Multiple site place-of-care manufactured anti-CD19 CAR-T cells induce high remission rates in B-cell malignancy patients.

Chimeric antigen receptor (CAR) T cells targeting the CD19 antigen are effective in treating adults and children with B-cell malignancies. Place-of-care manufacturing may improve performance and accessibility by obviating the need to cryopreserve and transport cells to centralized facilities. Here we develop an anti-CD19 CAR (CAR19) comprised of the 4-1BB co-stimulatory and TNFRSF19 transmembrane domains, showing anti-tumor efficacy in an in vivo xenograft lymphoma model. CAR19 T cells are manufactured under current good manufacturing practices (cGMP) at two disparate clinical sites, Moscow (Russia) and Cleveland (USA). The CAR19 T-cells is used to treat patients with relapsed/refractory pediatric B-cell Acute Lymphocytic Leukemia (ALL; n = 31) or adult B-cell Lymphoma (NHL; n = 23) in two independently conducted phase I clinical trials with safety as the primary outcome (NCT03467256 and NCT03434769, respectively). Probability of measurable residual disease-negative remission was also a primary outcome in the ALL study. Secondary outcomes include complete remission (CR) rates, overall survival and median duration of response. CR rates are 89% (ALL) and 73% (NHL). After a median follow-up of 17 months, one-year survival rate of ALL complete responders is 79.2% (95%CI 64.5‒97.2%) and median duration of response is 10.2 months. For NHL complete responders one-year survival is 92.9%, and median duration of response has not been reached. Place-of-care manufacturing produces consistent CAR-T cell products at multiple sites that are effective for the treatment of patients with B-cell malignancies.

Bispecific anti-CD20, anti-CD19 CAR T cells for relapsed B cell malignancies: a phase 1 dose escalation and expansion trial.

Chimeric antigen receptor (CAR) T cells targeting CD19 are a breakthrough treatment for relapsed, refractory B cell malignancies1-5. Despite impressive outcomes, relapse with CD19- disease remains a challenge. We address this limitation through a first-in-human trial of bispecific anti-CD20, anti-CD19 (LV20.19) CAR T cells for relapsed, refractory B cell malignancies. Adult patients with B cell non-Hodgkin lymphoma or chronic lymphocytic leukemia were treated on a phase 1 dose escalation and expansion trial (NCT03019055) to evaluate the safety of 4-1BB-CD3ζ LV20.19 CAR T cells and the feasibility of on-site manufacturing using the CliniMACS Prodigy system. CAR T cell doses ranged from 2.5 × 105-2.5 × 106 cells per kg. Cell manufacturing was set at 14 d with the goal of infusing non-cryopreserved LV20.19 CAR T cells. The target dose of LV20.19 CAR T cells was met in all CAR-naive patients, and 22 patients received LV20.19 CAR T cells on protocol. In the absence of dose-limiting toxicity, a dose of 2.5 × 106 cells per kg was chosen for expansion. Grade 3-4 cytokine release syndrome occurred in one (5%) patient, and grade 3-4 neurotoxicity occurred in three (14%) patients. Eighteen (82%) patients achieved an overall response at day 28, 14 (64%) had a complete response, and 4 (18%) had a partial response. The overall response rate to the dose of 2.5 × 106 cells per kg with non-cryopreserved infusion (n = 12) was 100% (complete response, 92%; partial response, 8%). Notably, loss of the CD19 antigen was not seen in patients who relapsed or experienced treatment failure. In conclusion, on-site manufacturing and infusion of non-cryopreserved LV20.19 CAR T cells were feasible and therapeutically safe, showing low toxicity and high efficacy. Bispecific CARs may improve clinical responses by mitigating target antigen downregulation as a mechanism of relapse.

Automated Manufacture of Autologous CD19 CAR-T Cells for Treatment of Non-hodgkin Lymphoma.

Chimeric antigen receptor T cells (CAR-T cell) targeting CD19 are effective against several subtypes of CD19-expressing hematologic malignancies. Centralized manufacturing has allowed rapid expansion of this cellular therapy, but it may be associated with treatment delays due to the required logistics. We hypothesized that point of care manufacturing of CAR-T cells on the automated CliniMACS Prodigy® device allows reproducible and fast delivery of cells for the treatment of patients with non-Hodgkin lymphoma. Here we describe cell manufacturing results and characterize the phenotype and effector function of CAR-T cells used in a phase I/II study. We utilized a lentiviral vector delivering a second-generation CD19 CAR construct with 4-1BB costimulatory domain and TNFRSF19 transmembrane domain. Our data highlight the successful generation of CAR-T cells at numbers sufficient for all patients treated, a shortened duration of production from 12 to 8 days followed by fresh infusion into patients, and the detection of CAR-T cells in patient circulation up to 1-year post-infusion.

Surgical Wound Misclassification to Clean From Clean-Contaminated in Common Abdominal Operations.

BACKGROUND: Wound classification helps predict wound-related complications and is useful in stratifying surgical site infection reporting. We sought to evaluate misclassification among commonly performed surgeries that are at least clean-contaminated. MATERIALS AND METHODS: The National Surgical Quality Improvement Program database was queried from 2005 to 2016 by Current Procedural Terminology codes identifying common surgeries that are, by definition, not clean: colectomy, cholecystectomy, hysterectomy, and appendectomy. Univariate analysis and multivariate logistic regression were performed. RESULTS: Of the 1,208,544 operative cases reviewed, 22,925 (1.90%) were misclassified as clean. Hysterectomy was the most commonly misclassified operation (3.11%), and colectomy the least (0.82%). Misclassification was higher in laparoscopic cases (1.92% versus 1.82%; P < 0.01). Misclassification increased from 2005 to 2016 (0.22% versus 3.11%; P < 0.01). Misclassified patients were younger (46.7 versus 47.7 y; P < 0.01); had lower rates of hypertension, chronic obstructive pulmonary disease, smoking history, and steroid use (P < 0.01); and had shorter length of stay (2.2 versus 3.2 d; P < 0.01), lower 30-d readmission rates (3.7% versus 5.0%; P < 0.01), and less surgical site infections (1.7% versus 3.4%; P < 0.01). Open hysterectomy was the most significant positive predictor for misclassification (odds ratio 3.34; P < 0.01). Open appendectomy was the most significant negative predictor (odds ratio 0.20; P < 0.01). CONCLUSIONS: There is an increasing trend of misclassifying wounds as clean. Misclassified patients have better outcomes, and misclassification may be affected by patient characteristics, operative approach, and type of procedure rather than reflecting the true infectious burden. Further research is warranted.

Factors Affecting Healing in the Treatment of Hidradenitis Suppurativa.

BACKGROUND: Hidradenitis suppurativa (HS) is a chronic debilitating condition. Treatment of HS depends on disease stage, goals of care, access to care, and frequency of symptoms. We present our experience with surgical treatment for patients with HS. METHODS: Patients were followed longitudinally for at least 2 years postsurgical intervention. Demographic data, participation in a multidisciplinary program, type of surgery, healing rates, and potential factors contributing to wound healing were retrospectively reviewed in all cases using multivariate analysis. RESULTS: Two hundred forty-eight patients met the inclusion criteria with a total of 810 involved sites. Overall, 59% of patients had Hurley stage 3 disease at the time of surgery. Healing rates of 80% were observed in stages 1 and 2, and 74% were observed in stage 3. Hurley stage was not a significant predictor of healing (P = 0.09). Surgical treatment consisted of 38% incision and drainage, 44% excision without closure, and 17% excision with primary closure. Incisional and excisional treatments healed 78% and 79%, respectively, at 2 years. Primarily repaired defects (grafts and flaps) were 68% healed at 2 years. Observed healing rates were uniform regardless of the number of sites involved (P = 0.959). Participation in the multidisciplinary program was the strongest predictor of healing (78% vs 45%, P = 0.004). Sex, age, body mass index, tobacco use, diabetes, presurgery hemoglobin, and family history of HS were statistically not significant. Continuation of immune modulating therapy within 2 weeks of surgery was a predictor of reduced healing (odds ratio, 0.23; P = 0.004), whereas holding biologics for at least 2 weeks was not significant (odds ratio, 1.99; P = 0.146). CONCLUSIONS: Participation in a multidisciplinary program is a strong predictor of long-term success when treating HS. Hurley score and number of involved sites did not correlate with successful healing after surgery. If taking biologics, we identified 2 weeks as an appropriate break from biologics before and after surgical intervention. Healing rates were highest with ablative procedures (incision and drainage, excision) alone.

Multispecific anti-HIV duoCAR-T cells display broad in vitro antiviral activity and potent in vivo elimination of HIV-infected cells in a humanized mouse model.

Adoptive immunotherapy using chimeric antigen receptor-modified T cells (CAR-T) has made substantial contributions to the treatment of certain B cell malignancies. Such treatment modalities could potentially obviate the need for long-term antiretroviral drug therapy in HIV/AIDS. Here, we report the development of HIV-1-based lentiviral vectors that encode CARs targeting multiple highly conserved sites on the HIV-1 envelope glycoprotein using a two-molecule CAR architecture, termed duoCAR. We show that transduction with lentiviral vectors encoding multispecific anti-HIV duoCARs confer primary T cells with the capacity to potently reduce cellular HIV infection by up to 99% in vitro and >97% in vivo. T cells are the targets of HIV infection, but the transduced T cells are protected from genetically diverse HIV-1 strains. The CAR-T cells also potently eliminated PBMCs infected with broadly neutralizing antibody-resistant HIV strains, including VRC01/3BNC117-resistant HIV-1. Furthermore, multispecific anti-HIV duoCAR-T cells demonstrated long-term control of HIV infection in vivo and prevented the loss of CD4+ T cells during HIV infection using a humanized NSG mouse model of intrasplenic HIV infection. These data suggest that multispecific anti-HIV duoCAR-T cells could be an effective approach for the treatment of patients with HIV-1 infection.

Zinc finger nuclease-mediated transgene deletion.

A transgene, flanked by zinc finger nuclease (ZFN) cleavage sites, was deleted from a stably transformed plant by crossing it with a second plant expressing a corresponding ZFN gene. A target construct, containing a GUS reporter gene flanked by ZFN cleavage sites, a GFP reporter gene and a PAT selectable marker gene, was transformed into tobacco. Basta-resistant plants were regenerated and screened for GUS and GFP expression. A second construct, containing a ZFN gene driven by the constitutive CsVMV promoter and an HPT selectable marker gene, was also transformed into tobacco. Selected T(0) plants were grown to maturity and allowed to self-pollinate. Homozygous target plants, which expressed GUS and GFP, were crossed with homozygous ZFN plants, which expressed the ZFN gene. Numerous GUS-negative plants were observed among the hybrids with one particular cross displaying approximately 35% GUS-negative plants. Evidence for complete deletion of a 4.3 kb sequence comprising the GUS gene was obtained and sequence confirmed. Co-segregation in F(2) progenies of 'truncated' and 'intact' target sequences with expected reporter gene phenotypes were observed. Since ZFNs can be designed to bind and cleave a wide range of DNA sequences, these results constitute a general strategy for creating targeted gene deletions.

Targeted transgene integration in plant cells using designed zinc finger nucleases.

Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research. Here it is reported that designed zinc finger nucleases (ZFNs) can drive site-directed DNA integration into transgenic and native gene loci. A dimer of designed 4-finger ZFNs enabled intra-chromosomal reconstitution of a disabled gfp reporter gene and site-specific transgene integration into chromosomal reporter loci following co-transformation of tobacco cell cultures with a donor construct comprised of sequences necessary to complement a non-functional pat herbicide resistance gene. In addition, a yeast-based assay was used to identify ZFNs capable of cleaving a native endochitinase gene. Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct comprising a pat herbicide resistance gene cassette flanked by short stretches of homology to the endochitinase locus yielded up to 10% targeted, homology-directed transgene integration precisely into the ZFN cleavage site. Given that ZFNs can be designed to recognize a wide range of target sequences, these data point toward a novel approach for targeted gene addition, replacement and trait stacking in plants.