Fresh Versus Cryopreserved/Thawed Bispecific Anti-CD19/CD20 CAR-T Cells for Relapsed, Refractory Non-Hodgkin Lymphoma
Authors: Shah Nirav N. , Zhu Fenlu , Schneider Dina , Krueger Winfried , Worden Andrew , Longo Walter L. , Hamadani Mehdi , Fenske Timothy S. , Dropulic Boro , Orentas Rimas J , Hari Parameswaran , Johnson Bryon 
Published: November 2019

Introduction
Chimeric Antigen Receptor modified T (CAR-T) cell therapies have revolutionized the relapsed, refractory B cell malignancy landscape. Due to the complex steps involved with cell production, some third-party companies require T cells to be cryopreserved prior to shipping, while most manufacturers deliver modified CAR-T cells to the treating center in a cryopreserved state. This is vastly different to the approach taken with traditional cell based therapies, specifically allogeneic transplant (allo-HCT), an immunological treatment that relies on a graft-versus-tumor (GVT) effect to prevent disease relapse. Historically, "fresh" stem cells were felt to be superior to cryopreserved products due to concerns that cryopreservation may damage T cells and other mononuclear cells delaying engraftment and limiting GVT reactivity. As a result, in clinical practice most allo-HCT products are still given as fresh infusions without cryopreservation.
In a Phase 1 clinical trial evaluating the safety of a bispecific anti-CD19, anti-CD20 CAR (LV20.19CAR), CAR-T cells were produced in a point-of-care fashion utilizing the CliniMACS Prodigy device. Local manufacturing allowed flexibility to administer either fresh LV20.19CAR-T cells without cryopreservation, or if indicated, thawed CAR-T cells post-cryopreservation.
Methods
Patients (pts) were treated on a Phase 1 dose escalation + expansion trial (NCT03019055) to demonstrate safety of 41BB/CD3z LV20.19CAR-modified T cells for adults with relapsed, refractory B cell NHL including DLBCL, MCL, FL, and CLL. The starting dose was 2.5x10^5 cells/kg with a target dose of 2.5x10^6 cells/kg. All pts received low dose fludarabine (30 mg/m2) x 3 days +cyclophosphamide (500 mg/m2) x 1 day for lymphodepletion. In the Phase 1 dose-escalation cohorts, pts received fractionated CAR-T cells over two days (30% on Day 0 and 70% on Day+1), while expansion cohort pts received CAR-T cells as a single infusion. The goal for all pts was to infuse fresh CAR-T cell prior to cryopreservation, however, CAR-T cell could be cryopreserved and infused at a later date for clinical / logistical reasons.
Results
A total of 20 pts received LV20.19CAR T cell therapy (Table 1). Fourteen pts received fresh CAR-T cells immediately post-harvest, 5 pts received post-thaw CAR-T cells, and 1 patient received a mixed fresh/cryopreserved product and was not included in this analysis. Reasons for cryopreserved administration was delay due to active infection (N=3), patient preference (N=1), and unexplained neutropenia (N=1).
Among 19 evaluable pts, the CR rate (79% vs 40%), mean ferritin, mean CRP, and incidence of CRS and neurotoxicity were all higher in the fresh infusion group (Table 1), but not statistically significant. In terms of LV20.19 CAR-T product characteristics, mean cell viability at infusion was 93% for the fresh infusion group versus 63% for cryopreserved pts (p<0.01). Point-of-care administration allowed final cell doses to be adjusted for diminished viability among pts receiving cryopreserved product. Figure 1 demonstrates the in-vivo expansion and persistence of LV20.19CART cells among fresh versus post-thaw pts. The peak percentage of CAR-T cells within the CD3 compartment was higher in pts given fresh cell infusions (Figure 2), but was not statistically significant (p=0.08).
Conclusions
Cryopreservation is known to diminish cell viability and increase clinical costs associated with freezing and storage. To date, there is limited clinical data evaluating outcomes of pts receiving fresh CAR-T cells compared to thawed CAR-T cells post-cryopreservation. Although it is presumed that in-vivo CAR-T cell activity is comparable in both scenarios, among our pts, both cell viability and in-vivo expansion favored pts who received a fresh infusion. Unlike third-party CAR-T cell products where viability is unknown at the time of infusion, we adjusted the final dose to accommodate decreased cell viability. CR rates and incidence of CRS and NTX were higher among fresh infused pts suggesting greater in-vivo activity, although findings were not statistically significant, partially a result of the small sample size. While our findings are limited by small numbers in each cohort and variability in cell dose and diagnosis, these data suggest that cryopreservation of CAR-T cells may impact clinical responses and is a logistical step that needs further investigation.

Disclosures
Shah: Cell Vault: Consultancy, Equity Ownership; Oncosec: Equity Ownership; Lentigen: Honoraria, Research Funding; Exelexis: Equity Ownership; Geron: Equity Ownership; Celgene: Other: Advisory Board; Incyte: Consultancy; Oncosec: Equity Ownership; Kite Pharma: Other: Advisory Board. Zhu:Miltenyi Biotec: Research Funding. Schneider:Lentigen Technology, A Miltenyi Biotec Company: Employment. Krueger:Lentigen Technology, A Miltenyi Biotec Company: Employment. Worden:Lentigen Technology, A Miltenyi Biotec Company: Employment. Hamadani:Sanofi Genzyme: Research Funding, Speakers Bureau; Otsuka: Research Funding; ADC Therapeutics: Consultancy, Research Funding; Takeda: Research Funding; Celgene: Consultancy; Janssen: Consultancy; Pharmacyclics: Consultancy; Merck: Research Funding; Medimmune: Consultancy, Research Funding. Dropulic:Lentigen Technology, A Miltenyi Biotec Company: Employment. Hari:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Research Funding; Janssen: Consultancy, Honoraria; Kite: Consultancy, Honoraria; Amgen: Research Funding; Spectrum: Consultancy, Research Funding; Sanofi: Honoraria, Research Funding; Cell Vault: Equity Ownership; AbbVie: Consultancy, Honoraria. Johnson:Miltenyi Biotec: Research Funding; Cell Vault: Equity Ownership.