mCRPC patients, treated with ipilimumab and GM-CSF.MethodsWe used next-generation sequencing of T-cell receptors (TCR) to compare the circulating T cells of melanoma and mCRPC patients receiving the same treatment with ipilimumab and GM-CSF by Wilcoxon rank sum test. Flow cytometry was utilized to investigate specific T-cell populations. TCR sequencing results were correlated with each T-cell subpopulation by Spearman’s rank correlation coefficient. Of note, 14 metastatic melanoma patients had samples available for TCR sequencing and 21 had samples available for flow cytometry analysis; 37 mCRPC patients had samples available for sequencing of whom 22 have TCR data available at both timepoints; 20 of these patients had samples available for flow cytometry analysis and 16 had data available at both timepoints.ResultsWhile melanoma and mCRPC patients had similar pretreatment circulating T-cell counts, treatment induces greater expansion of circulating T cells in melanoma patients. Metastatic melanoma patients have a higher proportion of clones that increased more than fourfold after the treatment compared with mCRPC patients (18.9% vs 11.0%, p=0.017). Additionally, melanoma patients compared with mCRPC patients had a higher ratio of convergent frequency (1.22 vs 0.60, p=0.012). Decreases in clonality induced by treatment are associated with baseline CD8+ T-cell counts in both patient groups, but are more pronounced in the melanoma patients (r=−0.81, pTrial registration numbersNCT00064129;NCT01363206.

187 Background: DNA damage repair mutations (DDM) are common in prostate cancer (PCa). Optimal treatment sequence and outcomes of androgen signaling inhibitors (ASIs) and chemotherapy in this population are unclear. Methods: A retrospective, single-institution study of patients (pts) with mPCa and DDM detected on next-generation sequencing between January 2016 and July 2019 was conducted. For pts with metastatic castration-resistant prostate cancer (mCRPC), chi-squared and Wilcoxon sum rank tests were used to compare PSA50 and Time to Next Treatment (TNT) among different treatment groups, respectively. Results: Among 70 pts with mPCa and DDM, the most common mutations were BRCA2 (24, 27%), ATM (20, 22%), CDK12 (19, 21%), and MLH1/MSH2/MSH6/PMS2 (10, 11%). Fifty-seven pts (81%) received systemic treatment for mCRPC and 68% received ≥3 mCRPC treatments. Among 57 pts with ≥1 mCRPC treatment, 19 (33%) received first abiraterone, and 18 (32%) first enzalutamide. There was a trend toward higher PSA50 (74% vs 47%, P=0.196) and longer TNT (55 vs 34 wk, P=0.286) with first abiraterone vs enzalutamide. Upon switching between ASIs, 0 of 10 pts had a PSA50 response. When given chemotherapy at any point during CRPC treatment, 16/27 (59%) pts had a PSA50 response to docetaxel alone and/or cabazitaxel alone, and 14/24 (58%) to carboplatin-based regimens. Conclusions: To our knowledge, this is the largest single-institution cohort providing real-world treatment data for pts with mPCa and DDM. In the frontline mCRPC setting, abiraterone had a trend suggesting increased activity over enzalutamide that was not statistically significant. Switching ASIs at progression produced no additional responses, suggesting cross-resistance. Responses to taxanes were similar to previously reported data in all-comers. Validation in a larger, prospective cohort is needed to confirm these preliminary findings.

502 Background: ICIs are effective agents in metastatic urothelial carcinoma in both platinum-refractory and frontline settings. Responses in patients (pts) with non-urothelial histological variants are not well defined. Methods: We undertook a retrospective analysis of pts with metastatic bladder cancer treated with ICI monotherapy. Pts were identified as having a variant histology if any non-urothelial component was present. Fisher’s exact test was used to assess differences in ORR by histology. Results: Between 12/2014 and 10/2019, 102 pts received ICI monotherapy, of whom 93 were evaluable for response and 33 had variant histology. Median age was 70 yrs, 66% were male, 50% received prior platinum-based chemotherapy. Most received pembrolizumab (66%) or atezolizumab (33%). ORR in the overall cohort was 26% (15% PR, 11% CR), with 12% having SD. Histology breakdown and responses are shown in Table. Although twice as many responses were seen in urothelial pts as in pts with variant histologies (ORR 31% vs 15%), this difference was non-significant (p = 0.14). Conclusions: In this large single-institution cohort, ORR in a heterogeneous population of bladder cancer pts was consistent with data previously reported in clinical trials. Pts with variant histologies had numerically lower responses relative to pure urothelial histology, but this difference was not statistically significant. Clinical benefit to ICIs was seen across multiple variant histologies suggesting potential efficacy in this patient population that should be confirmed prospectively.[Table: see text]

Sipuleucel-T is the only FDA-approved immunotherapy for metastatic castration-resistant prostate cancer. The mechanism by which this treatment improves survival is not fully understood. We have previously shown that this treatment can induce the recruitment of CD4 and CD8 T cells to the tumor microenvironment. In this study, we examined the functional state of these T cells through gene expression profiling. We found that the magnitude of T cell signatures correlated with the frequency of T cells as measured by immunohistochemistry. Sipuleucel-T treatment was associated with increased expression of Th1-associated genes, but not Th2-, Th17 – or Treg-associated genes. Post-treatment tumor tissues with high CD8+T cell infiltration was associated with high levels of CXCL10 expression. On in situ hybridization, CXCL10+ cells colocalized with CD8+T cells in post-treatment prostatectomy tumor tissue. Neoadjuvant sipuleucel-T was also associated with upregulation of immune inhibitory checkpoints, including CTLA4 and TIGIT, and downregulation of the immune activation marker, dipeptidylpeptidase, DPP4. Treatment-associated declines in serum PSA were correlated with induction of Th1 response. In contrast, rises in serum PSA while on treatment were associated with the induction of multiple immune checkpoints, including CTLA4, CEACAM6 and TIGIT. This could represent adaptive immune resistance mechanisms induced by treatment. Taken together, neoadjuvant sipuleucel-T can induce both a Th1 response and negative immune regulation in the prostate cancer microenvironment.

Multiplexed interrogation of tumor genomic and immune features in serial patient metastases reported in a recent issue of Cell (Angelova et al., 2018) reveals a pivotal role for immune editing in shaping clonal evolution of metastases.

While immune checkpoint blockade elicits efficacious responses in many patients with cancer, it also produces a diverse and unpredictable number of immune-related adverse events (IRAE). Mechanisms driving IRAEs are generally unknown. Because CTLA-4 blockade leads to proliferation of circulating T cells, we examined in this study whether ipilimumab treatment leads to clonal expansion of tissue-reactive T cells. Rather than narrowing the T-cell repertoire to a limited number of clones, ipilimumab induced greater diversification in the T-cell repertoire in IRAE patients compared with patients without IRAEs. Specifically, ipilimumab triggered increases in the numbers of clonotypes, including newly detected clones and a decline in overall T-cell clonality. Initial broadening in the repertoire occurred within 2 weeks of treatment, preceding IRAE onset. IRAE patients exhibited greater diversity of CD4+ and CD8+ T cells, but showed no differences in regulatory T-cell numbers relative to patients without IRAEs. Prostate-specific antigen responses to ipilimumab were also associated with increased T-cell diversity. Our results show how rapid diversification in the immune repertoire immediately after checkpoint blockade can be both detrimental and beneficial for patients with cancer. Cancer Res; 77(6); 1322–30. ©2016 AACR.

Resistance to checkpoint-blockade treatments is a challenge in the clinic. We found that although treatment with combined anti-CTLA-4 and anti-PD-1 improved control of established tumors, this combination compromised anti-tumor immunity in the low tumor burden (LTB) state in pre-clinical models as well as in melanoma patients. Activated tumor-specific Tcells expressed higher amounts of interferon-γ (IFN-γ) receptor and were more susceptible to apoptosis than naive Tcells. Combination treatment induced deletion of tumor-specific Tcells and altered the Tcell repertoire landscape, skewing the distribution of Tcells toward lower-frequency clonotypes. Additionally, combination therapy induced higher IFN-γ production in the LTB state than in the high tumor burden (HTB) state on a per-cell basis, reflecting a less exhausted immune status in the LTB state. Thus, elevated IFN-γ secretion in the LTB state contributes to the development of an immune-intrinsic mechanism of resistance to combination checkpoint blockade, highlighting the importance of achieving the optimal magnitude of immune stimulation for successful combination immunotherapy strategies.

Bladder cancer can be responsive to immunotherapies such as anti-PD-1 and anti-PD-L1 checkpoint inhibitors, but overall response rates are low. Tumor-resident T cells demonstrate considerable heterogeneity as far as antigenic repertoire and phenotype. Whether the bladder tumor environment is enriched for specific types of T cells with a particular functional profile and antigenic specificity, and whether these tumor-specific populations are associated with treatment or response to immunotherapy, remains unclear. We performed single-cell RNA sequencing of CD4+ and CD8+ T cells from localized human bladder tumors and paired adjacent non-malignant bladder. We also assessed resected bladder tumors treated with neoadjuvant chemotherapy or atezolizumab (anti-PD-L1) as part of an ongoing phase II trial. We find both known and unexpected CD4+ T cell functional populations that are specific to the tumor microenvironment, including regulatory T cells and a novel population of cytotoxic CD4+ T cells. Furthermore, by combining whole-transcriptome data with paired T cell receptor identification on single cells, we find that tumor-specific CD4+ populations are clonally expanded and utilize an antigenic repertoire which is distinct from uninvolved bladder. These findings provide evidence for specialization of both phenotype and antigenic specificity of CD4+ T cells in the bladder tumor environment, and indicate that a limited number of tumor-specific antigens may drive in situ expansion and differentiation of specialized CD4+ subsets whose function may be critical to tumor control. Citation Format: David Yoonsuk Oh, Serena S. Kwek, Serghei Mangul, Siddharth S. Raju, Sasha Targ, Arun Burra, Eric Chow, Dvir Aran, Sima Porten, Maxwell V. Meng, Terence W. Friedlander, Chun Jimmie Ye, Lawrence Fong. Single-cell discrimination of altered human T cell states in the bladder tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4688.

Although overall survival from colorectal cancer (CRC) has steadily improved over the past decade, there is still work to be done. The gains associated with improved detection and treatment paradigms with chemotherapy and biologics appear to have reached their ceiling. Immune-based therapies have recently demonstrated clinical benefit in other cancers, including CRC with microsatellite instability (MSI), but patients with CRC without MSI have not yet derived benefit. This article reviews the history of CRC immunotherapy trials, the conceptual basis for why the activity of the immune system may be relevant to survival in CRC, and current efforts in CRC immunotherapy, and speculates about future efforts in this area based on experience with immunotherapy efforts in other classes of solid tumors.

The three-dimensional thymic microenvironment and calcium signaling pathways are essential for driving positive selection of developing T cells. However, the nature of calcium signals and the diversity of their effects in the thymus are unknown. We describe here a thymic slice preparation for visualizing thymocyte motility and signaling in real time with two-photon microscopy. Naive thymocytes were highly motile at low intracellular calcium concentrations, but during positive selection cells became immobile and showed sustained calcium concentration oscillations. Increased intracellular calcium was necessary and sufficient to arrest thymocyte motility. The calcium dependence of motility acts to prolong thymocyte interactions with antigen-bearing stromal cells, promoting sustained signaling that may enhance the expression of genes underlying positive selection.

Granzyme A (GzmA) induces a caspase-independent cell death pathway characterized by single-stranded DNA nicks and other features of apoptosis. A GzmA-activated DNase (GAAD) is in an ER associated complex containing pp32 and the GzmA substrates SET, HMG-2, and Ape1. We show that GAAD is NM23-H1, a nucleoside diphosphate kinase implicated in suppression of tumor metastasis, and its specific inhibitor (IGAAD) is SET. NM23-H1 binds to SET and is released from inhibition by GzmA cleavage of SET. After GzmA loading or CTL attack, SET and NM23-H1 translocate to the nucleus and SET is degraded, allowing NM23-H1 to nick chromosomal DNA. GzmA-treated cells with silenced NM23-H1 expression are resistant to GzmA-mediated DNA damage and cytolysis, while cells overexpressing NM23-H1 are more sensitive.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is frequently utilized as an adjuvant in cancer immunotherapies, and has known effects as a growth factor. However the extent to which GM-CSF modulates the adaptive immune response, including possible effects on the antigenic repertoire, remains unclear. We used next-generation sequencing of T cell receptor (TCR) beta chain sequences amplified from total RNA from peripheral blood mononuclear cells using consensus primers to assess changes in the circulating antigenic repertoire of prostate cancer patients treated with GM-CSF in multiple clinical trials. Administration of systemic GM-CSF monotherapy to patients with localized prostate cancer prior to planned radical prostatectomy (NCT00305669) results in a significant decline in clonality from the pre-treatment timepoint to the 2-week timepoint on treatment, indicative of increased early repertoire diversity (p=0.039 by Wilcoxon signed rank test). In a separate clinical trial (NCT00064129), the combination of systemic GM-CSF (250 μg/m2/day on days 1-14 of each cycle) with ipilimumab in metastatic castrate-resistant prostate cancer (mCRPC) patients also resulted in a significant decline in clonality from pre-treatment samples to the 2-week timepoint on treatment (p=0.002). In contrast, mCRPC patients who received ipilimumab alone in a separate study (NCT00323882) did not experience a similar decline in clonality after 3 weeks on treatment (p=0.625). In addition, comparison of the dynamics of specific clonotypes between the paired timepoints in the two mCRPC studies demonstrates that while there is no significant difference in the ratios of post-treatment to pre-treatment clonality between studies, patients treated with the combination of iplilimumab and GM-CSF show more repertoire change, with lower Morisita’s distance for all clones found at either timepoint (p=0.023), smaller intraclass correlation coefficient (p=0.028), and a smaller proportion of clonotypes that remain unchanged (defined by +/- 2-fold change for clones found at both timepoints) (p=0.002). These results indicate increased repertoire turnover when GM-CSF is combined with checkpoint inhibition. Hence data from both localized and metastatic prostate cancer, and from monotherapy and combination therapy regimens, supports a role for GM-CSF in inducing early diversification of the TCR repertoire. Citation Format: David Y. Oh, Li Zhang, Jason Cham, Alan Paciorek, Mark Klinger, Malek Faham, Susan F. Slovin, Lawrence Fong. Systemic granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment increases T cell receptor diversity in localized and metastatic prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1694. doi:10.1158/1538-7445.AM2017-1694