Publications by authors named "Sarah Ahn"

10 Publications

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CAR T cells Targeting Human Immunoglobulin Light Chains Eradicate Mature B Cell Malignancies While Sparing a Subset of Normal B Cells.

Clin Cancer Res 2021 Apr 15. Epub 2021 Apr 15.

Lineberger Comprehensive Cancer Center, University of North Carolina.

Purpose: CD19-redirected chimeric antigen receptor (CAR.CD19) T cells promote clinical responses in patients with relapsed/refractory B cell non-Hodgkin lymphomas (NHL) and chronic lymphocytic leukemia (CLL). However, patients showing sustained clinical responses after CAR.CD19-T treatment show increased infection risk due to compromised B-lymphocyte recovery. Mature B-cell-derived malignancies express monoclonal immunoglobulins bearing either κ- or λ-light-chains. We initially constructed CAR-T targeting the κ-light-chain (CAR.κ) and established a clinical study with it. We then modified the CAR molecule and then developed CAR-T targeting the λ-light chain (CAR.λ) and explored its antitumor activity.

Experimental Design: Using Igλ lymphoma cell lines and patient-derived Igλ CLL cells, we evaluated the tumor cytotoxicity and cytokine profiles of CAR.λ. We also assessed the efficacy of CAR.λ in xenograft Igλ lymphoma models including a patient derived xenograft (PDX) of mantle cell lymphoma, and the effects of λ- or κ-light-chain specific CAR-T on normal B-lymphocytes in a humanized murine model.

Results: CAR.λ demonstrated antitumor effects against Igλ lymphoma cells and patient-derived CLL cells , and in xenograft and PDX Igλ lymphoma murine models. Antitumor activity of CAR.λ was superimposable to CAR.CD19. Furthermore, we demonstrated in the humanized murine model that λ- or κ-light-chain specific CAR-T cells only depleted the corresponding targeted light-chain expressing normal B cells, while sparing the reciprocal light-chain carrying B cells.

Conclusions: Adoptive transfer of CAR.λ and CAR.κ-T cells represents a useful and alternative modality to CAR.CD19-T cells in treating mature B-cell malignancies with minimal impact on humoral immunity.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-2754DOI Listing
April 2021

Preclinical Evaluation of B7-H3-specific Chimeric Antigen Receptor T Cells for the Treatment of Acute Myeloid Leukemia.

Clin Cancer Res 2021 Feb 2. Epub 2021 Feb 2.

Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina.

Purpose: The development of safe and effective chimeric antigen receptor (CAR) T-cell therapy for acute myeloid leukemia (AML) has largely been limited by the concomitant expression of most AML-associated surface antigens on normal myeloid progenitors and by the potential prolonged disruption of normal hematopoiesis by the immunotargeting of these antigens. The purpose of this study was to evaluate B7-homolog 3 (B7-H3) as a potential target for AML-directed CAR T-cell therapy. B7-H3, a coreceptor belonging to the B7 family of immune checkpoint molecules, is overexpressed on the leukemic blasts of a significant subset of patients with AML and may overcome these limitations as a potential target antigen for AML-directed CAR-T therapy.

Experimental Design: B7-H3 expression was evaluated on AML cell lines, primary AML blasts, and normal bone marrow progenitor populations. The antileukemia efficacy of B7-H3-specific CAR-T cells (B7-H3.CAR-T) was evaluated using coculture models and xenograft models of disseminated AML, including patient-derived xenograft models. The potential hematopoietic toxicity of B7-H3.CAR-Ts was evaluated using colony formation assays and in a humanized mouse model.

Results: B7-H3 is expressed on monocytic AML cell lines and on primary AML blasts from patients with monocytic AML, but is not significantly expressed on normal bone marrow progenitor populations. B7-H3.CAR-Ts exhibit efficient antigen-dependent cytotoxicity and in xenograft models of AML, and are unlikely to cause unacceptable hematopoietic toxicity.

Conclusions: B7-H3 is a promising target for AML-directed CAR-T therapy. B7-H3.CAR-Ts control AML and have a favorable safety profile in preclinical models.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-2540DOI Listing
February 2021

Activation and degranulation of CAR-T cells using engineered antigen-presenting cell surfaces.

PLoS One 2020 25;15(9):e0238819. Epub 2020 Sep 25.

FIT BEST Laboratory, Department of Chemical, Biological, and Bioengineering, North Carolina Agricultural and Technical State University, Greensboro, North Carolina, United States of America.

Adoptive cell transfer of Chimeric Antigen Receptor (CAR)-T cells showed promising results in patients with B cell malignancies. However, the detailed mechanism of CAR-T cell interaction with the target tumor cells is still not well understood. This work provides a systematic method for analyzing the activation and degranulation of second-generation CAR-T cells utilizing antigen-presenting cell surfaces. Antigen-presenting cell surfaces composed of circular micropatterns of CAR-specific anti-idiotype antibodies have been developed to mimic the interaction of CAR-T cells with target tumor cells using micro-contact printing. The levels of activation and degranulation of fixed non-transduced T cells (NT), CD19.CAR-T cells, and GD2.CAR-T cells on the antigen-presenting cell surfaces were quantified and compared by measuring the intensity of the CD3ζ chain phosphorylation and the Lysosome-Associated Membrane Protein 1 (LAMP-1), respectively. The size and morphology of the cells were also measured. The intracellular Ca2+ flux of NT and CAR-T cells upon engagement with the antigen-presenting cell surface was reported. Results suggest that NT and CD19.CAR-T cells have comparable activation levels, while NT have higher degranulation levels than CD19.CAR-T cells and GD2.CAR-T cells. The findings show that antigen-presenting cell surfaces allow a quantitative analysis of the molecules involved in synapse formation in different CAR-T cells in a systematic, reproducible manner.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0238819PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7518621PMC
November 2020

Scaffold-Mediated Static Transduction of T Cells for CAR-T Cell Therapy.

Adv Healthc Mater 2020 07 11;9(14):e2000275. Epub 2020 Jun 11.

Joint Department of Biomedical Engineering, University of North Carolina - Chapel Hill and North Carolina State University - Raleigh, 1840 Enterpreneur Way, Raleigh, NC, 27695, USA.

Chimeric antigen receptor T (CAR-T) cell therapy has produced impressive clinical responses in patients with B-cell malignancies. Critical to the success of CAR-T cell therapies is the achievement of robust gene transfer into T cells mediated by viral vectors such as gamma-retroviral vectors. However, current methodologies of retroviral gene transfer rely on spinoculation and the use of retronectin, which may limit the implementation of cost-effective CAR-T cell therapies. Herein, a low-cost, tunable, macroporous, alginate scaffold that transduces T cells with retroviral vectors under static condition is described. CAR-T cells produced by macroporous scaffold-mediated viral transduction exhibit >60% CAR expression, retain effector phenotype, expand to clinically relevant cell numbers, and eradicate CD19 lymphoma in vivo. Efficient transduction is dependent on scaffold macroporosity. Taken together, the data show that macroporous alginate scaffolds serve as an attractive alternative to current transduction protocols and have high potential for clinical translation to genetically modify T cells for adoptive cellular therapy.
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http://dx.doi.org/10.1002/adhm.202000275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7518635PMC
July 2020

Interleukin-23 engineering improves CAR T cell function in solid tumors.

Nat Biotechnol 2020 04 3;38(4):448-459. Epub 2020 Feb 3.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

Cytokines that stimulate T cell proliferation, such as interleukin (IL)-15, have been explored as a means of boosting the antitumor activity of chimeric antigen receptor (CAR) T cells. However, constitutive cytokine signaling in T cells and activation of bystander cells may cause toxicity. IL-23 is a two-subunit cytokine known to promote proliferation of memory T cells and T helper type 17 cells. We found that, upon T cell antigen receptor (TCR) stimulation, T cells upregulated the IL-23 receptor and the IL-23α p19 subunit, but not the p40 subunit. We engineered expression of the p40 subunit in T cells (p40-Td cells) and obtained selective proliferative activity in activated T cells via autocrine IL-23 signaling. In comparison to CAR T cells, p40-Td CAR T cells showed improved antitumor capacity in vitro, with increased granzyme B and decreased PD-1 expression. In two xenograft and two syngeneic solid tumor mouse models, p40-Td CAR T cells showed superior efficacy in comparison to CAR T cells and attenuated side effects in comparison to CAR T cells expressing IL-18 or IL-15.
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http://dx.doi.org/10.1038/s41587-019-0398-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7466194PMC
April 2020

THEMIS-SHP1 Recruitment by 4-1BB Tunes LCK-Mediated Priming of Chimeric Antigen Receptor-Redirected T Cells.

Cancer Cell 2020 02 30;37(2):216-225.e6. Epub 2020 Jan 30.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. Electronic address:

Chimeric antigen receptor (CAR) T cell costimulation mediated by CD28 and 4-1BB is essential for CAR-T cell-induced tumor regression. However, CD28 and 4-1BB differentially modulate kinetics, metabolism and persistence of CAR-T cells, and the mechanisms governing these differences are not fully understood. We found that LCK recruited into the synapse of CD28-encoding CAR by co-receptors causes antigen-independent CAR-CD3ζ phosphorylation and increased antigen-dependent T cell activation. In contrast, the synapse formed by 4-1BB-encoding CAR recruits the THEMIS-SHP1 phosphatase complex that attenuates CAR-CD3ζ phosphorylation. We further demonstrated that the CAR synapse can be engineered to recruit either LCK to enhance the kinetics of tumor killing of 4-1BB CAR-T cells or SHP1 to tune down cytokine release of CD28 CAR-T cells.
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http://dx.doi.org/10.1016/j.ccell.2019.12.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397569PMC
February 2020

Photothermal Therapy Promotes Tumor Infiltration and Antitumor Activity of CAR T Cells.

Adv Mater 2019 Jun 27;31(23):e1900192. Epub 2019 Mar 27.

Department of Bioengineering, California NanoSystems Institute, Jonsson Comprehensive Cancer Center and Center for Minimally Invasive Therapeutics, University of California, Los Angeles, CA, 90095, USA.

Chimeric antigen receptor (CAR)-redirected T lymphocytes (CAR T cells) show modest therapeutic efficacy in solid tumors. The desmoplastic structure of the tumor and the immunosuppressive tumor microenvironment usually account for the reduced efficacy of CAR T cells in solid tumors. Mild hyperthermia of the tumor reduces its compact structure and interstitial fluid pressure, increases blood perfusion, releases antigens, and promotes the recruitment of endogenous immune cells. Therefore, the combination of mild hyperthermia with the adoptive transfer of CAR T cells can potentially increase the therapeutic index of these cells in solid tumors. It is found that the chondroitin sulfate proteoglycan-4 (CSPG4)-specific CAR T cells infused in Nod scid gamma mice engrafted with the human melanoma WM115 cell line have superior antitumor activity after photothermal ablation of the tumor. The findings suggest that photothermal therapy facilitates the accumulation and effector function of CAR T cells within solid tumors.
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http://dx.doi.org/10.1002/adma.201900192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7262962PMC
June 2019

Cancer Immunotherapy with T Cells Carrying Bispecific Receptors That Mimic Antibodies.

Cancer Immunol Res 2019 05 6;7(5):773-783. Epub 2019 Mar 6.

Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

Tumors are inherently heterogeneous in antigen expression, and escape from immune surveillance due to antigen loss remains one of the limitations of targeted immunotherapy. Despite the clinical use of adoptive therapy with chimeric antigen receptor (CAR)-redirected T cells in lymphoblastic leukemia, treatment failure due to epitope loss occurs. Targeting multiple tumor-associated antigens (TAAs) may thus improve the outcome of CAR-T cell therapies. CARs developed to simultaneously target multiple targets are limited by the large size of each single-chain variable fragment and compromised protein folding when several single chains are linearly assembled. Here, we describe single-domain antibody mimics that function within CAR parameters but form a very compact structure. We show that antibody mimics targeting EGFR and HER2 of the ErbB receptor tyrosine kinase family can be assembled into receptor molecules, which we call antibody mimic receptors (amR). These amR can redirect T cells to recognize two different epitopes of the same antigen or two different TAAs and .
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http://dx.doi.org/10.1158/2326-6066.CIR-18-0636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6760241PMC
May 2019

Antitumor Responses in the Absence of Toxicity in Solid Tumors by Targeting B7-H3 via Chimeric Antigen Receptor T Cells.

Cancer Cell 2019 02;35(2):221-237.e8

Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA; Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599, USA. Electronic address:

The high expression across multiple tumor types and restricted expression in normal tissues make B7-H3 an attractive target for immunotherapy. We generated chimeric antigen receptor (CAR) T cells targeting B7-H3 (B7-H3.CAR-Ts) and found that B7-H3.CAR-Ts controlled the growth of pancreatic ductal adenocarcinoma, ovarian cancer and neuroblastoma in vitro and in orthotopic and metastatic xenograft mouse models, which included patient-derived xenograft. We also found that 4-1BB co-stimulation promotes lower PD-1 expression in B7-H3.CAR-Ts, and superior antitumor activity when targeting tumor cells that constitutively expressed PD-L1. We took advantage of the cross-reactivity of the B7-H3.CAR with murine B7-H3, and found that B7-H3.CAR-Ts significantly controlled tumor growth in a syngeneic tumor model without evident toxicity. These findings support the clinical development of B7-H3.CAR-Ts.
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http://dx.doi.org/10.1016/j.ccell.2019.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6645919PMC
February 2019

(p)ppGpp-Dependent Persisters Increase the Fitness of Escherichia coli Bacteria Deficient in Isoaspartyl Protein Repair.

Appl Environ Microbiol 2016 09 15;82(17):5444-54. Epub 2016 Aug 15.

Department of Biology, North Central College, Naperville, Illinois, USA

Unlabelled: The l-isoaspartyl protein carboxyl methyltransferase (PCM) repairs protein damage resulting from spontaneous conversion of aspartyl or asparaginyl residues to isoaspartate and increases long-term stationary-phase survival of Escherichia coli under stress. In the course of studies intended to examine PCM function in metabolically inactive cells, we identified pcm as a gene whose mutation influences the formation of ofloxacin-tolerant persisters. Specifically, a Δpcm mutant produced persisters for an extended period in stationary phase, and a ΔglpD mutation drastically increased persisters in a Δpcm background, reaching 23% of viable cells. The high-persister double mutant showed much higher competitive fitness than the pcm mutant in competition with wild type during long-term stationary phase, suggesting a link between persistence and the mitigation of unrepaired protein damage. We hypothesized that reduced metabolism in the high-persister strain might retard protein damage but observed no gross differences in metabolism relative to wild-type or single-mutant strains. However, methylglyoxal, which accumulates in glpD mutants, also increased fitness, suggesting a possible mechanism. High-level persister formation in the Δpcm ΔglpD mutant was dependent on guanosine pentaphosphate [(p)ppGpp] and polyphosphate. In contrast, persister formation in the Δpcm mutant was (p)ppGpp independent and thus may occur by a distinct pathway. We also observed an increase in conformationally unstable proteins in the high-persister strain and discuss this as a possible trigger for persistence as a response to unrepaired protein damage.

Importance: Protein damage is an important factor in the survival and function of cells and organisms. One specific form of protein damage, the formation of the abnormal amino acid isoaspartate, can be repaired by a nearly universally conserved enzyme, PCM. PCM-directed repair is associated with stress survival and longevity in bacteria, insects, worms, plants, mice, and humans, but much remains to be learned about the specific effects of protein damage and repair. This paper identifies an unexpected connection between isoaspartyl protein damage and persisters, subpopulations in bacterial cultures showing increased tolerance to antibiotics. In the absence of PCM, the persister population in Escherichia coli bacteria increased, especially if the metabolic gene glpD was also mutated. High levels of persisters in pcm glpD double mutants correlated with increased fitness of the bacteria in a competition assay, and the fitness was dependent on the signal molecule (p)ppGpp; this may represent an alternative pathway for responding to protein damage.
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http://dx.doi.org/10.1128/AEM.00623-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988189PMC
September 2016