Publication: Microfluidic Squeezing Enables MHC Class I Antigen Presentation by Diverse Immune Cells to Elicit CD8+ T Cell Responses with Antitumor Activity

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Microfluidic Squeezing Enables MHC Class I Antigen Presentation by Diverse Immune Cells to Elicit CD8+ T Cell Responses with Antitumor Activity

Matthew G. Booty, Kelan A. Hlavaty, Adam Stockmann, Emrah Ilker Ozay, Carolyne Smith, Lina Tian, Edylle How, Disha Subramanya, Anita Venkitaraman, Christian Yee, Olivia Pryor, Kelly Volk, Katarina Blagovic, Ildefonso Vicente-Suarez, Defne Yarar, Melissa Myint, Amy Merino, Jonathan Chow, Tarek Abdeljawad, Harry An, Sophia Liu, Shirley Mao, Megan Heimann, LeeAnn Talarico, Miye K. Jacques, Eritza Chong, Lucas Pomerance, John T. Gonzalez, Ulrich H. von Andrian, Klavs F. Jensen, Robert Langer, Hendrik Knoetgen, Christine Trumpfheller, Pablo Umaña, Howard Bernstein, Armon Sharei and Scott M. Loughhead

J Immunol published online 28 January 2022 http://www.jimmunol.org/content/early/2022/01/26/jimmun ol.2100656

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Published January 28, 2022, doi:10.4049/jimmunol.2100656

The Journal of Immunology

Microfluidic Squeezing Enables MHC Class I Antigen Presentation by Diverse Immune Cells to Elicit CD8+ T Cell Responses with Antitumor Activity

Matthew G. Booty,*,1 Kelan A. Hlavaty,*,1 Adam Stockmann,* Emrah Ilker Ozay,* Carolyne Smith,* Lina Tian,* Edylle How,* Disha Subramanya,* Anita Venkitaraman,* Christian Yee,* Olivia Pryor,* Kelly Volk,* Katarina Blagovic,* Ildefonso Vicente-Suarez,* Defne Yarar,* Melissa Myint,* Amy Merino,* Jonathan Chow,* Tarek Abdeljawad,*

Harry An,* Sophia Liu,† Shirley Mao,† Megan Heimann,† LeeAnn Talarico,* Miye K. Jacques,* Eritza Chong,* Lucas Pomerance,* John T. Gonzalez,* Ulrich H. von Andrian,‡,§,{

Klavs F. Jensen,† Robert Langer,† ,‖ Hendrik Knoetgen,# Christine Trumpfheller,** Pablo Umana,˜ ** Howard Bernstein,* Armon Sharei,* and Scott M. Loughhead*

CD8+ T cell responses are the foundation of the recent clinical success of immunotherapy in oncologic indications. Although checkpoint inhibitors have enhanced the activity of existing CD8+ T cell responses, therapeutic approaches to generate Ag-specific CD8+ T cell responses have had limited success. Here, we demonstrate that cytosolic delivery of Ag through microfluidic squeezing enables MHC class I presentation to CD8+ T cells by diverse cell types. In murine dendritic cells (DCs), squeezed DCs were ~1000-fold more potent at eliciting CD8+ T cell responses than DCs cross-presenting the same amount of protein Ag. The approach also enabled engineering of less conventional APCs, such as T cells, for effective priming of CD8+ T cells in vitro and in vivo. Mixtures of immune cells, such as murine splenocytes, also elicited CD8+ T cell responses in vivo when squeezed with Ag. We demonstrate that squeezing enables effective MHC class I presentation by human DCs, T cells, B cells, and PBMCs and that, in clinical scale formats, the system can squeeze up to 2 billion cells per minute. Using the human papillomavirus 16 (HPV16) murine model, TC-1, we demonstrate that squeezed B cells, T cells, and unfractionated splenocytes elicit antitumor immunity and correlate with an influx of HPV-specific CD8+ T cells such that >80% of CD8s in the tumor were HPV specific. Together, these findings demonstrate the potential of cytosolic Ag delivery to drive robust CD8+ T cell responses and illustrate the potential for an autologous cell-based vaccine with minimal turnaround time for patients. The Journal of Immunology, 2022, 208: 1;12.

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CD81 T cells are the driving force for adaptive immune responses against intracellular targets; hence, they play a key role in combating infectious diseases and cancer. The

success of checkpoint inhibitors in many cancer indications has generated excitement about the prospect of immunotherapy (1;3), and their impact in cancers with high mutational burden has correlated with the presence of CD81 T cells in the tumor (4). Although these approaches have enhanced the potential of existing CD81 T cell responses by combating immunosuppression, the ability to prime CD81 T cell responses in an Ag-specific manner has proved

challenging (5). Preclinical and clinical studies with mRNA, pep- tide, DNA, viral, and bacterial vaccination methods, which are normally effective for CD4 and humoral responses, have shown some promise but have not generated adequate CD81 T cell responses to merit widespread adoption in clinical practice (6). Although the potential impact of an effective vaccine that elicits potent tumor- specific CD81 T cells is immense, a potent approach has remained elusive.

The physiological interaction that underpins the priming of CD81 T cell responses is that between an APC and the T cell. A critical

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*SQZ Biotechnologies, Watertown, MA; † Department of Chemical Engineering, Massa- chusetts Institute of Technology, Cambridge, MA; ‡Department of Immunology, Har- vard Medical School, Boston, MA; xRagon Institute of MGH, MIT, and Harvard, Boston, MA; {Center for Immune Imaging at Harvard Medical School, Boston, MA; ‖David Koch Institute for Integrative Cancer Research, Massachusetts Institute of Tech- nology, Cambridge, MA; #Roche Innovation Center Basel, Roche Pharmaceutical Research and Early Development, Basel, Switzerland; and **Roche Innovation Center Zurich, Roche Pharmaceutical Research and Early Development, Schlieren, Switzerland

1M.G.B. and K.A.H. are co;first authors.

ORCIDs: 0000-0002-0835-3439(M.G.B.); 0000-0002-4464-0208(K.A.H.); 0000- 0002-5222-3156(A. Stockmann); 0000-0001-6132-6547(C.S.); 0000-0003-1665-9451(M.M.); 0000-0002-1041-8975(A.M.); 0000-0002-4321-2420(J.C.); 0000-0003-3614- 9731 (S.L.); 0000-0001-8973-6409(M.H.); 0000-0002-1020-8783(J.T.G.); 0000-0003-4231-2283(U.H.v.A.); 0000-0001-7192-580X(K.F.J.); 0000-0003-4255-0492(R.L.).

Received for publication July 6, 2021. Accepted for publication December 7, 2021.

This work was supported by Foundation for the National Institutes of Health Grant R44GM112259 (to H.B. and S.M.L.).

A. Stockmann, L. Tian, E.H., D.S., A.V., C.Y., E.I.O., O.P., C.S., K.V., K.B., M.K.J., A.M., L.P., J.T.G., E.C., H.A., S.L., S.M., and M.H. performed research; M.G.B., K.A.H., E.I.O., C.S., K.B., I.V.-S., L. Talarico, D.Y., M.M., J.C., T.A., U.H.v.A., H.K., C.T., P.U., H.B., A. Sharei, and S.M.L. designed research; S.M.L. wrote the manuscript; M.G.B., K.A.H., U.H.v.A., K.F.J., R.L., H.K., C.T., P.U., and A. Sharei reviewed and edited the manuscript.

Address correspondence and reprint requests to Dr. Scott M. Loughhead, SQZ Biotechnologies, 200 Arsenal Yards Boulevard, Watertown, MA 02472. E-mail address: [e-mail]

The online version of this article contains supplemental material.

Abbreviations used in this article: BMDC, bone marrow-derived dendritic cell; DC, dendritic cell; b2m−/−, MHC class I knockout; MFI, mean fluorescence intensity; MHC- I, MHC class I; moDC, monocyte-derived dendritic cell; SLP, synthetic long peptide; WT, wild type;

This article is distributed under The American Association of Immunologists, Inc., Reuse Terms and Conditions for Author Choice articles.

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www.jimmunol.org/cgi/doi/10.4049/jimmunol.2100656

2	SQUEEZING ENABLES PRIMING OF TUMORICIDAL CD8+ T CELLS

bottleneck for CD81 T cell activation is routing the desired Ag into the MHC class I (MHC-I) pathway of the APC to facilitate TCR stim- ulation. Peptides presented on MHC-I are derived from proteins that are degraded within the cytosol, transported into the endoplasmic reticulum, where they are loaded onto MHC-I, and then trafficked to the surface for display to CD81 T cells (7). Although the cytosolic presence of target Ag is critical to this mechanism, current vaccination approaches have by and large relied on introducing Ags into the extra- cellular vicinity of APCs or targeting their surface. In these contexts, APCs will endocytose the target material, thus segregating it from the cytosol in endosomal vesicles. There is, however, a process termed "cross-presentation" by which a fraction of Ags in endosomes escape into the cytosol and can be presented on MHC-I (8). Data from in vitro experimental models suggest that this transfer of Ags into the cytosol is a major rate-limiting step in cross-presentation (9). The process of cross-presentation is most often associated with dendritic cells (DCs), which has made them the favored cell type for cell-based approaches seeking to elicit CD81 T cell responses. Commercial and clinical success with this approach has been limited (10) due to the limited number of DCs in peripheral blood and the inefficiency with which Ags are cross-presented onto MHC-I by DCs.

Although delivery of materials to primary immune cells is challenging (11), microfluidic squeezing, which can deliver biomaterials directly into the cytosol of a wide array of cell types, represents an attractive approach for engineering Ag presentation to CD81 T cells. The squeezing process uses rapid mechanical deformation of cells to generate temporary pores that enable material delivery while minimally altering normal cellular function (12). In this work, we describe the implementation of the squeeze technology to facilitate direct cyto- solic delivery of Ags. We studied its impact across various primary human and murine immune cells, including DCs, T cells, B cells, and heterogeneous populations (splenocytes or PBMCs). Our results demonstrate that direct cytosolic loading dramatically improved Ag presentation across the tested cell types. In the context of DCs, for example, direct cytosolic delivery of protein Ag was shown to be ∼1000 times more effective than cross-presentation after endocytic uptake of protein Ag. These engineered cells also showed potent abilities to stimulate naive and previously activated CD81 T cells, both in vitro and in vivo. We also demonstrate that microfluidic squeezing can enable Ag presentation by human cells at manufacturing scale for potential clinical application. Finally, we show that immune responses elicited by squeezed cells, in combination with an adjuvant, are capable of driving antitumor effects that correlate with an influx of tumor- specific CD81 T cells. By overcoming the fundamental barrier to effective MHC-I presentation of Ag, squeeze-engineered cells could potentially be used as the basis for a potent, rapid turnaround, cell- based vaccine that is applicable across tumor types.

Materials and Methods

Ethics statement

All experimental methods were carried out in accordance with approved guidelines. The blood collection procedure was performed in accordance with guidelines approved by the New England Independent Review Board. All donors signed an informed consent for scientific research statement. All animal work was carried out in accordance with guidelines approved by the Institutional Animal Care and Use of Laboratory Animals and the US Government Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research and Training.

Cell isolation

Human PBMCs were isolated from fresh blood using Ficoll gradient centri- fugation. Human T cells were isolated using the EasySep Human T Cell Enrichment Kit (19051; STEMCELL Technologies). Human B cells were isolated using the EasySep Human B Cell Enrichment Kit (19054; STEM- CELL Technologies). Murine T cells were isolated directly from the spleen

using the EasySep Mouse T Cell Isolation Kit (19851; STEMCELL Tech- nologies). Murine B cells were isolated directly from the spleen using the EasySep Mouse Pan-B Cell Isolation Kit (19844; STEMCELL Technolo- gies). To generate a murine cell composition that approximates human PBMCs, a PBMC surrogate was generated with murine splenocytes by combining B cell;depleted splenocytes and untouched splenocytes in a ratio of 4:1. Four dissociated spleens were depleted of B cells using the EasySep Mouse CD19 Positive Selection Kit II (18954; STEMCELL Technologies) and combined with an untouched dissociated spleen before ammonium-chloride-potassium buffer RBC lysis. The method generates a cell composition of 70;80% CD31 cells and 10;20% B2201 cells, with smaller fractions of NK cells, monocytes, and neutrophils. For manufacturing scale, full Leuko- paks were enriched for WBCs by density centrifugation. T cells were puri- fied using a CliniMACS Plus (151-01; Miltenyi Biotec).

Cell squeezing and microfluidic devices

Cells were resuspended at 10;50 × 106 cells/ml in RPMI 1640 (11875-093; Life Technologies) as the delivery buffer. Cell squeezing was performed using previously established methods (13, 14) with the specifications described below. Human and murine cells were squeezed through microflui- dic channels containing a single 3.5;6-mm-wide,10;30-mm-long constriction at 45;60 pounds per square inch, depending on the cell type being squeezed. When appropriate, cargo was added to the cells and delivery buffer before squeezing. Delivery for each cell type was evaluated using 100 mg/ml 3 kDa Dextran Cascade Blue (D7132; Invitrogen). Viability was measured using the Zombie Yellow Fixable Viability Kit (423103; BioLegend). For manufacturing scale, 10 mg/ml 3 kDa Dextran Alexa Fluor 680 (D34681; Thermo Fisher Scientific) was used for squeezing.

Dendritic cell culture

Murine bone marrow-derived dendritic cells (BMDCs) were generated by culturing bone marrow cells in the presence of 55 mM 2-ME (Thermo Fisher Sci- entific), 20 ng/ml murine GM-CSF (R&D Systems), and 10 ng/ml murine IL-4 (R&D Systems). After 6;8 d, BMDCs were matured for 1 h with 100 international units of edotoxin/ml LPS (InvivoGen) and 100 ng/ml IFN-g (R&D Systems) before squeezing. Human monocyte-derived DCs (moDCs) were generated from human monocytes isolated from PBMCs using the Easy- Sep Human CD141 Enrichment Kit without CD16 Depletion (19058; STEM- CELL Technologies). Cells were cultured for 6 d in the presence of 800 U/ml

GM-CSF (R&D Systems) and 1000 U/ml IL-4 (R&D Systems). Flow cytometry was performed to confirm an immature DC phenotype of CD14−/low and CD11c1.

Immunization after adoptive transfer of TCR-transgenic cells

The following female 8;10-wk-old mice obtained from The Jackson Laboratory were used: C57BL/6J (000664), B6.SJL-Ptprca Pepcb/BoyJ (002014), B6.129P2-B2mtm1Unc/J (i.e., MHC-I knockout [b2m−/−]; 002087), C57BL/ 6-Tg(TcraTcrb)1100Mjb/J (i.e., OT-I; 003831), and B6.Cg-Thy1a/Cy-Tg(Tcra Tcrb)8Rest/J (i.e., pmel; 005023). CD81 T cells were isolated from OT-I or pmel mice using the EasySep Mouse CD81 T Cell Isolation Kit (19853; STEMCELL Technologies) and labeled with the CellTrace CFSE Cell Proliferation Kit (C34554; Invitrogen). CFSE-labeled CD81 T cells (2.5 × 106 per mouse) were administered i.v. on day 0 into either wild-type (WT) or b2m−/− recipients. Murine T or B cells were squeezed with 400 mg/ml EndoFit OVA (vac-pova-100; InvivoGen) as described above or incubated in the presence of OVA, and 5 × 106 cells were coinjected with 3 mg LPS i.v. on day 0 into the same WT or b2m−/− recipients. For studies using the gp100 Ag, 2.5 × 106 CFSE-labeled pmel CD81 T cells were transferred on day 0 along with B cells squeezed or incubated with 250 mg/ml gp100 synthetic long peptide

(SLP) (AVIGALLAVGALKVPRNQDWLGVSRQLRTKAWNRQ; BIO- SYNTAN). Epitope pulsed B cell conditions were prepared by incubating B cells with minimal epitope (SIINFEKL or KVPRNQDWL; AS-60193 and AS-62589, respectively; Anaspec) for 1 h at 37◦C, washing, and administering on day 0. After 3 d, lymph nodes were harvested, then processed into a single- cell suspension, and proliferation of OT-I cells was assessed by CFSE dilution using flow cytometry.

In vivo immunization and ex vivo restimulation

Cells of interest were cultured as described or harvested from the spleen and isolated accordingly. Cells were squeezed with OVA (vac-pova-100; Invivo- Gen) or E7 SLP (GQAEPDRAHYNIVTFSSKSDSTLRLSVQSTHVDIR; BIOSYNTAN). For DC immunizations, DCs were treated for 1 h with 100 international units of edotoxin/ml LPS (InvivoGen) and 100 ng/ml IFN-g (R&D Systems) before squeezing. B cells were incubated with 1 mM CpG 1826 (vac-1826-1; InvivoGen) for 16 h at 37◦C after squeezing. T cells were coinjected with 25 mg CpG. PBMC surrogate cells were incubated with 1 mM CpG for 4 h at 37◦C after squeezing. After 7 d, spleens were harvested and restimulated with 1 mg/ml SIINFEKL (AS-60193) or 1 mg/ml RAHYNIVTF

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The Journal of Immunology

(AS-61022) and 2 mg/ml anti-CD28(16-0281-86; eBioscience). After 1 h of incubation, GolgiPlug (555029; BD Biosciences) and GolgiStop (554724; BD Biosciences) were added for an additional 4 h. Cells were stained, fixed, and permeabilized according to the manufacturer's instructions (554714; BD Bio- sciences) and subsequently analyzed by flow cytometry.

In vitro stimulation of human T cell responders

CMV pp65-specific CD81 T cells that recognize the HLA-A*02 restricted peptide, NLVPMVATV, or the HLA-B*35 restricted peptide, IPSINVHHY, were purchased from Astarte Biologics. Human moDCs, T cells, B cells, or PBMCs squeezed with 50 mM SLP derived from CMV pp65 (HLA-A*02 restricted: PPWQAGILARNLVPMVATVQGQNLKYQEFFWDAND; BIO- SYNTAN) were cocultured with CMV-specific CD81 T cells in XVivo15 media (04-418Q; Lonza) supplemented with 5% Human AB Serum (H4522- 100ML; Corning) with 20 U/ml recombinant human IL-2(202-IL-010; R&D Systems) overnight. After overnight culture, supernatants were col- lected, and functional responses were assessed by production of IFN-g by ELISA (430104; BioLegend). For HLA-B*35 restricted responses, B cells were squeezed with 50 mM HLA-A*02 restricted pp65 SLP, 50 mM HLA- B*35 restricted pp65 SLP (HLA-B*35 restricted: LPLKMLNIPSINVHHYP- SAAERKHR; BIOSYNTAN), 50 mM of each SLP. Cryopreserved T cells were resuspended to 1 × 107 cells/ml in cryoprotectant media and frozen using a controlled rate freezer. T cells were thawed, and the buffer was exchanged before coculture with responder cells.

E.G7-OVA tumor studies

C57BL/6J mice were anesthetized using isoflurane and shaved on the right flank. E.G7-OVA cells (1 × 105, CRL-2113; American Type Culture Collec- tion) were s.c. injected into the flank with a 25-gauge needle. Mice were subsequently monitored twice weekly for tumor volume and body weight.

TC-1 tumor studies

TC-1 cells were obtained from Dr. T.C. Wu (Johns Hopkins University). C57BL/6J mice were anesthetized using isoflurane and shaved on the right flank. TC-1 cells (5 × 104) were s.c. injected into the flank with a 25-gauge needle. Mice were subsequently monitored twice weekly for tumor volume and body weight.

Tumor-infiltrating lymphocytes

Tumors were dissected from the right rear flank of the animal and weighed. Tumors were minced using scissors, then dissociated using a gentleMACS dissociator and incubated at 37◦C for 45 min with continuous rotation. The suspension was then filtered over a 70-mm cell strainer and processed for flow cytometric analysis. The E7 tetramer was from MBL International (TB-5008-1).

Flow cytometric analysis

Flow cytometry was performed using Attune NxT (Thermo Fisher Scien- tific). Data were examined using FlowJo (BD Biosciences). Fluorescently conjugated Abs were purchased from BioLegend.

Statistics

An unpaired two-tailed Student t test (for two-group comparisons) or a two- way ANOVA (for comparisons of more than two groups) was performed using Prism (GraphPad Software) to calculate statistical significance of the difference in mean values and p values. A p value <0.05 was considered statistically significant.

Results

Microfluidic squeezing enables Ag presentation in vivo by diverse immune cell types

Cellular approaches to priming CD81 T cell responses have traditionally used DCs, given their ability to cross-present Ags (8). To assess whether cytosolic delivery of Ag by microfluidic squeezing was superior to cross-presentation, mouse BMDCs were loaded with increasing concentrations of fluorescent OVA by either 30-min incubation (endocytosis) or squeezing. The mean fluorescence intensity (MFI) of fluorescent OVA showed that incubation alone resulted in approximately two to four times greater internalization of OVA within the range evaluated (Fig. 1A and 1B). However, the ineffi- ciency of escape from endosomes into the cytosol and subsequent presentation on MHC-I was demonstrated by coculture of these

3

BMDCs with OT-I CD81 T cells (specific for the OVA-derived epitope, SIINFEKL). Using CD69 upregulation on OT-I T cells as a measure for Ag presentation to CD81 T cells, squeezed BMDCs induced 70% upregulation of CD69 on OT-I T cells at a concentration of 5 mg/ml (MFI 170), whereas cross-presenting BMDCs induced similar CD69 upregulation at a concentration 1,000-fold higher (5,000 mg/ml OVA; MFI 42,000) (Fig. 1C). Presentation of OVA-derived SIINFEKL across the different concentrations was also quantified using the 25-D1.16 Ab, which recognizes SIINFEKL in the context of H-2Kb (15). This reinforced what was seen with activation of OT-I cells in that there was substantially greater 25- D1.16 staining on DCs that had been squeezed with OVA than on those that had been incubated with OVA (Fig. 1D and 1E). This suggests that cytosolic entry of endocytosed Ags is a major bottleneck for MHC-I presentation of proteins and that cytosolic delivery by squeezing bypasses this inefficient process.

Demonstrating superior activation of CD81 T cell responses by squeezed DCs in comparison with conventional cross-presenting DCs suggested that squeezing could enable presentation by other immune cells. B cells are attractive for use as APCs because they are abundant in the circulation and thus do not require expansion (up to 5 × 105/ml in human blood). Furthermore, B cells can migrate into lymphoid organs from the blood, which is where CD81 T cell responses are primed. Delivery of material to B cells was demonstrated using fluores- cent 3 kDa dextran (Supplemental Fig. 1A), as had been shown previously (16). To assess the kinetics of MHC-I presentation, OVA was delivered to B cells, and presentation was monitored using the 25-D1.16 Ab (15). SIINFEKL-MHC class I complexes could be found in as little as 2 h after squeeze and continued to accumulate for at least 4 h (Supplemental Fig. 1B). To evaluate the ability of squeezed B cells to prime CD81 T cell responses, endogenous CD81 T cell responses in naive mice were measured. B cells were squeezed with OVA and subsequently incubated with or without the TLR9 agonist, CpG, which will induce upregulation of costimulatory molecules as well as inflam- matory cytokines by B cells. In this context, the importance of CpG in generating a CD81 T cell response was apparent as B cells squeezed with OVA but incubated without the CpG failed to prime endogenous responses. Conversely, squeezed B cells incubated with CpG before injection induced robust CD81 T cell responses as measured by an increase in IFN-g;producing CD81 T cells in the spleen when restimulated ex vivo with the SIINFEKL epitope (Fig. 2A).

When considering cell subsets that are readily available to engineer as a cell therapy, T cells are the most abundant cell type in human PBMCs (up to 1.5 × 106 cells/ml of blood) and efficiently home to compartments and regions containing other T cells. However, T cells are poorly endocytic and rarely upregulate costimulatory molecules, thus making them poor candidates for Ag presentation by traditional methods. To test whether T cells could be used as APCs for priming CD81 T cell responses, delivery via squeeze was first optimized for murine T cells (Supplemental Fig. 1C). After OVA delivery to T cells, SIINFEKL-MHC complexes appeared on the surface within 2 h and continued to accumulate (Supplemental Fig. 1D). This was similar to the observed kinetics in B cells and DCs. To test the ability of squeezed T cells to elicit a naive CD81 T cell response, mice were immunized using squeezed T cell APCs with and without coinjected CpG. When OVA-squeezed T cells were injected alone, OVA-specific responses failed to be generated. However, when CpG was coinjected alongside OVA-squeezed T cells, endogenous OVA-specific responses were primed (Fig. 2B), demonstrating the need for an inflammatory context for effective priming.

Given that unconventional cell types could elicit a CD81 T cell response and that squeezing could mediate delivery to different immune cells, murine splenocytes were evaluated for their Ag- presenting capacity after squeezing. When unfractionated murine

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