DNA Sequencing to Predict Leukemia Relapse After CAR-T Therapy

Introduction: Adoptive infusion of CD19-targeted chimeric antigen receptor T (CAR-T) cells has showed promising treatment effects for relapsed/refractory (R/R) acute lymphoblastic leukemia (ALL). While immune response induced by murine single-chain variable fragment (scFv) of the CAR leads to its premature elimination, and thus increases the risk of leukemia relapse. Humanized scFv is expected to reduce the immunogenicity of CAR, thereby promoting the survival time as well as improving the therapeutic efficacy of CAR-T treatment. We developed a humanized anti-CD19 scFv domain and now report on treatment with humanized CD19 CAR-T cells (hCART19s). Methods: A pilot phase 1 study of CAR-modified T cells containing a humanized anti-CD19 scFv domain (hCART19s) was performed with recruitment of children and adults with R/R B-ALL with or without prior exposure to a murine CD19 CAR-T cell product. Patient-derived T cells were transduced ex vivo with a lentiviral vector encoding a CAR composed of humanized anti-CD19 scFv, the human CD8 transmembrane, CD8 hinge, 4-1BB costimulatory domain, CD3ζ intracellular regions and T2A-EGFRt sequence. After lymphodepletion chemotherapy with cyclophosphamide (750 mg/m2, day -5) and fludarabine (30 mg/m2/day, days -5 to -2), patients received a single dose of autologous hCART19s infusion at a dose of 1×106 cells/kg (body weight) on day 0. The post-infusion responses, toxicities, expansion and persistence of hCART19s in patients were observed and monitored. Results: 51 R/R B-ALL patients aged 3-69 yr were treated with hCART19s. 5 patients was diagnosed as Ph+ B-ALL. 2 patients had received prior allogeneic stem cell transplant (SCT). A total of 8 patients had central nervous system leukemia (CNSL), and 1 had testicular leukemia (TL). Among 46 patients without previous CAR-T therapy, 38 (82.6%) achieved complete remission (CR) or CR with incomplete count recovery (CRi) on day 30, while 2 of 5 patients, who relapsed after murine CAR-T infusion, achieved CR after hCART19s infusion. The rates of event-free survival and overall survival were 63% (95% confidence interval [CI], 46 to 75) and 79% (95% CI, 64 to 88), respectively, at 6 months and 44% (95% CI, 27 to 59) and 67% (95% CI, 51 to 79) at 12 months. Among the 40 patients with CR or CRi, 17 had a relapse before receiving additional anticancer therapy. 12 patients underwent allogeneic hematopoietic stem-cell transplantation (HSCT) while in remission, 10 were alive without relapse and 2 had a relapse after HSCT. During treatment, 37 (72.5%) patients developed grade 1-2 cytokine release syndrome (CRS), and 11 (21.6%) patients developed grade 3-5 CRS. Neurologic events occurred in 4 (7.8%) patients within 8 weeks after infusion. 2 (3.9%) patients had grade 3 neurologic events; no grade 4 events or cerebral edema were reported. Conclusions: This study demonstrated that hCART19s have high therapeutic efficacy and safety in children and adults with R/R B-ALL. More importantly, hCART19s was confirmed to exert anti-leukemic activities in patients who relapsed after murine CAR-T infusion. HSCT is a potential approach to reduce leukemia relapse in patients who achieved CR after CAR-T therapy. Disclosures No relevant conflicts of interest to declare.

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Successful treatment of acute B lymphoblastic leukemia relapse in the skin and testicle by anti-CD19 CAR-T with IL-6 knocking down: a case report

Biomarker Research ◽

10.1186/s40364-020-00193-5 ◽

2020 ◽

Vol 8 (1) ◽

Cited By ~ 1

Author(s):

Ze-Fa Liu ◽

Li-Yun Chen ◽

Jin Wang ◽

Li-qing Kang ◽

Hua Tang ◽

...

Keyword(s):

Case Report ◽

Successful Treatment ◽

Lymphoblastic Leukemia ◽

Car T ◽

Leukemia Relapse

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CAR T Cell–Driven Trogocytosis Induces Antigen-Low Leukemia Relapse

Cancer Discovery ◽

10.1158/2159-8290.cd-rw2019-053 ◽

2019 ◽

Vol 9 (6) ◽

pp. OF10-OF10

Keyword(s):

T Cell ◽

Car T Cell ◽

Car T ◽

Leukemia Relapse

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Precision Co-Targeting of the Thymic Stromal Lymphopoietin Receptor in Childhood CRLF2-Rearranged Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2021-148602 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1705-1705

Author(s):

Asen Bagashev ◽

Joseph Patrick Loftus ◽

Savannah Ross ◽

Lisa M Niswander ◽

Haiying Qin ◽

...

Keyword(s):

T Cell ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Cytokine Release ◽

Cytokine Release Syndrome ◽

Car T ◽

Leukemia Relapse ◽

Cell Functionality ◽

Pdx Models

Abstract Introduction : Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is associated with high rates of chemoresistance and relapse. CRLF2 (cytokine receptor-like factor 2) rearrangements occur in 50% of Ph-like and 60% of Down Syndrome (DS)-associated ALL and induce constitutive JAK/STAT and other kinase signaling. Current clinical trials are studying chemotherapy with the JAK inhibitor ruxolitinib in patients with CRLF2-rearranged Ph-like ALL, but results are not yet known. While chimeric antigen receptor T-cell (CART) immunotherapies have induced remarkable remissions in children with relapsed/refractory B-ALL, approximately 50% of CD19CART-treated patients will relapse again, many with CD19 antigen loss. New therapies are needed to prevent relapse and overcome immunotherapeutic resistance. Methods : We previously developed CAR T cells targeting the thymic stromal lymphopoietin receptor (TSLPR; encoded by CRLF2) and demonstrated potent preclinical activity in Ph-like ALL models (Qin Blood 2015), which has led to a soon-to-open phase 1 clinical trial for patients with relapsed/refractory CRLF2-overexpressing ALL. In the current preclinical studies, we hypothesized that combinatorial targeting with bispecific TSLPRxCD19CART or TSLPRxCD22CART (Ross Cancer Res 2020) or with TSLPRCART + ruxolitinib will have superior activity against CRLF2-rearranged Ph-like and DS-ALL. Results : TSLPRCART treatment of CRLF2-rearranged ALL cell line (n=1) and patient-derived xenograft (PDX) models potently inhibited leukemia proliferation in vitro and in vivo and induced long-term 'cure' of xenograft mice. However, co-administration of TSLPRCART + ruxolitinib markedly diminished in vivo T cell numbers, blunted cytokine production, and facilitated leukemia relapse, which could be abrogated by delaying ruxolitinib. Importantly, ruxolitinib co-treatment prevented severe TSLPRCART-induced cytokine release syndrome (CRS) and animal death. Interestingly, ruxolitinib withdrawal led to return of T-cell functionality with re-detection of TSLPRCART in peripheral blood, induction of IFN-γ production, and leukemia clearance upon CRLF2+ ALL rechallenge (Figure 1). Conclusions: In these preclinical studies, we report potent activity of TSLPRCART in cell line (n=1) and PDX models of childhood CRLF2-rearranged Ph-like ALL (n=2) and DS-ALL (n=2) and, interestingly, deleterious effects of concomitant JAK inhibition upon CAR T cell functionality. We demonstrated that ruxolitinib co-administration impaired in vivo TSLPRCART-induced ALL cell killing but was also beneficial in protection against life-threatening cytokine release syndrome in co-treated animals. Importantly, TSLPRCART was not eliminated, only suppressed, by JAKi co-treatment with restoration of T cell functionality upon ruxolitinib removal and/or leukemia relapse/rechallenge studies. Ongoing studies are defining optimal TSLPRCART + ruxolitinib sequence(s) to maximize both anti-leukemia efficacy and potential CRS mitigation, as well as assessing in vivo efficacy of bispecific TSLPRCARTs in CRLF2-R Ph-like ALL and DS-ALL PDX models for future translation and clinical evaluation in next-generation trials. Figure 1 Figure 1. Disclosures Fry: ElevateBio: Research Funding; Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Tasian: Aleta Biotherapeutics: Consultancy; Kura Oncology: Consultancy; Gilead Sciences: Research Funding; Incyte Corporation: Research Funding.

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Imaging polymers, proteins and dna in aqueous solutions with the atomic force microscope

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100152136 ◽

1989 ◽

Vol 47 ◽

pp. 32-33

Author(s):

S.A.C. Gould ◽

B. Drake ◽

C.B. Prater ◽

A.L. Weisenhorn ◽

S.M. Lindsay ◽

...

Keyword(s):

Amino Acids ◽

Dna Sequencing ◽

Aqueous Solutions ◽

Atomic Force Microscope ◽

Piezoelectric Crystal ◽

Atomic Force ◽

Structure Of Molecules ◽

Optical Lever

The atomic force microscope (AFM) is an instrument that can be used to image many samples of interest in biology and medicine. Images of polymerized amino acids, polyalanine and polyphenylalanine demonstrate the potential of the AFM for revealing the structure of molecules. Images of the protein fibrinogen which agree with TEM images demonstrate that the AFM can provide topographical data on larger molecules. Finally, images of DNA suggest the AFM may soon provide an easier and faster technique for DNA sequencing.The AFM consists of a microfabricated SiO2 triangular shaped cantilever with a diamond tip affixed at the elbow to act as a probe. The sample is mounted on a electronically driven piezoelectric crystal. It is then placed in contact with the tip and scanned. The topography of the surface causes minute deflections in the 100 μm long cantilever which are detected using an optical lever.

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