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Immune checkpoint inhibitors have shown significant therapeutic responses against tumors containing increased mutation-associated neoantigen load. We have examined the evolving landscape of tumor neoantigens during the emergence of acquired resistance in patients with non–small cell lung cancer after initial response to immune checkpoint blockade with anti–PD-1 or anti–PD-1/anti–CTLA-4 antibodies. Analyses of matched pretreatment and resistant tumors identified genomic changes resulting in loss of 7 to 18 putative mutation-associated neoantigens in resistant clones. Peptides generated from the eliminated neoantigens elicited clonal T-cell expansion in autologous T-cell cultures, suggesting that they generated functional immune responses. Neoantigen loss occurred through elimination of tumor subclones or through deletion of chromosomal regions containing truncal alterations, and was associated with changes in T-cell receptor clonality. These analyses provide insight into the dynamics of mutational landscapes during immune checkpoint blockade and have implications for the development of immune therapies that target tumor neoantigens. Significance: Acquired resistance to immune checkpoint therapy is being recognized more commonly. This work demonstrates for the first time that acquired resistance to immune checkpoint blockade can arise in association with the evolving landscape of mutations, some of which encode tumor neoantigens recognizable by T cells. These observations imply that widening the breadth of neoantigen reactivity may mitigate the development of acquired resistance. Cancer Discov; 7(3); 264–76. ©2017 AACR. See related commentary by Yang, p. 250. This article is highlighted in the In This Issue feature, p. 235

Although immune checkpoint inhibitors (ICIs) have shown promise in treating various cancers, fewer than half of patients with most tumor types experience a durable response. Thus, there is a need for biomarkers to better predict outcomes. Recent studies suggest that the presence of a handful of microbial species and greater alpha-diversity in the gut may serve as a biomarker for and might facilitate ICI responses. However, the specific bacteria, or bacterial communities, that associate with improved ICI responses vary across study populations, and the factors that contribute to these discrepant findings remain elusive. Thus, a standardized method by which biospecimens may be collected, transported, and stored for gut microbiome studies in the context of ICI therapy is needed. In this study, we evaluated a method for shipping fecal samples using a low-cost (1 year of durable tumor response after ICI therapy. Patients were provided with stool collection kits and either asked to collect fecal samples at home within 48 hours of their clinic visit and store at 4°C (fresh) or asked to place stool in preservative and ship at ambient temperature to our laboratory (fixed). For fresh samples, a portion of each sample was frozen and another portion placed into preservative as a paired control. DNA was extracted using Zymo Quick-DNA™ Fecal/Soil Microbe kit. For fixed samples, methanol was removed by evaporation prior to DNA extraction. Microbial composition was analyzed with 16S rRNA amplicon sequencing with V1-V2 primers with 150bp paired-end sequencing using an Illumina platform. Among n=10 samples collected in clinic, fixed portions demonstrated decreased alpha-diversity and a uniform shift in beta-diversity compared to the paired frozen portions. In all fixed samples, Faecalibacterium and Roseburia relative abundance decreased with a corresponding increase in Bacteroides. Among a second set of n=11 samples that were placed in preservative by patients and then shipped to our laboratory, we analyzed the effects of shipping by comparing fresh stool samples and shipped fixed samples collected by the same patient within one month. The data revealed similar trends, suggesting that fixation, rather than shipping, drives the overall effect. We are currently verifying the relative abundance of specific bacterial species in both sets of samples using quantitative RT-PCR. Our data show that methanol-based preservation must be optimized prior to clinical utilization for accurate assessment. If optimized, we have identified a low-cost method to collect fecal samples that could be adopted in community practices and low-income areas. Ongoing work from our group includes optimization of processing procedures, ratio of fecal matter to preservative, and storage conditions. Citation Format: Kimberly E. Peloza, Joell J. Gills, Fyza Y. Shaikh, James R. White, Sara Glass, Carisse Lansiquot, Courtney Stevens, William Assan, William H. Sharfman, Dung T. Le, Jarushka Naidoo, Evan J. Lipson, Drew M. Pardoll, Cynthia L. Sears. Development of a low-cost method for collecting fecal samples in clinical trials [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B13.

Introduction: Nivolumab and ipilimumab (NIVO+IPI) in combination was more efficacious than nivolumab (NIVO) alone in advanced melanoma patients (pts) in the phase 3 CheckMate 067 study (Larkin et al, NEJM. 2015). To elucidate the differential mechanisms of action of NIVO+IPI vs NIVO, we assessed biomarkers in pts with advanced melanoma treated with these agents in the CheckMate 038 study. Methods: IPI-naive pts without brain metastases from parts 1-3 of the phase 1b CA209-038 study (NCT01621490) who received NIVO+IPI Q3W (1 mg/kg+3 mg/kg, n=53) or NIVO alone Q2W (3 mg/kg, n=52) were included in the analysis. Tumor and peripheral immune markers were assessed (Table), and compared across treatment groups and by best overall response (BOR) per RECIST v1.1. Results: Increases in tumor-infiltrating CD8 T cells were observed with both NIVO+IPI and NIVO treatment, with greatest increases in NIVO+IPI pts with CR/PR or SD vs other BOR groups (Table). Increases in tumor PD-L1 expression were observed with both treatments, with 10-fold greater increases in pts with CR/PR to NIVO+IPI vs pts with PD/NE to NIVO+IPI or CR/PR to NIVO alone. Reductions in circulating MDSCs were observed with NIVO+IPI treatment across BOR groups, but only in pts with CR/PR in the NIVO-treated group. Increases in CXCL9, CXCL10 and IFN-gamma were observed irrespective of BOR in pts treated with both NIVO+IPI and NIVO alone, with the greatest increases with NIVO+IPI treatment. Conclusion: NIVO+IPI vs NIVO alone was associated with consistent reductions in MDSCs and greater increases in IFN-gamma signaling. Pts with CR/PR to NIVO+IPI had the greatest increases in CD8 T cells and PD-L1 expression, suggesting enhanced T-cell effector function. This may partly explain the higher ORR observed for NIVO+IPI vs NIVO alone in CheckMate 067. Further analyses will elucidate the differential immunomodulatory effects of NIVO+IPI in combination vs NIVO monotherapy in pts with advanced melanoma. Table.Summary of results dataMedian change from baselinea (min, max)NIVO+IPI, n=53NIVO alone, n=52CR/PRSDPD/NECR/PRSDPD/NETumor immune markerbn%n%n%n%n%n%CD8 T cells1013.8 (1.3, 40.1)612.6 (0.3, 39.6)132.9 (-16.8, 33.0)34.3 (-17.3, 26.2)3-0.4 (-3.0, 7.8)40.5 (-1.3, 24.6)PD-L11041.0 (5.0, 93.0)51.0 (0.0, 55.0)94.0 (-1.0, 57.0)94.0 (-17.0, 30.0)54.0 (2.0, 70.0)142.0 (-53.0, 70.0)Peripheral immune markercn%n%n%n%n%n%MDSCs13-1.4 (-12.6, 3.7)7-5.8 (-9.9, 3.3)12-1.5 (-7.7, 4.5)3-4.0 (-5.2, 2.8)22.8 (-0.4, 5.9)55.6 (-7.8, 14.2)Immune serum cytokinednpg/mLnpg/mLnpg/mLnpg/mLnpg/mLnpg/mLCXCL9164294.5 (840, 53653)53670 (2153, 26270)152354 (402, 23188)14934 (85, 8970)9748 (190, 1765)17762 (-1250, 4490)CXCL1016410 (2, 4775)5345 (131, 2403)15369 (-20, 1706)14187 (-27, 768)995 (50, 932)17155 (-172, 1099)IFN-gamma160.40 (-0.02, 38.93)50.09 (-0.14, 0.48)150.11 (-0.41, 42.97)140.02 (-0.02, 0.64)90.02 (-0.16, 4.33)170.04 (-0.08, 0.19)CR/PR=complete response/partial response; SD=stable disease; PD/NE=progressive disease/not evaluable for BOR, but otherwise response evaluablea Baseline is defined as the last evaluable data prior to first dose treatment within 29 daysb Tumor biopsies collected day 8-36 on-treatment, assessed by immunohistochemistryc Whole-blood samples collected prior to third-dose NIVO+IPI or prior to fourth-dose NIVO alone, assessed by flow cytometryd Serum samples collected day 15+/-3 on-treatment, assessed by immunoassays Citation Format: Antoni Ribas, Salvador Martín-Algarra, Shailender Bhatia, Wen-Jen Hwu, Craig L. Slingluff, William H. Sharfman, F. Stephen Hodi, Walter J. Urba, Jason J. Luke, John B. Haanen, Margaret K. Callahan, Jedd D. Wolchok, Scott D. Chasalow, Petra Ross-Macdonald, Tina C. Young, Anila Qureshi, Christine E. Horak. Immunomodulatory effects of nivolumab and ipilimumab in combination or nivolumab monotherapy in advanced melanoma patients: CheckMate 038 [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 CT073. doi:10.1158/1538-7445.AM2017-CT073

Background: NIVO (anti-PD-1) is approved for the treatment of several cancers including advanced melanoma, but its efficacy in other types of skin cancer has not yet been evaluated. MCC is a rare and aggressive form of skin cancer, with most tumors being associated with the Merkel cell polyomavirus. MCCs frequently express PD-L1, and MCC-reactive T cells express PD-1. Methods: In CheckMate 358 (NCT02488759), patients (pts) with 5 types of advanced virus-associated cancers who had received ≤2 prior therapies, with an ECOG PS of 0-1, were eligible to receive NIVO 240 mg Q2W until progression or unacceptable toxicity. Key exclusion criteria were active brain metastases, autoimmune disease, hepatitis, and HIV infection. Primary endpoints included objective response rate (ORR by RECIST v1.1) and safety; secondary endpoints were duration of response, progression-free survival (PFS), and overall survival (OS). Twenty-five pts with MCC were treated with a median follow-up of 26 wks (range: 5-35). Results: Among 25 treated pts, median age was 66 yrs, 68% were male, and 60% were treatment-naive; 12 of 18 (67%) tested tumors were virus-positive. In 22 response-evaluable pts, ORR was 68% (table) with ongoing responses in 13 of 15 (87%) pts. Responses occurred in treatment-naive pts (71%), in pts with 1-2 prior systemic therapies (63%), and in both virus-positive and virus-negative tumors; 67% of responses occurred at ~8 weeks. At 3 months, PFS and OS rates were 82% and 92%, respectively. Treatment-related adverse events of any grade and grade 3/4 occurred in 68% and 20% of pts; 12% of pts had treatment-related AEs that led to NIVO discontinuation. Response to treatmentResponse-evaluable pts (N=22)Treatment-naive pts (n=14)Pts with 1-2 prior systemic therapies (n=8)Best overall response – n (%)Complete response3 (14)3 (21)0Partial response12 (55)7 (50)5 (63)Stable disease4 (18)3 (21)1 (13)Progressive disease3 (14)1 (7)2 (25)ORR – % (95% CI)68 (45–86)71 (42–92)63 (25–92)Time to response, months; median (range)2.0 (1.8–5.3)––Duration of response, months; median (range)Not reached (0.0–5.6)–– Conclusions: NIVO induces rapid and durable tumor regressions in the majority of treatment-naive and treatment-experienced pts with advanced MCC, with a manageable safety profile. Updated clinical response and biomarker data will be presented. Citation Format: Suzanne L. Topalian, Shailender Bhatia, Antoine Hollebecque, Ahmad Awada, Jan Paul De Boer, Ragini R. Kudchadkar, Anthony Goncalves, Jean-Pierre Delord, Uwe M. Martens, Jose Maria Lopez Picazo, Ana Oaknin, William C. Spanos, Raid Aljumaily, William H. Sharfman, Shangbang Rao, Ibrahima Soumaoro, Alexander Cao, Paul Nghiem, Dirk Schadendorf. Non-comparative, open-label, multiple cohort, phase 1/2 study to evaluate nivolumab (NIVO) in patients with virus-associated tumors (CheckMate 358): Efficacy and safety in Merkel cell carcinoma (MCC) [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 CT074. doi:10.1158/1538-7445.AM2017-CT074

Background: Response to checkpoint blockade may be dependent on tumor mutational load and the presence of antigen-specific effector T cells in the tumor microenvironment; however, how blockade modulates these features during therapy is unclear. We assessed genomic changes in tumors from patients (pts) with advanced melanoma receiving nivolumab (nivo) who progressed on ipilimumab (ipi-P) or were ipi-naive (ipi-N). Methods: Tumor biopsies were collected pretreatment and 4 weeks post first nivo dose from ipi-N or ipi-P pts treated with nivo 3 mg/kg Q2W in the phase 1 open-label CA209-038 study (NCT01621490). Biopsies from 68 pts were analyzed by whole exome, transcriptome, and/or TCR sequencing (paired biopsies from 41, 42, and 34 pts, respectively). Results: Objective response rate (ORR) in the overall cohort (n=85) was 27% with similar ORR in ipi-N and ipi-P cohorts. In the genomic cohort (n=68), ORR was 23% with a similar number of complete or partial responses (CR/PR) in ipi-N and ipi-P pts (n=7 and n=8, respectively). Prior to treatment, mutational and neoantigen load were comparable, regardless of previous treatment. Following nivo treatment, both mutational and neoantigen load were reduced 5-fold in pts who responded (CR/PR; n=9) and 1.2-fold in pts with stable disease (SD, n=13) compared with a 1.1-fold increase in pts with progressive disease (PD, n=19). Intratumoral heterogeneity analysis before and after nivo demonstrated that CR/PR pts generally lost tumor mutation clones/subclones. Novel tumor mutation clones were observed in on-treatment samples from 2 CR/PR pts and all pts who progressed on nivo. Transcriptome analyses revealed significant increases in distinct tumor immune cell subsets (CD8+ T cells and NK cells) and immune checkpoint gene expression (LAG3, CTLA4, PCDC1, and CD274 [PD-L1]) following nivo, which were more pronounced in pts with CR/PR vs PD (log2 fold-changes of 1.24, 1.07, 1.71, and 0.74, respectively). Consistent with the transcriptome analyses, tumor-infiltrating lymphocytes, as assessed by immunohistochemistry, generally increased following nivo in pts who responded: 2.8 vs 1.9-fold change in CR/PR/SD vs PD in the ipi-P cohort; 4.8 vs 1.8-fold change in CR/PR/SD vs PD in the ipi-N cohort. Differences in treatment-related TCR repertoire diversity changes were apparent between pts who responded within the ipi-N and ipi-P cohorts: a decrease in the evenness of T-cell clonotype distribution was observed among pts with CR/PR/SD relative to pts with PD in the ipi-N cohort (P=0.036), but not in the ipi-P cohort. Conclusion: Nivo and ipi modulate T-cell repertoire and tumor mutational heterogeneity in pts with advanced melanoma, presenting potential mechanisms of action underlying successful nivo therapy. These data also show that prior ipi treatment may influence biological response to nivo, but further investigation is warranted. Citation Format: Timothy A. Chan, Nadeem Riaz, Jonathan J. Havel, Vladimir Makarov, Alexis Desrichard, Jennifer S. Sims, F. Stephen Hodi, Salvador Martín-Algarra, William H. Sharfman, Shailender Bhatia, Wen-Jen Hwu, Thomas F. Gajewski, Craig L. Slingluff, Sviatoslav M. Kendall, Han Chang, John-William Sidhom, Jonathan P. Schneck, Nils Weinhold, Christine E. Horak, Walter J. Urba. Immunogenomic analyses of tumor cells and microenvironment in patients with advanced melanoma before and after treatment with nivolumab [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 2988. doi:10.1158/1538-7445.AM2017-2988

Background: In previously treated MEL patients (pts), the results of an early phase I trial with NIVO monotherapy (CA209-003) demonstrated tumor responses that were durable even after treatment discontinuation (Topalian et al. J Clin Oncol 2014;32:1020). We report extended follow-up with 5-year overall survival (OS) data from this study. Methods: IPI-naïve pts (N = 107) who had received 1-5 prior systemic therapies for MEL were treated with NIVO (0.1, 0.3, 1, 3, or 10 mg/kg) every 2 weeks for ?96 weeks. Pts were followed for OS, progression-free survival (PFS), long-term safety, and response duration after discontinuing NIVO treatment. Pts began treatment in October 2008, and current data were analyzed in October 2015 with a minimum follow-up of 45 months (time from when the last pt received his or her first dose of NIVO). Results: Median age of the pts was 61 years, 67% were male, 97% had an ECOG performance status of 0 or 1, 62% had received ?2 prior systemic therapies, and 36% had elevated lactate dehydrogenase levels at baseline. In all 107 pts, the 60-month OS rate was 34% (95% confidence interval [CI]: 25-43) and median OS was 17.3 months (95% CI: 12.5-37.8) (Table). OS rates appeared to plateau at ∼48 months, although further follow-up is needed. Similar results were observed with NIVO at 3 mg/kg, the currently approved monotherapy dose (Table). At the last timepoint for tumor assessment, PFS rates at 30 months were 18.6% and 25.7% for all pts and those who received NIVO at 3 mg/kg, respectively. Conclusions: This analysis represents the longest survival follow-up of pts who received anti-PD-1 therapy in a clinical study. In this heavily pretreated population of MEL pts, these results suggest durable, long-term survival following NIVO monotherapy, with 34% of pts alive at 5 years. Characteristics of long-term survivors and updated safety data will also be presented. OS ratesNIVO 3 mg/kg (n=17)All Patients (N=107)OS rate, % (95% CI)*12-month64.7 (37.7-82.3)62.7 (52.6-71.2)24-month47.1 (23.0-68.0)48.0 (38.1-57.2)36-month41.2 (18.6-62.6)42.1 (32.4-51.4)48-month35.3 (14.5-57.0)34.8 (25.7-44.1)60-month35.3 (14.5-57.0)33.6 (24.6-42.9)Median OS, months (95% CI)20.3 (7.2-NR)17.3 (12.5-37.8)*Based on Kaplan-Meier estimates.NR, not reached. Citation Format: F. Stephen Hodi, Harriet Kluger, Mario Sznol, Richard Carvajal, Donald Lawrence, Michael Atkins, John Powderly, William Sharfman, Igor Puzanov, David Smith, Philip Leming, Evan Lipson, Janis Taube, Robert Anders, Christine Horak, Joel Jiang, David McDermott, Jeffrey Sosman, Julie Brahmer, Drew Pardoll, Suzanne Topalian. Durable, long-term survival in previously treated patients with advanced melanoma (MEL) who received nivolumab (NIVO) monotherapy in a phase I trial. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT001.

Background: Merkel cell carcinoma (MCC) is an aggressive skin cancer linked to UV exposure and the Merkel cell polyomavirus (MCPyV). Cytotoxic chemotherapy is associated with high response rates in patients (pts) with metastatic MCC, although responses are seldom durable and median progression-free survival is only 13 weeks. MCC may be responsive to PD-1 pathway blockade as tumors often express PD-L1 and MCPyV-specific T cells express the inhibitory co-receptor PD-1. Methods: In this open-label, phase II trial by the Cancer Immunotherapy Trials Network (NCT02267603), adults with advanced/metastatic MCC and no prior systemic therapy were enrolled. Pembrolizumab, a humanized IgG4 anti-PD-1 monoclonal antibody, was given IV every 3 weeks (2 mg/kg). Response was assessed by RECIST 1.1 every 9-12 weeks. Presence of MCPyV was assessed by measuring serum antibody titers to the MCPyV oncoprotein and/or quantifying tumor oncoprotein expression by immunohistochemistry (IHC). Results: Twenty-six pts were treated (median duration 22 weeks, range 4 to 49 weeks as of 1/8/16 dataset). Twenty-three pts had at least one documented radiologic or clinical assessment of response. Fifteen of 23 evaluable pts (65%) responded (4 complete responses and 11 partial responses); 2 additional pts had stable disease and 6 had progressive disease as best response. Fourteen of 15 responses (93%) were ongoing at last follow-up. Ten responders had at least one follow-up evaluation with response duration ranging from 8+ to 27+ weeks beyond initial documented response. Treatment was generally well tolerated with mostly grade 1 or 2 adverse events. Two pts developed severe drug-related toxicities including grade 4 myocarditis (n = 1) and grade 4 transaminase elevation (n = 1), both of which improved with corticosteroid treatment. Both pts have ongoing tumor responses despite discontinuation of pembrolizumab. Sixteen of 26 pts (62%) were classified as having virus-positive MCC. Among the 23 evaluable pts, 9 of 14 (64%) of those with virus positive tumors responded, while 6 of 9 (67%) of pts with virus negative tumors responded. Analyses of T cells specific to MCPyV and titers of oncoprotein antibodies during therapy, as well as IHC characterization of tumor biopsies including PD-L1 expression are ongoing. Conclusions: First-line therapy with pembrolizumab in pts with advanced MCC is associated with frequent (65%) and durable responses. Notably, the response rate was similar among pts with virus-positive and virus-negative tumors. While virus-positive tumors are known to express highly antigenic MCPyV oncoproteins, virus-negative tumors are typically UV-induced and display strikingly elevated mutational burdens generating putative neo-antigens. Thus, potentially through distinct mechanisms, both virus-positive and virus-negative MCC tumors appear to be immunogenic and frequently responsive to anti-PD-1 therapy. Citation Format: Paul Nghiem, Shailender Bhatia, Evan Lipson, Ragini Kudchadkar, Natalie Miller, Adil Daud, Steven Fling, Philip Friedlander, Harriet Kluger, Holbrook Kohrt, Lisa Lundgren, Kim Margolin, Thomas Olencki, Sunil Reddy, Erica Shantha, William Sharfman, Elad Sharon, Michi Shinohara, John Thompson, Jennifer Yearley, Suzanne Topalian, Martin Cheever. Clinical activity, immune and viral correlates of PD-1 blockade with pembrolizumab as first systemic therapy in patients with advanced Merkel cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT096.

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Nivolumab, a fully human anti-programmed death-1 (PD-1) immune checkpoint inhibitor antibody, has shown encouraging clinical activity in melanoma. We evaluated the pharmacodynamic effects of nivolumab on immune endpoints in patients (pts) with advanced melanoma in an exploratory phase 1 study ([NCT01621490][1]). Methods: 85 pts (41 and 44 anti-CTLA-4 therapy-naive and refractory, respectively) received nivolumab 3 mg/kg IV Q2W in 56-day cycles. Tumor biopsies were obtained at baseline (B/L) and on treatment during Cycle 1, Week 4 (C1Wk4). Response was assessed during the last week of each cycle. The primary objective was to evaluate the pharmacodynamic activity of nivolumab on immune biomarkers, including circulating T-cell subsets (by multiparametric flow cytometry), serum interferon (IFN) and IFN-induced factors (by multiplex immunoassay), and T-cell infiltration in the tumor microenvironment (by immunohistochemistry for CD3, CD4, CD8, PD-1 and FOXP3). Secondary objectives included safety, efficacy, and the association between tumor PD ligand-1 (PD-L1) expression and clinical responses. Results: Across all 85 pts, the objective response rate was 25%, with 2 pts having a confirmed complete response. The safety profile was consistent with prior nivolumab monotherapy trials. Of 85 pts, 34 had evaluable biopsies at B/L and C1Wk4. Increases in tumor infiltrating lymphocytes (TILs) at C1Wk4 relative to B/L were seen in most pts; 65% of pts had an increase in CD3+ T cells. Expression of other TIL markers CD4, CD8, FOXP3 and PD-1, correlated well with one another and CD3 after treatment (C1Wk4; r = 0.69-0.93, P