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Improved Antitumor Efficacy of Chimeric Antigen Receptor T Cells That Secrete Single-Domain Antibody Fragments

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Improved Antitumor Efficacy of Chimeric Antigen Receptor T Cells That Secrete Single-Domain Antibody Fragments Published OnlineFirst February 4, 2020; DOI: 10.1158/2326-6066.CIR-19-0734 CANCER IMMUNOLOGY RESEARCH | RESEARCH ARTICLE Improved Antitumor Efficacy of Chimeric Antigen Receptor T Cells that Secrete Single-Domain Antibody Fragments Yushu Joy Xie1,2, Michael Dougan3, Jessica R. Ingram4,†, Novalia Pishesha1,2, Tao Fang1, Noor Momin2,5, and Hidde L. Ploegh1 ABSTRACT ◥ Chimeric antigen receptor (CAR) T-cell therapy is effective in the that can endow CAR T cells with desirable properties. The secretion treatment of cancers of hematopoietic origin. In the immunosup- of an anti-CD47 VHH by CAR T cells improves engagement of the pressive solid tumor environment, CAR T cells encounter obstacles innate immune system, enables epitope spreading, and can enhance that compromise their efficacy. We developed a strategy to address the antitumor response. CAR T cells that secrete anti–PD-L1 or these barriers by having CAR T cells secrete single-domain antibody anti–CTLA-4 nanobodies show improved persistence and demon- fragments [variable heavy domain of heavy chain antibodies (VHH) strate the versatility of this approach. Furthermore, local delivery of or nanobodies] that can modify the intratumoral immune landscape secreted anti-CD47 VHH-Fc fusions by CAR T cells at the tumor and thus support CAR T-cell function in immunocompetent ani- site limits their systemic toxicity. CAR T cells can be further mals. VHHs are small in size and able to avoid domain swapping engineered to simultaneously secrete multiple modalities, allowing when multiple nanobodies are expressed simultaneously—features for even greater tailoring of the antitumor immune response. Introduction cytokines such as IL12, IL15, and IL18 can modify the tumor micro- environment, make it more hospitable for T-cell activity, and thus Chimeric antigen receptor (CAR) T-cell therapy relies on T cells promote an antitumor response (9–11). Other means of manipulating that have been redirected with a receptor designed to recognize an the tumor microenvironment, for example by having CAR T cells antigen of choice. CAR T cells have been used to successfully treat release proteins that affect cell–cell interactions, therefore deserve hematologic cancers (1, 2). In the case of tumors of B-cell origin, consideration as well. CD19-targeted CAR T cells lead to remission in patients refractory to Variable heavy domain of heavy chain antibodies (VHH), also several other lines of therapy (3). CAR T cells have shown less success referred to as nanobodies, are single-domain antibody fragments in the treatment of solid tumors. Obstacles include a paucity of tumor- derived from the variable region of camelid heavy-chain-only anti- specific targets and a high rate of antigen escape (4, 5). A dense bodies (12). They are small, standalone proteins of approximately extracellular matrix, characteristic of many solid tumors, and the 15 kDa that retain binding affinities comparable with full sized presence of inhibitory checkpoint signals both blunt the immune mAbs (13). VHHs are stable, soluble, and can be expressed in excellent response (6–8). Suppressive cell types, such as T-regulatory cells or yields without the need for extensive optimization (14). For use as myeloid-derived suppressor cells, can further inhibit or cause therapeutic agents, their small size and sequence similarity to human exhaustion not only of CAR T cells but also of endogenous T cells immunoglobulin V regions render them less immunogenic than that have infiltrated the tumor (6). This combination of factors murine mAbs, and humanization of VHHs is possible (15). CAR T prevents CAR T cells from recognizing and attacking the tumor. To cells can be engineered so that they express and secrete VHHs with overcome some of these challenges, “armored” CAR T cells that secrete immunomodulatory properties to enhance their antitumor effect. Current immunotherapy approaches aim to improve adaptive immune recognition and killing of tumor cells, but the importance 1Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, of engaging the innate immune system for enhanced antigen presen- 2 Massachusetts. Department of Biological Engineering, Massachusetts Institute tation and epitope spreading is increasingly recognized (16, 17). For of Technology, Cambridge, Massachusetts. 3Division of Gastroenterology, example, the CD47 protein delivers a “don't eat me signal” to pha- Massachusetts General Hospital, Boston, Massachusetts. 4Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachu- gocytes. Consequently, a blockade of this signal synergizes with the setts. 5Koch Institute for Integrative Cancer Research, Massachusetts Institute of efficacy of mAb therapy for a variety of cancers in preclinical Technology, Cambridge, Massachusetts. models (18–21). Here, we show that engagement of the innate immune Note: Supplementary data for this article are available at Cancer Immunology system through blockade of CD47 by anti-CD47 VHH-secreting CAR Research Online (http://cancerimmunolres.aacrjournals.org/). T cells improves their antitumor effect. – †Deceased. We further demonstrate the modularity of VHH and VHH fusion secreting CAR T cells by developing CAR T cells that secrete VHHs Corresponding Author: Hidde L. Ploegh, Boston Children's Hospital, 1 Blackfan and VHH fusion proteins specific for multiple checkpoints. Such CAR Circle, RB09213, Boston, MA 02115-5713. Phone: 617-919-1613; Fax: 617-432-4775; E-mail: [email protected] T cells show less exhaustion and improved persistence when compared with nonsecreting CAR T cells. Because CAR T cells traffic to and Cancer Immunol Res 2020;8:518–29 persist at sites where their antigen is present (in our case, the tumor doi: 10.1158/2326-6066.CIR-19-0734 microenvironment), this strategy can limit immune-related adverse Ó2020 American Association for Cancer Research. effects associated with checkpoint blockade (22). Furthermore, we AACRJournals.org | 518 Downloaded from cancerimmunolres.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst February 4, 2020; DOI: 10.1158/2326-6066.CIR-19-0734 VHH-Secreting CAR T Cells show that, owing to their modest size, multiple VHH and VHH-fu- spinfected twice in 6-well plates at 2,000 g with the virus and polybrene sions can be expressed from the same vector in the same cell, under- mixture for 90 minutes. Fresh media was added after each spinfection, scoring the flexibility of this approach. as described previously (27, 30). After two spinfections, cells were Using CAR T cells as a vehicle for local delivery of VHH and VHH expanded for 2 days in complete RPMI IL2 and used for in vitro and fusion proteins should limit systemic exposure to immune modulators in vivo assays. such as anti-CD47 mAbs that have off-tumor toxicity. Anti-CD47 blocks engagement of the CD47 ligand, SIRP1a, while enhancing Anti-HA immunoprecipitation and immunoblots FcgR-mediated phagocytosis (23). The ubiquitous expression of Anti-HA immunoprecipitations (IP) were performed on culture CD47, especially on red blood cells (RBC), complicates systemic supernatants of transduced cells using the Pierce HA-Tag IP/Co-IP Kit administration of anti-CD47. The anemia that results from such (Thermo Fisher Scientific). HA tags were located at the C-terminus of treatment is sufficiently severe to have led to interruption of several the secreted VHHs. Supernatant (850 mL) was incubated with anti-HA clinical trials because of safety concerns (24, 25). Even so, CD47 agarose beads overnight, and eluates were run on a 12% SDS PAGE gel blockade remains a promising treatment if the toxicity concerns can and transferred to a PDVF membrane (Bio-Rad TurboBlot). Mem- be dealt with (26). We demonstrate that an anti-CD47 VHH, fused to branes were blocked in 5% milk in TBST and incubated with anti-HA- an Fc portion and secreted by CAR T cells, provides a stimulus HRP mAb overnight. Membranes were developed with western ECL for macrophage engulfment. CAR T cells that secrete anti-CD47 Fc substrate (PerkinElmer) and imaged on a Bio-Rad ChemiDoc imager. fusions show superior antitumor efficacy compared with CAR T-cell For immunoblots on cell lysates, cells were lysed with RIPA buffer therapy without such supplementation. Toxicity associated with sys- (25 mmol/L Tris, 150 mmol/L NaCl, 1% NP-40, 0.5% sodium temic exposure to anti-CD47 Fc is thus avoided. deoyxcholate, and 0.1% SDS) and protease inhibitor (complete mini protease inhibitor, Sigma). DNA was removed through benzonase digestion, and lysates were boiled with Laemmli sample buffer and Materials and Methods 2-mercaptoethanol. VHH-CAR retroviral design and construction Vectors used for the generation of CAR T cells were comprised of a In vitro cytotoxicity and activation assays fluorescent protein tracer linked to a P2A sequence followed by a CD8 Cytotoxicity of CAR T cells was measured using various coculture signal sequence and the VHH domain linked to a CD8 hinge and assays. The cytotoxicity of the PD-L1–targeted CAR T cells was shown transmembrane domain. The VHH sequences used are as follows: A4 – by coculture with B16F10 melanoma cells, which express PD-L1. B16 QVQLVESGGGLVEPGGSLRLSCAASGIIFKINDMGWYRQAPGK- cells were plated in complete RPMI (Corning) and recombinant RREWVAASTGGDEAIYRDSVKDRFTISRDAKNSVFLQMNSLKP- murine Il2, and A12 or A12 A4 CAR T cells were added to the EDTAVYYCTAVISTDRDGTEWRRYWGQGTQVTVSS; A12 – QV- B16F10 cells for a 24-hour incubation. T
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