Publications by authors named "Craig Aarts"

8 Publications

  • Page 1 of 1

Manufacturing T cells in hollow fiber membrane bioreactors changes their programming and enhances their potency.

Oncoimmunology 2021 9;10(1):1995168. Epub 2021 Nov 9.

McMaster Immunology Research Centre, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada.

Engineered T cell therapies have revolutionized modern oncology, however processes for manufacturing T cell therapies vary and the impact of manufacturing processes On the cell product is poorly understood. Herein, we have used a commercially available hollow fiber membrane bioreactor (HFMBR) operated in a novel mode to demonstrate that T cells can be engineered with lentiviruses, grown to very high densities, and washed and harvested in a single, small volume bioreactor that is readily amenable to automation. Manufacturing within the HFMBR dramatically changed the programming of the T cells and yielded a product with greater therapeutic potency than T cells produced using the standard manual method. This change in programming was associated with increased resistance to cryopreservation, which is beneficial as T cell products are typically cryopreserved prior to administration to the patient. Transcriptional profiling of the T cells revealed a shift toward a glycolytic metabolism, which may protect cells from oxidative stress offering an explanation for the improved resistance to cryopreservation. This study reveals that the choice of bioreactor fundamentally impacts the engineered T cell product and must be carefully considered. Furthermore, these data challenge the premise that glycolytic metabolism is detrimental to T cell therapies.
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http://dx.doi.org/10.1080/2162402X.2021.1995168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8583081PMC
November 2021

Development of a B-cell maturation antigen-specific T-cell antigen coupler receptor for multiple myeloma.

Cytotherapy 2021 09 1;23(9):820-832. Epub 2021 Jul 1.

Department of Pathology and Molecular Medicine, McMaster Immunology Research Centre, McMaster University, Hamilton, Canada. Electronic address:

Background Aims: T cells engineered with synthetic receptors have delivered powerful therapeutic results for patients with relapsed/refractory hematologic malignancies. The authors have recently described the T-cell antigen coupler (TAC) receptor, which co-opts the endogenous T-cell receptor (TCR) and activates engineered T cells in an HLA-independent manner. Here the authors describe the evolution of a next-generation TAC receptor with a focus on developing a TAC-engineered T cell for multiple myeloma.

Methods: To optimize the TAC scaffold, the authors employed a bona fide antigen-binding domain derived from the B-cell maturation antigen-specific monoclonal antibody C11D5.3, which has been used successfully in the clinic. The authors first tested humanized versions of the UCHT1 domain, which is used by the TAC to co-opt the TCR. The authors further discovered that the signal peptide affected surface expression of the TAC receptor. Higher density of the TAC receptor enhanced target binding in vitro, which translated into higher levels of Lck at the immunological synapse and stronger proliferation when only receptor-ligand interactions were present.

Results: The authors observed that the humanized UCHT1 improved surface expression and in vivo efficacy. Using TAC T cells derived from both healthy donors and multiple myeloma patients, the authors determined that despite the influence of receptor density on early activation events and effector function, receptor density did not impact late effector functions in vitro, nor did the receptor density affect in vivo efficacy.

Conclusions: The modifications to the TAC scaffold described herein represent an important step in the evolution of this technology, which tolerates a range of expression levels without impacting therapeutic efficacy.
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http://dx.doi.org/10.1016/j.jcyt.2021.05.007DOI Listing
September 2021

Antagonists of the serotonin receptor 5A target human breast tumor initiating cells.

BMC Cancer 2020 Aug 5;20(1):724. Epub 2020 Aug 5.

Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main Street West, Hamilton, ON, L8S 4L8, Canada.

Background: Breast tumor initiating cells (BTIC) are stem-like cells that initiate and sustain tumor growth, and drive disease recurrence. Identifying therapies targeting BTIC has been hindered due primarily to their scarcity in tumors. We previously reported that BTIC frequency ranges between 15% and 50% in multiple mammary tumors of 3 different transgenic mouse models of breast cancer and that this frequency is maintained in tumor cell populations cultured in serum-free, chemically defined media as non-adherent tumorspheres. The latter enabled high-throughput screening of small molecules for their capacity to affect BTIC survival. Antagonists of several serotonin receptors (5-HTRs) were among the hit compounds. The most potent compound we identified, SB-699551, selectively binds to 5-HT5A, a Gα protein coupled receptor (GPCR).

Methods: We evaluated the activity of structurally unrelated selective 5-HT5A antagonists using multiple orthogonal assays of BTIC frequency. Thereafter we used a phosphoproteomic approach to uncover the mechanism of action of SB-699551. To validate the molecular target of the antagonists, we used the CRISPR-Cas9 gene editing technology to conditionally knockout HTR5A in a breast tumor cell line.

Results: We found that selective antagonists of 5-HT5A reduced the frequency of tumorsphere initiating cells residing in breast tumor cell lines and those of patient-derived xenografts (PDXs) that we established. The most potent compound among those tested, SB-699551, reduced the frequency of BTIC in ex vivo assays and acted in concert with chemotherapy to shrink human breast tumor xenografts in vivo. Our phosphoproteomic experiments established that exposure of breast tumor cells to SB-699551 elicited signaling changes in the canonical Gα-coupled pathway and the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) axis. Moreover, conditional mutation of the HTR5A gene resulted in the loss of tumorsphere initiating cells and BTIC thus mimicking the effect of SB-699551.

Conclusions: Our data provide genetic, pharmacological and phosphoproteomic evidence consistent with the on-target activity of SB-699551. The use of such agents in combination with cytotoxic chemotherapy provides a novel therapeutic approach to treat breast cancer.
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http://dx.doi.org/10.1186/s12885-020-07193-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7404930PMC
August 2020

A Cross-Reactive Small Protein Binding Domain Provides a Model to Study Off-Tumor CAR-T Cell Toxicity.

Mol Ther Oncolytics 2020 Jun 14;17:278-292. Epub 2020 Apr 14.

Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON L8S 4K1, Canada.

Tumor-targeted chimeric antigen receptor (CAR)-engineered T lymphocytes (CAR-T cells) have demonstrated striking clinical success, but their use has been associated with a constellation of toxicities. A better understanding of the pathogenesis of these toxicities is required to improve the safety profile of CAR-T cells. Herein, we describe a xenograft model of off-tumor CAR-T cell-associated toxicity. Human CAR-T cells targeted against HER2 using a small-protein binding domain induced acute, dose-dependent toxicities in mice. The inclusion of a CD28 or 4-1BB co-stimulatory domain in the CAR was required to produce toxicity; however, co-stimulation through CD28 was most toxic on a per-cell basis. CAR-T cell activation in the lungs and heart was associated with a systemic cytokine storm. The severity of observed toxicities was dependent upon the peripheral blood mononuclear cell (PBMC) donor used as a T cell source and paralleled the CD4-to-CD8 T cell ratio in the adoptive transfer product. CD4 CAR-T cells were determined to be the primary contributors to CAR-T cell-associated toxicity. However, donor-specific differences persisted after infusion of a purified CD4 CAR-T cell product, indicating a role for additional variables. This work highlights the contributions of CAR-T cell-intrinsic variables to the pathogenesis of off-tumor toxicity.
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http://dx.doi.org/10.1016/j.omto.2020.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7191649PMC
June 2020

The chimeric TAC receptor co-opts the T cell receptor yielding robust anti-tumor activity without toxicity.

Nat Commun 2018 08 3;9(1):3049. Epub 2018 Aug 3.

Department of Pathology and Molecular Medicine, McMaster University, 1280 Main St W, Hamilton, ON, L8S 4L8, Canada.

Engineering T cells with chimeric antigen receptors (CARs) is an effective method for directing T cells to attack tumors, but may cause adverse side effects such as the potentially lethal cytokine release syndrome. Here the authors show that the T cell antigen coupler (TAC), a chimeric receptor that co-opts the endogenous TCR, induces more efficient anti-tumor responses and reduced toxicity when compared with past-generation CARs. TAC-engineered T cells induce robust and antigen-specific cytokine production and cytotoxicity in vitro, and strong anti-tumor activity in a variety of xenograft models including solid and liquid tumors. In a solid tumor model, TAC-T cells outperform CD28-based CAR-T cells with increased anti-tumor efficacy, reduced toxicity, and faster tumor infiltration. Intratumoral TAC-T cells are enriched for Ki-67 CD8 T cells, demonstrating local expansion. These results indicate that TAC-T cells may have a superior therapeutic index relative to CAR-T cells.
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http://dx.doi.org/10.1038/s41467-018-05395-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6076291PMC
August 2018

Ex vivo expanded natural killer cells from breast cancer patients and healthy donors are highly cytotoxic against breast cancer cell lines and patient-derived tumours.

Breast Cancer Res 2017 Jul 1;19(1):76. Epub 2017 Jul 1.

Department of Pathology and Molecular Medicine, McMaster Immunology Research Centre, McMaster University, 1280 Main Street West, MDCL 4015, Hamilton, ON, Canada.

Background: Natural killer (NK) cells play a critical role in cancer immunosurveillance. Recent developments in NK cell ex-vivo expansion makes it possible to generate millions of activated NK cells from a small volume of peripheral blood. We tested the functionality of ex vivo expanded NK cells in vitro against breast cancer cell lines and in vivo using a xenograft mouse model. The study aim was to assess functionality and phenotype of expanded NK cells from breast cancer patients against breast cancer cell lines and autologous primary tumours.

Methods: We used a well-established NK cell co-culture system to expand NK cells ex vivo from healthy donors and breast cancer patients and examined their surface marker expression. Moreover, we tested the ability of expanded NK cells to lyse the triple negative breast cancer and HER2-positive breast cancer cell lines MDA-MB-231 and MDA-MB-453, respectively. We also tested their ability to prevent tumour growth in vivo using a xenograft mouse model. Finally, we tested the cytotoxicity of expanded NK cells against autologous and allogeneic primary breast cancer tumours in vitro.

Results: After 3 weeks of culture we observed over 1000-fold expansion of NK cells isolated from either breast cancer patients or healthy donors. We also showed that the phenotype of expanded NK cells is comparable between those from healthy donors and cancer patients. Moreover, our results confirm the ability of ex vivo expanded NK cells to lyse tumour cell lines in vitro. While the cell lines examined had differential sensitivity to NK cell killing we found this was correlated with level of major histocompatibility complex (MHC) class I expression. In our in vivo model, NK cells prevented tumour establishment and growth in immunocompromised mice. Finally, we showed that NK cells expanded from the peripheral blood of breast cancer patients show high cytotoxicity against allogeneic and autologous patient-derived tumour cells in vitro.

Conclusion: NK cells from breast cancer patients can be expanded similarly to those from healthy donors, have a high cytotoxic ability against breast cancer cell lines and patient-derived tumour cells, and can be compatible with current cancer treatments to restore NK cell function in cancer patients.
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http://dx.doi.org/10.1186/s13058-017-0867-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5493877PMC
July 2017

Serotonergic system antagonists target breast tumor initiating cells and synergize with chemotherapy to shrink human breast tumor xenografts.

Oncotarget 2017 May;8(19):32101-32116

Department of Biochemistry and Biomedical Sciences, McMaster University, Canada.

Breast tumors comprise an infrequent tumor cell population, termed breast tumor initiating cells (BTIC), which sustain tumor growth, seed metastases and resist cytotoxic therapies. Hence therapies are needed to target BTIC to provide more durable breast cancer remissions than are currently achieved. We previously reported that serotonergic system antagonists abrogated the activity of mouse BTIC resident in the mammary tumors of a HER2-overexpressing model of breast cancer. Here we report that antagonists of serotonin (5-hydroxytryptamine; 5-HT) biosynthesis and activity, including US Federal Food and Drug Administration (FDA)-approved antidepressants, targeted BTIC resident in numerous breast tumor cell lines regardless of their clinical or molecular subtype. Notably, inhibitors of tryptophan hydroxylase 1 (TPH1), required for 5-HT biosynthesis in select non-neuronal cells, the serotonin reuptake transporter (SERT) and several 5-HT receptors compromised BTIC activity as assessed by functional sphere-forming assays. Consistent with these findings, human breast tumor cells express TPH1, 5-HT and SERT independent of their molecular or clinical subtype. Exposure of breast tumor cells ex vivo to sertraline (Zoloft), a selective serotonin reuptake inhibitor (SSRI), reduced BTIC frequency as determined by transplanting drug-treated tumor cells into immune-compromised mice. Moreover, another SSRI (vilazodone; Viibryd) synergized with chemotherapy to shrink breast tumor xenografts in immune-compromised mice by inhibiting tumor cell proliferation and inducing their apoptosis. Collectively our data suggest that antidepressants in combination with cytotoxic anticancer therapies may be an appropriate treatment regimen for testing in clinical trials.
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http://dx.doi.org/10.18632/oncotarget.16646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5458271PMC
May 2017

Kaiso depletion attenuates the growth and survival of triple negative breast cancer cells.

Cell Death Dis 2017 03 23;8(3):e2689. Epub 2017 Mar 23.

Department of Biology, McMaster University, Hamilton, Ontario L8S 4K1, Canada.

Triple negative breast cancers (TNBC) are highly aggressive and lack specific targeted therapies. Recent studies have reported high expression of the transcription factor Kaiso in triple negative tumors, and this correlates with their increased aggressiveness. However, little is known about the clinical relevance of Kaiso in the growth and survival of TNBCs. Herein, we report that Kaiso depletion attenuates TNBC cell proliferation, and delays tumor onset in mice xenografted with the aggressive MDA-231 breast tumor cells. We further demonstrate that Kaiso depletion attenuates the survival of TNBC cells and increases their propensity for apoptotic-mediated cell death. Notably, Kaiso depletion downregulates BRCA1 expression in TNBC cells expressing mutant-p53 and we found that high Kaiso and BRCA1 expression correlates with a poor overall survival in breast cancer patients. Collectively, our findings reveal a role for Kaiso in the proliferation and survival of TNBC cells, and suggest a relevant role for Kaiso in the prognosis and treatment of TNBCs.
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http://dx.doi.org/10.1038/cddis.2017.92DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5386582PMC
March 2017
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