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• W2007753570 abstract "Autologous T cells genetically modified to express a chimeric antibody receptor (CAR) against carboxy-anhydrase-IX (CAIX) were administered to 12 patients with CAIX-expressing metastatic renal cell carcinoma (RCC). Patients were treated in three cohorts with a maximum of 10 infusions of a total of 0.2 to 2.1 × 109 CAR T cells. CTC grade 2–4 liver enzyme disturbances occurred at the lowest CAR T cell doses, necessitating cessation of treatment in four out of eight patients in cohorts 1 and 2. Examination of liver biopsies revealed CAIX expression on bile duct epithelium with infiltration of T cells, including CAR T cells. Subsequently four patients were pre-treated with CAIX monoclonal antibody (mAb) G250 to prevent CAR-specific toxicity and showed no liver toxicities and indications for enhanced peripheral T cell persistence. No clinical responses were recorded. This report shows that CAIX-targeting CAR T cells exerted antigen-specific effects in vivo and induced liver toxicity at the lowest dose of 0.2 × 109 T cells applied, illustrating the potency of receptor-modified T cells. We provide in-patient proof that the observed “on-target” toxicity is antigen-directed and can be prevented by blocking antigenic sites in off-tumor organs and allowing higher T cell doses. Autologous T cells genetically modified to express a chimeric antibody receptor (CAR) against carboxy-anhydrase-IX (CAIX) were administered to 12 patients with CAIX-expressing metastatic renal cell carcinoma (RCC). Patients were treated in three cohorts with a maximum of 10 infusions of a total of 0.2 to 2.1 × 109 CAR T cells. CTC grade 2–4 liver enzyme disturbances occurred at the lowest CAR T cell doses, necessitating cessation of treatment in four out of eight patients in cohorts 1 and 2. Examination of liver biopsies revealed CAIX expression on bile duct epithelium with infiltration of T cells, including CAR T cells. Subsequently four patients were pre-treated with CAIX monoclonal antibody (mAb) G250 to prevent CAR-specific toxicity and showed no liver toxicities and indications for enhanced peripheral T cell persistence. No clinical responses were recorded. This report shows that CAIX-targeting CAR T cells exerted antigen-specific effects in vivo and induced liver toxicity at the lowest dose of 0.2 × 109 T cells applied, illustrating the potency of receptor-modified T cells. We provide in-patient proof that the observed “on-target” toxicity is antigen-directed and can be prevented by blocking antigenic sites in off-tumor organs and allowing higher T cell doses. Adoptive transfer of antigen-specific T cells has shown therapeutic successes in the treatment of viral infections and tumors.1Butler MO Friedlander P Milstein MI Mooney MM Metzler G Murray AP et al.Establishment of antitumor memory in humans using in vitro-educated CD8+ T cells.Sci Transl Med. 2011; 3: 80ra34Crossref PubMed Scopus (92) Google Scholar,2Savoldo B Goss JA Hammer MM Zhang L Lopez T Gee AP et al.Treatment of solid organ transplant recipients with autologous Epstein Barr virus-specific cytotoxic T lymphocytes (CTLs).Blood. 2006; 108: 2942-2949Crossref PubMed Scopus (222) Google Scholar,3Yee C Thompson JA Byrd D Riddell SR Roche P Celis E et al.Adoptive T cell therapy using antigen-specific CD8+ T cell clones for the treatment of patients with metastatic melanoma: in vivo persistence, migration, and antitumor effect of transferred T cells.Proc Natl Acad Sci USA. 2002; 99: 16168-16173Crossref PubMed Scopus (1002) Google Scholar,4Walter EA Greenberg PD Gilbert MJ Finch RJ Watanabe KS Thomas ED et al.Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor.N Engl J Med. 1995; 333: 1038-1044Crossref PubMed Scopus (1592) Google Scholar,5Dudley ME Wunderlich JR Yang JC Sherry RM Topalian SL Restifo NP et al.Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma.J Clin Oncol. 2005; 23: 2346-2357Crossref PubMed Scopus (1370) Google Scholar Treatment of patients with gene-engineered T cells equipped with either chimeric antigen receptors (CARs) or T cell receptors (TCRs) provides an attractive strategy to provide therapeutic immunity. Despite some marked successes,6Kalos M Levine BL Porter DL Katz S Grupp SA Bagg A et al.T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia.Sci Transl Med. 2011; 3: 95ra73Crossref PubMed Scopus (1807) Google Scholar,7Robbins PF Morgan RA Feldman SA Yang JC Sherry RM Dudley ME et al.Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1.J Clin Oncol. 2011; 29: 917-924Crossref PubMed Scopus (1237) Google Scholar gene-engineered T cells failed to yield antitumor responses in a substantial number of patients.8Lamers CH Sleijfer S Vulto AG Kruit WH Kliffen M Debets R et al.Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience.J Clin Oncol. 2006; 24: e20-e22Crossref PubMed Scopus (700) Google Scholar,9Till BG Jensen MC Wang J Chen EY Wood BL Greisman HA et al.Adoptive immunotherapy for indolent non-Hodgkin lymphoma and mantle cell lymphoma using genetically modified autologous CD20-specific T cells.Blood. 2008; 112: 2261-2271Crossref PubMed Scopus (558) Google Scholar,10Morgan RA Dudley ME Wunderlich JR Hughes MS Yang JC Sherry RM et al.Cancer regression in patients after transfer of genetically engineered lymphocytes.Science. 2006; 314: 126-129Crossref PubMed Scopus (2120) Google Scholar,11Johnson LA Morgan RA Dudley ME Cassard L Yang JC Hughes MS et al.Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen.Blood. 2009; 114: 535-546Crossref PubMed Scopus (1092) Google Scholar,12Parkhurst MR Yang JC Langan RC Dudley ME Nathan DA Feldman SA et al.T cells targeting carcinoembryonic antigen can mediate regression of metastatic colorectal cancer but induce severe transient colitis.Mol Ther. 2011; 19: 620-626Abstract Full Text Full Text PDF PubMed Scopus (718) Google Scholar One of the main challenges in the field of T cell engineering is receptor specificity as engineered T cells endowed with high-affinity receptors proved significantly toxic when tumor-associated antigens were targeted that are also expressed, even at low level, on normal tissue,8Lamers CH Sleijfer S Vulto AG Kruit WH Kliffen M Debets R et al.Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience.J Clin Oncol. 2006; 24: e20-e22Crossref PubMed Scopus (700) Google Scholar,11Johnson LA Morgan RA Dudley ME Cassard L Yang JC Hughes MS et al.Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen.Blood. 2009; 114: 535-546Crossref PubMed Scopus (1092) Google Scholar,12Parkhurst MR Yang JC Langan RC Dudley ME Nathan DA Feldman SA et al.T cells targeting carcinoembryonic antigen can mediate regression of metastatic colorectal cancer but induce severe transient colitis.Mol Ther. 2011; 19: 620-626Abstract Full Text Full Text PDF PubMed Scopus (718) Google Scholar,13Morgan RA Yang JC Kitano M Dudley ME Laurencot CM Rosenberg SA Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2.Mol Ther. 2010; 18: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1765) Google Scholar,14Gilham DE Debets R Pule M Hawkins RE Abken H CAR-T cells and solid tumors: tuning T cells to challenge an inveterate foe.Trends Mol Med. 2012; 18: 377-384Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar,15Kochenderfer JN Dudley ME Feldman SA Wilson WH Spaner DE Maric I et al.B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells.Blood. 2012; 119: 2709-2720Crossref PubMed Scopus (1149) Google Scholar so-called “on-target” toxicity. We have designed a CAR-directed against carboxy-anhydrase-IX (CAIX) and treated patients with CAIX-expressing metastatic renal cell carcinoma (RCC).16Weijtens ME Willemsen RA Valerio D Stam K Bolhuis RL Single chain Ig/gamma gene-redirected human T lymphocytes produce cytokines, specifically lyse tumor cells, and recycle lytic capacity.J Immunol. 1996; 157: 836-843Crossref PubMed Google Scholar In a previous report on the first three patients treated in this clinical trial, we reported (i) limiting liver enzyme disturbances in two patients, most likely caused by on-target toxicity; (ii) a limited peripheral persistence of transferred CAR T cells; and (iii) immunogenicity of the CAIX CAR receptor.8Lamers CH Sleijfer S Vulto AG Kruit WH Kliffen M Debets R et al.Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience.J Clin Oncol. 2006; 24: e20-e22Crossref PubMed Scopus (700) Google Scholar We have now extended our observations based on an amended clinical protocol in nine additional patients in which we addressed two therapy-related questions related to on-target toxicity. First, can on-target toxicity be prevented or diminished when treating patients with lower doses of CAR T cells and can a maximum tolerated dose (MTD) of CAR T cells be determined? Second, can on-target toxicity be prevented or diminished by shielding the CAIX sites in the liver but not tumor by applying a parental CAIX monoclonal antibody (mAb) before T cell treatment and will such a pre-treatment enhance the MTD of CAR T cells? Previously, administration of CAIX mAb was shown to be well tolerated,17Bleumer I Knuth A Oosterwijk E Hofmann R Varga Z Lamers C et al.A phase II trial of chimeric monoclonal antibody G250 for advanced renal cell carcinoma patients.Br J Cancer. 2004; 90: 985-990Crossref PubMed Scopus (145) Google Scholar and to saturate liver uptake of further CAIX mAb at a single low dose of 5 mg and leaving CAIX antigen in RCC metastasis accessible.18Brouwers AH Mulders PF de Mulder PH van den Broek WJ Buijs WC Mala C et al.Lack of efficacy of two consecutive treatments of radioimmunotherapy with 131I-cG250 in patients with metastasized clear cell renal cell carcinoma.J Clin Oncol. 2005; 23: 6540-6548Crossref PubMed Scopus (67) Google Scholar,19Steffens MG Boerman OC Oyen WJ Kniest PH Witjes JA Oosterhof GO et al.Intratumoral distribution of two consecutive injections of chimeric antibody G250 in primary renal cell carcinoma: implications for fractionated dose radioimmunotherapy.Cancer Res. 1999; 59: 1615-1619PubMed Google Scholar,20Steffens MG Oosterwijk-Wakka JC Zegwaart-Hagemeier NE Boerman OC Debruyne FM Corstens FH et al.Immunohistochemical analysis of tumor antigen saturation following injection of monoclonal antibody G250.Anticancer Res. 1999; 19: 1197-1200PubMed Google Scholar Here, we provide in-patient proof that the observed on-target toxicity is antigen-directed and that effective blocking of a CAR-specific antigen expressed on normal (off-tumor) tissue resulted in an improved toxicity profile and allowed higher T cell doses. Detailed pre-infusion characteristics of CAIX CAR T cells for patient treatment are summarized in Table 1. Of the administered T cells to the 12 patients, a median of 61% were CD8+ (range, 18–83%) and 53% (range, 24–65%) expressed the CAIX CAR, with similar expression on both CD4 and CD8 T cell subsets. The CAR T cells had incorporated a median of 2.6 copies of the CAR transgene in their DNA (range, 1.2–12.9). We report a median CAR-mediated net cytolytic activity of 107 LU20/106 CAR T cells (range, 18–372) and a CAR-mediated net interferon-γ (IFN-γ) production of 29 ng/24 hours/106 CAR T cells (range, 1–47). Specific IFN-γ production by samples from therapeutic infusions was at least 20-fold higher than production of interleukin-5 (IL-5), tumor necrosis factor-α, and IL-4.Table 1C haracteristics of pre-infusion CAIX CAR T cellsCAIX-specific functionIFN-γ?(ng/24 hours)eCAIX-specific IFN-γ production after 24 hours co-culture with CAIX-expressing renal cancer cell line SKRC17 clone 4; presented as ng/24 hours per 106 CAIX+ T cells and per total infused cell number.Cytolysis (LU20)fCAIX-specific cytolysis in co-culture with CAIX-expressing renal cancer cell line SKRC17 clone 4; presented as LU20 per 106 CAR T cells and per total infused cell number. 23CohortPatient numberNumber of cells (× 109)aTreatment schedule: CAR T cell infusion days: cohort 1, days 1-5 and days 17-19, cohort 2 and 3, days 1-5 and days 29-33 (patient 11, days 36-40); IL-2 doses: 5 × 105 IU/m2/12 hours at days 1-10 (patient 1-12) and days 17-25 (patient 1-3) or days 29-38 (patient 4-10, 12; patient 11, days 36-5); second treatment cycle patient 11 was postponed for 7 days due to a line infection.Percentage of CAR T cellsbCAR expression was assessed by anti-G250-Id mAb flow cytometry: mean of 2-10 infusions.Number of CAR T cells (× 109)cTotal number of infused CAR T cells in 2-10 infusions.Percentage of CD4/CD8IL-2 dosesCAR DNA copiesdNumber of CAR DNA copies per CAR T cells; mean of 2-10 infusions.Per 106TotalPer 106Total114. 0532. 136/619g2. 33370, 720372792, 20421. 4610. 8542/61365. 52924, 1449378, 77430. 6630. 3861/419g2. 82810, 6828833, 274241. 7601. 066/38406. 93535, 094120119, 70052. 5411. 056/484012. 94039, 4887978, 71861. 9531. 026/7535gTreatment stopped early due to CTC ≥ grade 3 liver toxicities.2. 04747, 039123123, 14972. 1471. 017/83402. 311, 0051817, 97080. 8240. 238/635g2. 5183, 50421743, 340392. 0501. 050/50402. 61616, 040134134, 292101. 8541. 026/75402. 62323, 078186185, 928112. 2451. 085/18401. 21312, 5404039, 625123. 4582. 026/76402. 72957, 49862123, 328Abbreviations: CAIX, carboxy-anhydrase-IX; CAR, chimeric antibody receptor; CTC, common toxicity criteria; IFN, interferon; IL, interleukin; mAb, monoclonal antibody.a Treatment schedule: CAR T cell infusion days: cohort 1, days 1-5 and days 17-19, cohort 2 and 3, days 1-5 and days 29-33 (patient 11, days 36-40); IL-2 doses: 5 × 105 IU/m2/12 hours at days 1-10 (patient 1-12) and days 17-25 (patient 1-3) or days 29-38 (patient 4-10, 12; patient 11, days 36-5); second treatment cycle patient 11 was postponed for 7 days due to a line infection.b CAR expression was assessed by anti-G250-Id mAb flow cytometry: mean of 2-10 infusions.c Total number of infused CAR T cells in 2-10 infusions.d Number of CAR DNA copies per CAR T cells; mean of 2-10 infusions.e CAIX-specific IFN-γ production after 24 hours co-culture with CAIX-expressing renal cancer cell line SKRC17 clone 4; presented as ng/24 hours per 106 CAIX+ T cells and per total infused cell number.f CAIX-specific cytolysis in co-culture with CAIX-expressing renal cancer cell line SKRC17 clone 4; presented as LU20 per 106 CAR T cells and per total infused cell number. 23g Treatment stopped early due to CTC ≥ grade 3 liver toxicities. Open table in a new tab Abbreviations: CAIX, carboxy-anhydrase-IX; CAR, chimeric antibody receptor; CTC, common toxicity criteria; IFN, interferon; IL, interleukin; mAb, monoclonal antibody. Between March 2002 and December 2010, 12 metastatic RCC patients were treated, Table 2 provides patient characteristics and disease history, and consort diagram (Figure 1) showing compliance to eligibility criteria and protocol treatment. Patients had their diseased kidney removed and presented with metastasis, primarily in lung and a wide range of anatomical sites and were refractory to prior treatment with IFN-α and/or tyrosine kinase inhibitors. Patients were treated with CAIX CAR T cells in three cohorts, as further illustrated in Supplementary Figure S1. In cohort 1, an in-patient uptitration scheme was applied of a maximum of eight infusions at days 1–5 and days 17–19 with 2 × 107 to 2 × 109 CAR T cells per infusion; three patients were treated in this cohort with an actual cumulative dose of 0.38 to 2.1 × 109 CAR T cells (Table 1). Two out of three patients developed dose-limiting toxicities of liver enzymes (see below) and the treatment protocol was amended (see Supplementary Figures S1 and S2). In cohort 2, patients were treated according a conventional 3 × 3 phase I approach, starting at a dose of 1 × 108 CAR T cells per infusion and applying a maximum of 10 infusions at days 1–5 and days 29–33. At the starting dose, a liver enzyme dose-limiting toxicity was obtained in the third and a second in the fifth patient (i.e., patient 6 and 8, Table 3); patients received an actual cumulative dose of 0.2 to 1.0 × 109 CAR T cells (Table 1). Therefore, cohort 2 was closed without applying a dose escalation and assessment of a MTD of CAIX CAR T cells. In cohort 3, patients were treated as in cohort 2, but with pre-treatment of an intravenous infusion of 5 mg of the anti-CAIX mAb G250 3 days before start of each series of CAR T cell infusions, in order to block CAIX in liver and prevent liver enzyme toxicity. Three patients were treated at the starting (cumulative) dose of 1 × 109 CAR T cells without toxicity and CAR T cell dose was escalated to 2 × 109 CAR T cells (cumulative) for next patients.Table 2Patient characteristics and disease history before CAIX CAR T cell therapyCohortPatient numberAge/sexNephrectomy (months from primary diagnosis till metastasis)aNephrectomy left (L), right (R) (months till appearance of metastatic disease).Prior treatmentbFirst line treatment: IFN-a; Sur, surgical metastasectomy; Su, sunitinib; second line treatment: Sor, sorafinib; Sor + IL2. (months till progression)Sites of evaluable diseasecSite of metastasis: Ad, adrenal; Bo, bone; Li, liver; Ln, lymphnode; Ki, kidney; Lu, lung; Med, medastinum; Sub, subcuaneous; Subpl, subpleural area.1155/FR (124, 134)Sur, IFN (7)Ad, Lu268/FR (35)IFN (9)Lu346/MR (63)IFN (18)Lu2463/ML (33)IFN (17)Lu554/ML (58, 85)Sur, IFN (9)Lu, Sub654/MR (6)Su (8)Ln, Lu769/ML (0, 5)SurdThrombectomy of vena cava inferior b/o local tumor spread., Su (3)Ad, Bo, Li854/FL (0, 5)Sur, Su (6)Bo, Li, Ln, Lu3964/FL (0)IFN (3); Sor + IL2 (14)Lu1074/ML (12)IFN (17), Sor (17)Lu, Med, Subpl1167/ML (0)Su (6)Lu1262/MR (48, 72, 102)SureMetastasectomy in parotid gland and thyroid gland., Su (26), Sor + IL2 (3)Ad, Bo, KiAbbreviations: CAIX, carboxy-anhydrase-IX; CAR, chimeric antibody receptor; IFN, interferon; IL, interleukin.a Nephrectomy left (L), right (R) (months till appearance of metastatic disease).b First line treatment: IFN-a; Sur, surgical metastasectomy; Su, sunitinib; second line treatment: Sor, sorafinib; Sor + IL2.c Site of metastasis: Ad, adrenal; Bo, bone; Li, liver; Ln, lymphnode; Ki, kidney; Lu, lung; Med, medastinum; Sub, subcuaneous; Subpl, subpleural area.d Thrombectomy of vena cava inferior b/o local tumor spread.e Metastasectomy in parotid gland and thyroid gland. Open table in a new tab Table 3T oxicities and blocking of CAIX antigen sites by CAIX mAb during T cell therapyToxicityBlood levels of CAIX mAb at start ofLiver enzymesbMaximum grade CTC reached by any of the individual liver enzymes (according to CTCAE, version 4), see Supplementary Table S1 for details.Tx-cycle 1 (day 1)Tx -cycle 2 (day 29)CohortPatient numberPre-treatment CAIX mAbaInfusion of 5 mg CAIX mAb at days −3 and 26 (patient 11, day 33), i. e., 3 days before start of T cell infusions in cycles 1 and 2, respectively.Pre-TxCycle 1Cycle 2ng/mlT1/2 (d)ng/mlT1/2 (d)11No04—2No0113No13—24No1115No1116No1237No0108No23—39Yes111157—cToo few data points to calculate mAb half-life (T1/2) in the circulation.1592. 310Yes0112062. 92362. 711Yes1113201. 72671. 712Yes1111011. 3981. 5Abbreviations: CAIX, carboxy-anhydrase-IX; CTC, common toxicity criteria; mAb, monoclonal antibody.a Infusion of 5 mg CAIX mAb at days −3 and 26 (patient 11, day 33), i. e., 3 days before start of T cell infusions in cycles 1 and 2, respectively.b Maximum grade CTC reached by any of the individual liver enzymes (according to CTCAE, version 4), see Supplementary Table S1 for details.c Too few data points to calculate mAb half-life (T1/2) in the circulation. Open table in a new tab Abbreviations: CAIX, carboxy-anhydrase-IX; CAR, chimeric antibody receptor; IFN, interferon; IL, interleukin. Abbreviations: CAIX, carboxy-anhydrase-IX; CTC, common toxicity criteria; mAb, monoclonal antibody. From 2005 onwards several new, highly active treatment options were introduced for RCC.21Escudier B Szczylik C Porta C Gore M Treatment selection in metastatic renal cell carcinoma: expert consensus.Nat Rev Clin Oncol. 2012; 9: 327-337Crossref PubMed Scopus (115) Google Scholar Consequently, patient accrual gradually diminished as patients were in a too poor condition to meet eligibility criteria after these multilineage treatments. The study was terminated after one patient in the second dose level of cohort 3 was treated. No clinical responses were obtained and the median overall survival was 9.5 months (range: 3–33 months) for cohorts 1 (n = 3 patients) and 2 (n = 5 patients), and 12.5 months (6–24 months) for cohort 3 (n = 4 patients). We have previously reported8Lamers CH Sleijfer S Vulto AG Kruit WH Kliffen M Debets R et al.Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience.J Clin Oncol. 2006; 24: e20-e22Crossref PubMed Scopus (700) Google Scholar that two out of three patients in cohort 1 developed transient liver enzyme disturbances (CTC grades 3–4). This observation necessitated cessation of treatment in patients 1 and 3, corticosteroid treatment in patient 1 and reduction of maximal T cell dose to 2 × 108 T cells in patients 2 and 3.8Lamers CH Sleijfer S Vulto AG Kruit WH Kliffen M Debets R et al.Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience.J Clin Oncol. 2006; 24: e20-e22Crossref PubMed Scopus (700) Google Scholar The toxicity was likely to be caused by an antigen-specific encounter of the CAR T cells with CAIX expressing bile duct epithelial cells, so-called “on-target toxicity”. Here, we add to these data our observations in nine more patients treated according to an amended protocol according a conventional phase I strategy without (cohort 2) and with (cohort 3) pre-treatment with anti-CAIX mAb (see Supplementary Figures S1 and S2). In cohort 2, the patients 6 and 8 developed transient CTC grade 3 liver enzyme disturbances after 10 and 2 T cell infusions, respectively. Again, treatment was halted and corticosteroids were applied. Of note, patient 8 presented with CTC grade 2 liver enzyme levels before treatment, but was eligible because of the presence of liver metastasis (Tables 2 and 3, and Supplementary Table S1). Liver biopsies were taken from patient 6 at day 40 and from patient 8 at day 4, i.e., 7 and 2 days after the last T cell infusion, respectively. Pathological examination showed intact liver architecture, minor (patient 6) to purulent cholangitis (patient 8), limited (patient 6) to massive (patient 8) T cell infiltrates around the bile ducts with sporadic bile duct epithelium infiltration, and clear CAIX antigen expression on the bile duct epithelial cells (Figure 2). Remarkably, in patient 8, the portal T cell infiltrate consisted predominantly of CD4 T cells (CD4:CD8 ratio, 4.0) while the CAR T cell infusions consisted predominantly of CD8 T cells (ratio, 0.6; Table 1). Flow cytometry analysis confirmed presence of CAR T cells in this biopsy and revealed that 0.07% of total T cells were in fact CAR T cells, whereas the percentage of CAR T cells in a corresponding blood sample was 0.15 (Supplementary Figure S3). In addition, staining of cryosections using G250 anti-idiotype mAb NuH82 suggested the presence of CAR T cells, but poor morphology prohibited the assessment of their location. In cohort 3, patients were pre-treated with 5 mg CAIX mAb followed by a cumulative dose of 1 × 109 (three patients) or 2 × 109 CAR T cells (one patient). Dose-limiting toxicities or higher than grade 1 liver toxicity were not observed (Table 3 and Supplementary Table S1). Blood concentrations of CAIX mAb at start of CAR T cell infusions were similar for treatment cycles 1 and 2 (i.e., days 1 and 29) of individual patients and ranged from 98 to 320 ng/ml with a T1/2 of 1.7 days (median; range 1.3–2.9), Table 3. As a result, CAIX mAb was detectable in blood up to 6–10 days after start of CAR T cell infusions. CAR T cells were detectable in peripheral blood by flow cytometry during a median of 39 days (range, 12–74; n = 12 patients).22Lamers CH Willemsen R van Elzakker P van Steenbergen-Langeveld S Broertjes M Oosterwijk-Wakka J et al.Immune responses to transgene and retroviral vector in patients treated with ex vivo-engineered T cells.Blood. 2011; 117: 72-82Crossref PubMed Scopus (267) Google Scholar The number of circulating CAR T cells peaked at a median of 2.8/µl (range, 0.8–10.0) and 2.6/µl (range, 1.3–9.0) for treatment cycles 1 and 2, respectively, at 6 days (range, 3–16) after start of infusions in each treatment (Table 4). We analyzed CAR T cell persistence, defined as the last day of flow cytometry CAR T cell detection relative to start of treatment cycle 2, in patients receiving an equal cumulative dose of 1 × 109 CAR T cells in cohort 2 (patients 4–7) versus cohort 3 (patients 9–11). Persistence of CAR T cells tend to be somewhat longer in cohort 3 (21, 23, 37 days) when compared with cohort 2 (10, 5, 14, 21 days; P = 0.06 using Student's t-test). Patient 12, treated in cohort 3 with a total of 2 × 109 CAR T cells developed a fast and vigorous anti-CAIX CAR T cell response, and CAR T cells were detected for only 6 days relative to start of treatment cycle 2 (Supplementary Data).Table 4Characteristics of post-infusion CAIX CAR T cellsCirculating CAR T cellsEx vivo CAIX-specific reactivity of patient PBMCPeak level (cells/µl; day)aPeak level of CAR T cell numbers per µl in blood; between brackets, peak day relative to start of treatment; nt, no treatment in cycle 2; see also legend to Table 1.IFN-γctionbNet CAIX-specific IFN-γ production by post-infusion PBMC after 24 hours co-culture with CAIX-expressing renal cancer cell line SKRC17 clone 4; presented as pg/106 PBMC/24 hours., peak level (pg; day)CytotoxicitycNet CAIX-specific cytolysis by post-infusion PBMC in 4 hours co-culture with CAIX-expressing renal cancer cell line SKRC17 clone 4; presented as % cytolysis at PBMC: target cell ratio of 40: 1, i. e., % cytolysis in 2, 500 target cells by 105 PBMC in 4 hours., peak level (%CTX; day)CohortPatient numberCycle 1Cycle 2Cycle 1Cycle 2Cycle 1Cycle 2117. 3 (7)nt30 (8)nt13 (8)nt210. 0 (16)9. 0 (16)84 (8)123 (5)13 (5)19 (3)30. 8 (3)nt31 (5)nt31 (5)nt241. 4 (5)1. 5 (8)67 (5)51 (5)11 (5)8 (5)52. 2 (4)1. 3 (5)45 (5)20 (8)3 (8)<1 (-)61. 3 (7)3. 0 (6)48 (8)27 (8)14 (8)8 (8)71. 7 (5)1. 7 (3)14 (8)3 (5)<1 (-)<1 (-)80. 9 (4)nt6 (5)nt<1 (-)nt395. 3 (11)5. 4 (5)44 (5)63 (5)5 (5)6 (5)105. 0 (6)4. 3 (8)43 (5)39 (5)2 (8)3 (8)113. 4 (7)2. 6 (10)11 (5)6 (5)19 (8)12 (8)125. 8 (8)1. 7 (5)50 (5)17 (5)6 (5)15 (5)Abbreviations: CAIX, carboxy-anhydrase-IX; CAR, chimeric antibody receptor; IFN, interferon; PBMC, peripheral blood mononuclear cell.a Peak level of CAR T cell numbers per µl in blood; between brackets, peak day relative to start of treatment; nt, no treatment in cycle 2; see also legend to Table 1.b Net CAIX-specific IFN-γ production by post-infusion PBMC after 24 hours co-culture with CAIX-expressing renal cancer cell line SKRC17 clone 4; presented as pg/106 PBMC/24 hours.c Net CAIX-specific cytolysis by post-infusion PBMC in 4 hours co-culture with CAIX-expressing renal cancer cell line SKRC17 clone 4; presented as % cytolysis at PBMC: target cell ratio of 40: 1, i. e., % cytolysis in 2, 500 target cells by 105 PBMC in 4 hours. Open table in a new tab Abbreviations: CAIX, carboxy-anhydrase-IX; CAR, chimeric antibody receptor; IFN, interferon; PBMC, peripheral blood mononuclear cell. CAIX-specific IFN-γ production by post-treatment peripheral blood mononuclear cell (PBMC), expressed as pg IFN-γ/106 PBMC/24 hours, is presented in Table 4 and further specified in Supplementary Table S2a. CAIX-specific IFN-γ production peaked at 5 days (median, range: 5–8) after start of each treatment cycle, with a median IFN-γ production of 43 pg/106 PBMC/24 hours (range 3–123 pg). The IFN-γ production by post-infusion PBMC correlates with pre-infusion CAIX CAR T cell IFN-γ production potency (Pearson's correlation r = 0.45, P = 0.04; Supplementary Table S2b and Figure 3). Post-treatment PBMC exerted CAIX-specific cytolysis in 17 out of 21 treatment cycles, peaking again at 5 days (range, 3–8) after start of treatment are presented in Table 4 and further specified in Supplemental Table S3a. CAIX-specific cytolysis by post-treatment PBMC did not correlate with pre-infusion CAIX CAR T cell cytolytic (Supplementary Table S3b). Pre-treatment with CAIX mAb does not seem to affect the ex vivo activity of patients' PBMC in cohort 3 when compared with cohort 2. Blood cytokine levels were assessed in plasma samples obtained at regular intervals before, during, and after CAR T cell infusions and are presented in Figure 4 for individual patients and treatment cycles at day 1 (pre-treatment) and at the days that levels peaked. Blood cytokin" @default.
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