cytotoxic t lymphocytes , na qiao , mao zhang , madhushree ... · fucosylation of selectin ligands...

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Fucosylation Enhances the Efficacy of Adoptively Transferred Antigen-specific

Cytotoxic T Lymphocytes

Gheath Alatrash*1, Na Qiao2†, Mao Zhang1†, Madhushree Zope1, Alexander A.

Perakis1, Pariya Sukhumalchandra1, Anne V. Philips2, Haven R. Garber1, Celine

Kerros1, Lisa S. St. John1, Maria R. Khouri1, Hiep Khong3, Karen Clise-Dwyer1,

Leonard P. Miller4, Steve Wolpe4, Willem W. Overwijk3, Jeffrey J. Molldrem1,

Qing Ma1, Elizabeth J. Shpall1, Elizabeth A. Mittendorf*5

Affiliations:

Departments of 1Stem Cell Transplantation and Cellular Therapy, 2Breast

Surgical Oncology, 3Melanoma Medical Oncology, The University of Texas MD

Anderson Cancer Center, Houston, TX, USA; 4Targazyme Inc., Carlsbad, CA,

USA; 5Surgical Oncology, Dana-Farber/Brigham and Women’s Cancer Center,

Boston, MA, USA

†Contributed equally to this manuscript

Running Title: Fucosylation enhances T cell anti-tumor efficacy

Key Words: Immunotherapy, T cells, Breast Cancer, Leukemia

Financial Support: This work was supported by a grant from the Leukemia

Research Foundation (G.A.); Leukemia & Lymphoma Society (G.A.); Nancy

Owens Memorial Foundation and Pink Ribbons Project (EAM) and Cancer

Center Support Grant NCI #CA16672. E.A. Mittendorf is an R. Lee Clark Fellow

at The University of Texas MD Anderson Cancer Center, supported by the

Jeanne F. Shelby Scholarship Fund.

Research. on June 13, 2020. © 2019 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 15, 2019; DOI: 10.1158/1078-0432.CCR-18-1527

http://clincancerres.aacrjournals.org/

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Correspondence: Gheath Alatrash, Department of Stem Cell Transplantation

and Cellular Therapy, University of Texas MD Anderson Cancer Center, 1515

Holcombe Blvd, Unit 900, Houston, TX 77030, USA. Phone: (713) 563-3334; Fax

(713) 563-3364; Email: [email protected]; and Elizabeth A. Mittendorf,

Robert and Karen Hale Distinguished Chair in Surgical Oncology, Dana-

Farber/Brigham and Women’s Cancer Center, 450 Brookline Ave, Suite 1220,

Boston, MA 02215, USA. Phone: (617) 582-9980; Fax: (617) 632-2495; Email:

[email protected]

Conflict of Interest: The authors have no conflict of interest to disclose.

Abstract word count: 250; Text word count: 4,439; Figures: 6; Tables: 0;

Supplementary Figures: 5; Supplementary table: 1; References: 52

Statement of Translational Relevance: Adoptive cellular therapy (ACT),

including tumor-specific cytotoxic T lymphocytes (CTLs) and chimeric antigen

receptor (CAR)-T cells, has shown promise in the treatment of a number of

malignancies. One limitation to ACT is the inadequacy of immune cell homing to

the tumor site. To overcome this obstacle, we investigated the role of ex

vivo fucosylation of antigen-specific CTLs as a mechanism to boost CTL efficacy

against solid tumors and leukemia. Our results show that ex vivo fucosylation of

CTLs enhances their homing and cytolytic machinery in the setting of breast

cancer and leukemia. These findings are highly valuable from a clinical

perspective because they identify a novel and simple approach to improve the

efficacy of ACT, circumventing the standard labor-intensive and costly

engineering methodologies to improve the potency and homing of therapeutic T

cells. Clinically, fucosylation could prove to be a valuable tool to improve ACT for

cancer.



mailto:[email protected]

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Abstract

Purpose: Inefficient homing of adoptively transferred cytotoxic T lymphocytes

(CTLs) to tumors is a major limitation to the efficacy of adoptive cellular therapy

(ACT) for cancer. However, through fucosylation, a process whereby

fucosyltransferases (FTs) add fucose groups to cell surface glycoproteins, this

challenge may be overcome. Endogenously fucosylated CTLs and ex vivo

fucosylated cord blood stem cells and regulatory T cells were shown to

preferentially home to inflamed tissues and marrow. Here we show a novel

approach to enhance CTL homing to leukemic marrow and tumor tissue.

Experimental Design: Using the enzyme FT-VII, we fucosylated CTLs that

target the HLA-A2-restricted leukemia antigens CG1 and PR1, the HER2-derived

breast cancer antigen E75, and the melanoma antigen gp-100. We performed in

vitro homing assays to study the effects of fucosylation on CTL homing and

target killing. We used in vivo mouse models to demonstrate the effects of ex

vivo fucosylation on CTL anti-tumor activities against leukemia, breast cancer

and melanoma.

Results: Our data show that fucosylation increases in vitro homing and

cytotoxicity of antigen specific CTLs. Furthermore, fucosylation enhances in vivo

CTL homing to leukemic bone marrow, breast cancer and melanoma tissue in

NOD/SCID gamma (NSG) and immunocompetent mice, ultimately boosting the

anti-tumor activity of the antigen-specific CTLs. Importantly, our work

demonstrates that fucosylation does not interfere with CTL specificity.




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Conclusion: Together, our data establish ex vivo CTL fucosylation as a novel

approach to improving the efficacy of ACT, which may be of great value for the

future of ACT for cancer.




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Introduction

The success of adoptive cellular therapy (ACT) for cancer has been impeded by

the paucity of T cells homing to tumor tissue (1,2). A number of molecules,

including T cell adhesion molecules, chemokine receptors, and selectins, have

been shown to play important roles in the homing of T cells to malignant tissues

(3-5). Of these, selectins appear to be the major molecules involved in T cell

homing into tumors and inflamed tissues (6-10). T cell expression of selectin

ligands is critical for T cell efficacy in providing long-term protection against tumor

recurrence. Selectins are a family of three C-type lectins (P-selectin, E-selectin,

and L-selectin) expressed by hematopoietic, immune, and endothelial cells.

Selectins have been shown to play a role in T cell function, including

trafficking (6,7). They bind to the tetrasaccharide sialyl-Lewis X (sLeX), which

decorates certain proteins that are expressed on T cells and tumor endothelial

cells. sLeX is the moiety found on selectin ligands such as PSGL-1 and CD44,

which are the critical binding units that mediate the interaction between

circulating cells with E- and P-selectins expressed on vascular endothelial cells

and inflamed tissues (11,12). This cooperative binding leads to the attachment,

rolling, and adherence of cytotoxic T lymphocytes (CTLs) to vascular endothelial

cells, supporting the first critical step in the homing process.

sLeX formation is controlled in part by the expression of

fucosyltransferases (FTs) that attach a terminal fucose to trisaccharide acceptor

molecules. In T cells, this activity is regulated by FT-IV and FT-VII (13).

Fucosylation of T cell surface molecules has been shown to facilitate the




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migration of T cells into tissues (13-18). In contrast, defects in fucosylation

contribute to decreased T cell homing (19-21).

Currently available data highlight the importance of endogenous

fucosylation of selectin ligands in T cell homing and trafficking; however, to date,

ex vivo fucosylation has been studied only in the setting of allogeneic stem cell

transplantation (allo-SCT) (22-24). After validating the effects of ex vivo

fucosylation in animal models, one study showed that ex vivo fucosylation of cord

blood hematopoietic stem cells shortened time to engraftment following allo-SCT

in 22 patients (24). Moreover, Parmar et al. showed that ex vivo fucosylation of

regulatory T cells (T-regs) enhances homing into inflamed tissues affected by

graft-versus-host disease (GvHD) in a xenograft mouse model (25). In these

studies, fucosylation was achieved by a simple reaction involving a short

incubation of cells with the substrate guanosine diphosphate-fucose (GDP-

fucose) and FT-VI (TZ-101: FT-VI + GDP fucose). Since FT-VII fucosylates

CTLs more efficiently than FT-VI, we used FT-VII (TZ102: FT-VII + GDP fucose)

to fucosylate CTLs ex vivo in this study (22-25). Incubating cells with TZ102

results in an enzymatically mediated, site- and stereo-specific addition of fucose

to form the tetrasaccharide sLeX.

We hypothesized that fucosylation of antigen-specific CTL in the setting of

leukemia and breast cancer enhances their homing into tumor tissues and their

anti-tumor activities. Using CTL that target the human leukemia antigens PR1

and CG1 (PR1- and CG1-CTL)(26-30), the human breast cancer antigen E75

(E75-CTL)(31,32), and the mouse melanoma antigen gp-100 (pmel-1 CD8+ T




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cell)(33), we show that fucosylation of CTLs results in: (A) increased migration

and cytotoxicity of antigen-specific CTLs following fucosylation using in vitro

assays; (B) favorable changes in the expression of CTL adhesion molecules, co-

stimulatory receptors, CTL cytolytic granules and CTL:target synapse formation;

(C) enhanced killing of leukemia, breast cancer and melanoma by CG1-CTL,

PR1-CTL, E75-CTL and pmel-1 CD8+ T cells in vivo; and that (D) fucosylation

does not alter the specificity of the antigen-specific CTL for their targets and does

not increase homing of CTL to normal mouse tissue.




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Materials and methods

Cells and cell culture

The U937 (histiocytic leukemia), SKBR3 (breast cancer), and T2 (HLA-A2+ T/B

cell hybridoma) cell lines were obtained from American Type Culture Collection

(ATCC, Manassas, VA, USA). U937 and T2 cell lines were grown in RPMI-1640

supplemented with 10% fetal bovine serum (FBS) (Gemini Bio-Products,

Sacramento, CA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin

(Cellgro, Manassas, VA, USA); SKBR3 breast cancer cell line was grown in

Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with the same. All

cell lines were cultured and maintained in 5% CO2 at 37°C. The HLA-A2- cell line

U937 and the HLA-A2low SKBR3 (SKBR3 is genotypically one allele HLA-A2

positive, but phenotypically HLA-A2 low) were transduced with HLA-A*0201 (i.e.

HLA-A2) using lentiviral transduction as previously described (28,34). Cell lines

were validated using short tandem repeat DNA fingerprinting by our institutional

sequencing facility. After thawing, cells were used for experiments within 5

passages. Mycoplasma testing was performed quarterly using the MycoAlert Kit

(Lonza Inc., Allendale, NJ, USA). Patient peripheral blood and apheresis

samples were obtained through an Institutional Review Board approved protocol.

Peptide-specific CTL generation

CG1-CTLs, PR1-CTLs, and E75-CTLs were generated using the modified

dendritic cell (DC)-based CTL expansion method (35). HLA-A2+ peripheral blood

mononuclear cells (PBMCs) from healthy donors were isolated by Histopaque

(Sigma Aldrich, St. Louis, MO) density gradient cell separation. An enriched




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population of CD14 monocytes was prepared by magnetic bead depletion of

CD2, CD19, and CD8-expressing cells (Dynal-Invitrogen, Carlsbad, CA).

Enriched monocytes were then cultured in Macrophage Serum-Free Media

(Gibco-Invitrogen, Carlsbad, CA) with 100 ng/mL GM-CSF and 100 ng/mL IL4

(R&D Systems, Minneapolis, MN). TNFα (R&D Systems) was added at 10 ng/mL

after 48 hours to mature the DC population. Antigen-specific CD8+ T cells were

prepared from PBMCs by supplementing complete media with 10 ng/ml of IL7

(R&D Systems) and pulsing the cells with 40 µg/ml of peptide (CG1-

FLLPTGAEA/ PR1-VLQELNVTV/ E75-KIFGSLAFL, Biosynthesis, Lewisville,

TX). DCs were harvested on day 5 and pulsed with the above peptides for 2

hours. Once pulsed, the DCs were then co-cultured with autologous CD8+ T cells

in complete media containing 10 ng/ml of IL7 and 50 IU/mL of IL2. All cultures

were maintained in a humidified incubator in 5% CO2 at 37°C. On day 14, CTLs

were harvested and used in in vitro assays and in vivo studies. Prior to use,

CTLs were passed through a negative selection column (MACS Miltenyi Biotec-

CD8+ T Cell Isolation Kit, Auburn, CA).

Fucosylation of CTLs

Ex vivo expanded T cells were incubated in fucosylation solution: 20 g/mL of

FT-VII in 1 mM GDP Fucose in phosphate-buffered saline (PBS) with 1% human

serum albumin (Targazyme Inc, Carlsbad, California) at room temperature for 30

minutes, as previously described (25). FT-VII was used since it fucosylates CTLs

at a much higher efficiency than FT-VI. Cells were then re-suspended in PBS.

Fucosylation was confirmed using flow cytometry (LSR Fortessa; BD




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Biosciences, San Jose, CA) after the cells were stained with the FITC-conjugated

HECA-452 antibody (BD Biosciences), which targets cutaneous lymphocyte

antigen (CLA), shown to be sLeX on PSGL-1 (14).

CTL Migration Assay

CTL migration was assessed using a CytoSelect Leukocyte Transmigration

assay (Cell Biolabs, Inc., San Diego, CA). Human umbilical vein endothelial cells

(HUVECs) (1 x 105) were cultured in each of 24 trans-well inserts for 24 hours.

Antigen-specific CTLs labeled with LeukoTracker dye were then placed into each

inner well, in contact with full serum media below. Cells that had migrated

through the membrane and into the media were lysed with specific lysis buffer,

and the fluorescence was measured with a plate reader at 480/520 nm (BioTek

Cytation3, Winooski, VT).

CTL Phenotypic Analysis

CTL (1.5 x 106) were stained for molecules that modulate T cell trafficking,

including CD49d (clone 9F10; BioLegend, San Diego, CA, USA), CD162 (PSGL-

1; clone KPL-1; BioLegend), CD183 (CXCR3; clone 1C6/CXCR3; BD

Biosciences), and CD195 (CCR5; clone 2D7/CCR; BD), as well as molecules

involved in co-stimulation/inhibition, including CD137 (41BB; clone 5F4;

BioLegend), CD279 (PD1; clone EH12.2H7; BioLegend), and CD357 (GITR;

eBioAITR; eBioscience, San Diego, CA), within 2 hours after fucosylation. The

LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Life Technologies, Eugene, OR)

was used to assess cell viability. Flow cytometry was done on live cells using a




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BD LSR Fortessa, and data was analyzed using FlowJo software (FlowJo,

Ashland, OR).

CTL Cytotoxicity Assay

Antigen-specific CTL were harvested on days 12-14 and passed through a

magnetic separation column by human CD8+ T cell Isolation kit (Miltenyi Biotec).

Peptide-specific cytotoxicity was assessed with a standard 4-hour calcein-AM

release assay as previously described (27,36,37). Target cells included peptide-

pulsed T2, SKBR3-A2 breast cancer cells, primary patient acute myeloid

leukemia (AML) cells, or U937-A2 leukemia cell line. Target cells (1 x 103) were

fluorescently labeled with calcein-AM (Invitrogen) for 15 min at 37°C and washed

with RMPI-1640 to remove free calcein-AM. These cells were seeded into 60-

well Tarasaki plates at indicated effector-to-target (E:T) ratios for 4 hours at

37°C. The reaction was quenched with trypan blue, and fluorescence was

measured using a microplate fluorescence reader (BioTek Cytation3, Winooski,

VT). The percent specific cytotoxicity was calculated using the following formula:

([1 − 𝑓𝑙𝑢𝑜𝑟𝑒𝑠𝑒𝑛𝑐𝑒𝑡𝑎𝑟𝑔𝑒𝑡+𝑒𝑓𝑓𝑒𝑐𝑡𝑜𝑟 − 𝑓𝑙𝑢𝑜𝑟𝑒𝑠𝑒𝑛𝑐𝑒𝑚𝑒𝑑𝑖𝑎]/[𝑓𝑙𝑢𝑜𝑟𝑒𝑠𝑒𝑛𝑐𝑒𝑡𝑎𝑟𝑔𝑒𝑡 𝑎𝑙𝑜𝑛𝑒 −

𝑓𝑙𝑢𝑜𝑟𝑒𝑠𝑒𝑛𝑐𝑒𝑚𝑒𝑑𝑖𝑎]) × 100

In vivo Mouse Methods

NOD/SCID gamma (NSG) and NSG-HLA-A2 transgenic mice (NSG-A2), 4-6-

week-old female mice (28,38), were purchased from Jackson Laboratory (Bar

Harbor, ME). For AML experiments, HLA-A*0201 primary AML samples with a

high leukemia burden or U937-A2 (U937-A2+/GFP+) cells were administered via




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tail vein at doses of 1 x 106 to 1 x 107 cells to sublethally irradiated (250 cGy)

mice. Mice were monitored 2 times/week for AML engraftment by peripheral

blood analysis and GVHD by clinical assessment. Fucosylated or non-

fucosylated CG1- or PR1-CTLs (0.5 x 105) (28,39) were administered to mice

intravenously (IV) via tail vein 2 days after leukemia injection. Mice were

sacrificed on day 3 to evaluate CTL homing and on week 2 to evaluate for tumor

killing. Bone marrow was stained with human (h)CD13, hCD33, hCD3, hCD8 (BD

Biosciences), hCD45 (BioLegend) and mouse (m)CD45 antibodies

(eBioscience), and assessed for GFP to quantify xenograft disease and CTL

homing into tumor (28,40). Residual AML was classified as

hCD33+/hCD45+/mCD45- and antigen-specific CTL were classified as hCD33-

/hCD45+/mCD45-/hCD13-/hCD3+/hCD8+.

For breast cancer, 1.5 x 107 luciferase-expressing SKBR3-A2 breast

cancer cells were injected into NSG mice orthotopically on day 0. On days 7 and

10, fucosylated or non-fucosylated E75-CTLs (2 x 106) were injected IV via tail

vein. Mice were followed for weight loss, tumor size, survival, and tumor

bioluminescence every three days. Mice were sacrificed on week 5, and tumor-

infiltrating lymphocytes (TILs) were extracted from primary tumors using a tumor

dissociation kit (Miltenyl Biotec, Auburn, CA). TILs were stained with the same

panel used for bone marrow samples in AML mice, and E75-CTL were classified

as mCD45-/hCD45+/ hCD3+/ hCD8+.

For melanoma, immunocompetent mouse model, provided by Dr. Willem

Overwijk, pmel-1 T cell receptor (TCR) transgenic mice on C57BL/6 background




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(The Jackson Laboratory) were crossed with CD90.1 congenic mice to yield

pmel-1+/+CD90.1+/+ mice (referred to as pmel-1 mice)(33). Splenocytes from

pmel-1 mice were extracted and then expanded using standard protocol (33).

Concurrently, 0.3 x 106 B16-F10 melanoma cells (ATCC) were inoculated

subcutaneously into C57BL/6 mice (Charles River Laboratories, Wilmington,

MA). After one week of pmel-1 CD8+ T cell ex vivo expansion, tumor bearing

mice received IV tail vein injection of 5 x 106 fucosylated or non-fucosylated

Pmel-1 T cells (day 0). Mice underwent intraperitoneal injections of IL-2 (0.1 x

106 IU/mice; Prometheus Therapeutics & Diagnostics, San Diego, CA) twice daily

for two days. Mice were observed daily for tumor size, weight loss and survival.

Mice were sacrificed on Day 9, and TILs were enriched from tumors, stained with

mCD3, mCD4, mCD8 (BioLegend), mCD45, CD90.1 (eBioscience), and Ghost

Dye Violet 510 (Tonbo Biosciences, San Diego, CA), and were analyzed by flow

cytometry. Pmel-1 CD8+ T cells were identified as mCD3+, mCD8+,mCD45+ and

CD90.1+.

The experiments were executed in compliance with institutional guidelines

and regulations and after approval from the MD Anderson Cancer Center

Institutional Animal Care and Use Committee.

Activation and Proliferation Assays

CTL activation was analyzed by measuring the expression of Fas ligand (FasL)

perforin and granzyme B, as previously described (41,42).Fucosylated and non-

fucosylated CTLs were co-cultured with T2 cells that were pulsed with E75, CG1,

or PR1 (40 ug/mL) at a ratio of 1:1 overnight at 37oC. At the end of the




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incubation period, the cells were stained with fluorescently-conjugated antibodies

targeting CD3, CD8, FasL (BioLegend), CLA, and Ghost Dye Violet 510. After

staining for cell surface markers, cells were permeabilized and stained with

fluorescently-conjugated antibodies targeting granzyme B and perforin

(BioLegend). Staining was analyzed using flow cytometry (BD LSR Fortessa).

To test proliferation, we performed standard CFSE assays. Briefly,

fucosylated and non-fucosylated CTLs were labeled with CFDA SE Cell tracer

reagent (Invitrogen) following manufacturer’s protocols. Cells were cultured in

96-well round bottom plates at a density of 0.3 x 106 cells in RPMI + 10% FBS in

the presence or absence of anti-CD3 and CD28 antibodies (BD Biosciences) to

stimulate proliferation. Cells were harvested on days 2, 4 and 5 and stained with

anti-CD8, anti-CD4 (Biolegend) and Ghost Dye Violet 510. Live, CD8+/CD4- cells

were gated on CFSE-positivity, and percent proliferation was calculated by gating

on CD8+ cells and comparing the CFSE+ populations between samples.

Colony Forming Unit (CFU) Assay

Fucosylated CTLs were co-cultured with HLA-A2+ healthy donor bone marrow to

determine the effects of fucosylation on CTL specificity. We used CG1- and PR1-

CTLs as we have an established in vitro model using CFU assays with normal

donor bone marrow hematopoietic stem cells (HSC) to test the specificity of

these CTLs (28). Normal HSC were thawed and cultured for 24 hours in RPMI

with 1% Penicillin/Streptomycin (P/S). Bone marrow cells and CTLs were co-

cultured at a 1:5 ratio in RPMI (10% FBS + 1% P/S) for 4 hours in a 48-well plate.




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The incubated cultures were resuspended in Iscove's Modified Dulbecco's Media

(IMDM) + 2% FBS and added into settled MammoCultTM H4034 Optimum

Human Medium (STEMCELL Technologies, Cambridge, MA). These mixes were

transferred to 6-well plates to incubate for 10-14 days. CFUs were imaged (Leica

DMi8) and counted on Day 14. To confirm cell phenotype, wells were incubated

at 4°C for 2-hours and washed with IMDM and PBS to prepare for antibody

staining. Cells were stained with: fluorescently conjugated CD3, CD4, CD8,

CD14, CD16, CD19, CD33, CD34 and live/dead Aqua (BD Biosciences).

Histology

To determine the effects of fucosylation on the specificity of antigen-specific

CTLs, mice were sublethally irradiated and on the following day were treated with

2.5 x 106 antigen-specific CTLs, and twice weekly. After 4 weeks, mouse tissues

including spleen, liver, kidney, bone marrow, intestine, brain, heart, and lung

were harvested and fixed in 10% formalin. The fixed tissue samples were

embedded in paraffin, sectioned, and stained with hematoxylin and eosin prior to

histological examination.

Immune synapse studies

T2 cells were pulsed with E75, PR1 and CG1 peptides (40 ug/mL), were washed

and labeled with CellTracker Deep Red Dye (Life Technologies, Thermo Fisher

Scientific, Waltham, MA), and were co-cultured with fucosylated and non-

fucosylated CTLs at a 1:1 ratio at 37oC. Cells were then fixed in 2%

paraformaldehyde, washed, and stained with anti-CD8-FITC (Biolegend) to




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identify CTLs. Following surface staining, cells were washed, permeabilized

using 0.1% Triton-X (Sigma, St. Louis, MO), and stained with phalloidin

AlexaFlour 594 (Life Technologies, Thermo Fisher) to visualize actin filaments at

the synapse between CTLs and T2 cells. Data were collected on an imaging

flow cytometer (ImageStreamXMarkII, Millipore Sigma, Burlington, MA) and

analyzed using Ideas Software (Millipore Sigma).

Statistical analysis

Statistical analyses were performed using GraphPad Prism 7.0 software. P

values less than 0.05 were considered significant.




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Results

Fucosylated CTLs demonstrate enhanced endothelial transmigration in

vitro

Fucosylation of antigen specific-CTLs with FT-VII was verified by staining cells

with HECA-452 antibody, which targets CLA (Fig. 1A and 1B) (14). Non-

fucosylated groups showed 0% CLA+ CG1- and PR1-CTLs and 1.1% E75-CTLs,

whereas CTLs incubated with FT-VII showed 96.7%, 94.7%, and 99.5 CLA+

CG1-, PR1-, and E75-CTLs, respectively.

Using a trans-well assay, we showed that fucosylated CG1-, PR1-, and E75-

CTLs had increased ability to migrate through the HUVEC barrier, as indicated

by the increased cell count and relative fluorescence in the lower chamber (Fig.

1C). Fucosylated CTLs specific for CG1, PR1, and E75 had 1.7, 1.3, and 1.5

fold-increased migration, respectively, in comparison to non-fucosylated CTLs

(all p < 0.05).

Fucosylation alters the expression of CTL surface markers

Differences in cell surface marker expression post-fucosylation were determined

by cell surface staining and flow cytometry (Fig. 2). Fucosylated antigen-specific

CTLs showed increased surface expression of the trafficking molecule

CD162/PSGL-1 and CD183 (CXCR3), and the co-regulatory molecule CD137

(41BB) (Fig. 2).




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Fucosylation enhances CTL cytotoxicity in vitro

To study the efficacy of fucosylated CTL-induced killing of targets expressing

cognate peptide-MHC, we utilized calcein-AM assays at multiple E:T ratios.

Fucosylated antigen-specific CTLs demonstrated higher cytotoxicity against

corresponding target cells than non-fucosylated CTLs (Fig. 3). CG1-CTLs

showed increased killing of CG1 pulsed T2 cells and primary patient AML

samples (Fig. 3A; Supplementary Table S1). Similarly, fucosylated PR1-CTLs

had increased killing of PR1 peptide-pulsed T2 cells and the U937-A2 AML cell

line in comparison to non-fucosylated PR1-CTLs (Fig. 3B). Likewise, fucosylation

of E75-CTLs was associated with significantly increased killing of E75 pulsed T2

cells and SKBR3-A2 breast cancer cells (Fig. 3C).

In addition, we studied the CTL cytolytic machinery after fucosylation by

analyzing the intracellular expression of granzyme B and perforin, and surface

expression of FasL, after co-culturing CTLs with peptide-pulsed targets. Data

demonstrate an increase in the percentage of CTLs expressing all three markers

after fucosylation (Supplementary Fig. S1). We also investigated whether

fucosylation enhances the binding of CTLs to target cells by analyzing synapse

formation between CTLs and peptide-pulsed target cells (Supplementary Fig.

S2). Our data show that fucosylation enhances the frequency of CTLs forming

conjugates with target cells compared with non-fucosylated CTLs.

Fucosylation enhances the efficacy and homing of antigen-specific CTLs in

vivo




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The efficacy of antigen specific CTLs was tested in vivo using NSG xenograft

mouse models for AML and breast cancer. Treatment of patient AML- or SKBR3-

A2-engrafted NSG mice with the corresponding fucosylated antigen-specific

CTLs resulted in a decreased disease burden in comparison with non-

fucosylated antigen specific CTLs (Fig. 4). Specifically, mice that received

fucosylated CG1-CTLs (n=12) showed significantly greater inhibition of leukemia

than mice that received non-fucosylated CG1-CTLs (n=13) (p<0.05) (Fig. 4A;

Supplementary Table S1). Similarly, treatment of the same patient primary AML

with fucosylated PR1-CTLs (n=13) resulted in better disease inhibition when

compared to treatment with non-fucosylated PR1-CTLs (n=13) (p<0.01) (Fig.

4B). Similar results were also obtained in the setting of breast cancer, where

fucosylated E75-CTLs (n=6) demonstrated significantly improved disease

inhibition in SKBR3-A2 engrafted mice compared with non-fucosylated E75-CTLs

(n=6) (p<0.05) (Fig. 4C). Likewise, in the immunocompetent C57BL/6 mouse

model, mice treated with fucosylated pmel-1 CD8+ T cells (n=9) (Supplementary

Fig. S3) demonstrated a significant decrease in tumor volume growth (p<0.05), in

comparison with mice treated with non-fucosylated pmel-1 CD8+ T cells (n=8)

and untreated mice (n=9) (Fig. 4D).

These findings were corroborated by evidence of increased homing of

fucosylated antigen specific CTLs into the leukemic bone marrow and tumor in

comparison with non-fucosylated antigen-specific CTLs (Fig. 5). Frequencies of

total live human CD3+ CG1-CTLs (n=11) in mouse bone marrow were

significantly higher than for non-fucosylated CG1-CTLs (n=10) (p<0.001) (Fig.




20

5A). Fucosylated E75-CTLs (n=15) showed similar trends in homing to SKBR3-

A2 primary tumor when compared with non-fucosylated E75-CTLs (n=13)

(p=0.05) (Fig. 5B). These findings were also confirmed in the immunocompetent

C57BL/6 mice. Mice treated with fucosylated pmel-1 CD8+ T cells demonstrated

higher frequencies of pmel-CD8+ T cells (live, mCD3+ mCD8+, CD90.1+)

compared with mice that were treated with non-fucosylated pmel-1 CD8+ T cells

(p<0.05) (Fig. 5C).

CFSE assays showed that fucosylation does not affect CTL proliferation

(Supplementary Fig. S4); however, we acknowledge that the in vitro proliferation

assay does not conclusively exclude an effect of fucosylation on CTL proliferation

in vivo.

Fucosylated CTL do not impair healthy HSC growth in vitro

CFU assays were used to verify that fucosylation does not interfere with CTL

specificity. We have an established normal hematopoiesis model that tests the

specificity of CG1- and PR1-CTL (26,28). This model takes advantage of the fact

that CG1 and PR1 are both processed from azurophil granule proteases normally

expressed by progenitor cells, although at lower levels than what is found in

leukemia (26-28,40). We show that human HLA-A2+ normal bone marrow (BM)

was not affected when co-cultured with fucosylated or non-fucosylated CG1-

(Fig. 6A) or PR1-CTLs (Supplementary Fig. S5). Non-fucosylated and

fucosylated CG1-CTLs co-cultured with BM resulted in an average of 388 CFUs

and 448 CFUs, respectively (not significant). The same conditions with PR1-

CTLs resulted in 140 and 137 CFUs, respectively (not significant). BM co-




21

cultured with 1mM cytarabine was used as a positive control. BM co-cultured with

leukemia specific CTLs also did not result in any significant differences in CFUs

when compared to BM co-cultured with HIV-CTLs, an irrelevant control for

antigen specificity.

Fucosylation does not affect CTL homing into normal tissues

Fucosylation is believed to enhance the interaction of cell surface expressed

selectin ligands with selectins, which are expressed on tumor and endothelial

tissues and are unregulated during inflammatory states (6-10). To confirm that

fucosylation does not increase homing of CTLs to normal (i.e. non-inflamed)

mouse tissues, we administered CG1-, PR1-, and E75-CTLs to normal NSG and

NSG-A2 transgenic mice. Histologic evaluation of various tissues confirmed that

fucosylation does not increase the homing of antigen-specific CTLs to normal

tissue (Fig. 6B).




22

Discussion

We have shown that fucosylation enhances the homing of antigen specific CTLs

to malignant niches, resulting in increased anti-tumor efficacy. Importantly,

fucosylation does not increase CTL homing to normal tissues. Furthermore,

fucosylation enhances CTL efficacy, in part by altering CTL trafficking, cytolytic

machinery, synapse formation, and expression of distinct activating surface

molecules. Taken together, our data illustrate a novel, robust, and simple

approach to enhance CTL homing to hematologic and solid tumor malignancies.

Additionally, our data indicate ex vivo CTL fucosylation is an effective method to

enhance T cell efficacy against tumor cells.

T cell homing to tumor sites following adoptive transfer is critical to

achieve desired anti-tumor immune responses (43). Leukocytes migrate to

inflamed tissues via interactions between T cell ligands and specific molecules

expressed by the tumor and the endothelia. Most immunotherapeutic approaches

have focused on the expression of chemokines within the tumor tissues and their

cognate receptors by T cells. Numerous studies have employed methodologies

to virally transduce chemokine receptors into effector cells to enhance homing. In

one study by Garetto et al., the C-C chemokine receptor type 2 (CCR2) was

transduced into CD8+ T cells that were specific for the SV40 large T antigen

(SV40Tag) in a murine prostate cancer mouse model that expressed the

chemokine (C-C motif) ligand 2 (CCL2) (44). Increased expression of CCR2

resulted in enhanced T cell homing and significantly improved anti-tumor activity.

These data were corroborated by Craddock et al. in a neuroblastoma model




23

using GD2-specific chimeric antigen receptor (CAR)-T cells retrovirally-

transduced with CCR2b (45). A similar approach was employed using the

chemokine (C-X-C motif) ligand 1 (CXCL1) and its receptor CXCR2 (46). In that

study, CXCR2 virally-transduced gp100 TCR transgenic T cells were adoptively

transferred into CXCL1-expressing melanoma-bearing mice. CXCR2-transduced

T cells showed increased migration and anti-tumor activity following adoptive

transfer.

Another approach to enhance T cell homing focused on the extracellular

matrix (ECM) components of the tumor microenvironment, which can pose an

impediment to the efficacy of the anti-tumor immune response (47). Caruana et

al. virally transduced GD2-specific CAR T cells with the enzyme heparanase

(HPSE), which degrades heparan sulfate proteoglycans, a main component of

the subendothelial basement membrane (48). In this neuroblastoma model, the

investigators demonstrated enhanced tumor infiltration and anti-tumor activity by

the HPSE-transduced CAR T cells. Collectively, these studies have validated the

potential to improve immunotherapy by modulating chemokine receptor- and

ECM-degrading protease-expression by effector T cells. However, both

approaches mandate prior knowledge of the tumor microenvironment, including

the chemokine milieu of the tumor, the ECM components, and the expression of

cognate receptors and proteases by the effector T cells. Furthermore, these

approaches require a viral transduction step that could interfere with T cell

functions and adds expense and technical challenges.




24

Other methodologies to enhance the homing of CTLs to tumor tissues

have relied on the use of reagents that modulate the tumor vasculature (49,50).

In a study by Calcinotto et al., a TNF-fusion protein, NGR-TNF, was utilized to

increase T cell homing in mouse prostate and melanoma tumor models (49).

Administration of NGR-TNF altered the endothelial layer of the tumor

microvasculature, increased adhesion molecule expression by endothelial cells,

and increased cytokine and chemokine release within the tumor

microenvironment. Together, these modifications enhanced the extravasation of

immune cells to tumors, resulting in reduced tumor burden. A similar result was

shown using angiogenesis inhibitors and paclitaxel, where treatment of tumor-

bearing mice with angiogenesis inhibitors increased the leukocyte-endothelium

interactions and expression of endothelial cell adhesion molecules, and

enhanced CD8+ CTL infiltration of the tumor (50). However, one significant

drawback of treatment with angiogenesis inhibitors is that it requires an intact

host adaptive cellular immune system, including endogenous antigen-specific

CTLs that are capable of tumor elimination. It is possible that the co-

administration of angiogenesis inhibitors with fucosylated antigen-specific CTLs

could synergistically enhance T cell infiltration of tumor and further boost the anti-

tumor immune response.

Our study validates the novel approach of fucosylation to enhance CTL

homing and anti-tumor efficacy. Although the changes in in vivo antitumor

effects were small in some of our experiments, this study provides a proof of

principal for the potential of fucosylation to enhance the efficacy of ACT for




25

cancer. This approach has broad implications for the field of cellular

immunotherapy for cancer. Vaccines are effective in the setting of minimal

tumor burden, however, for patients with metastatic disease, the efficacy of

vaccines is limited by the number of antigen-specific CTLs that vaccination

can generate, the TCR avidity of vaccine-induced CTLs for the target antigen,

the activation status of vaccine-induced CTLs, and the ability of CTLs to

home to tumor tissue (51). ACT approaches can address many of these

shortcomings, including CTL number, TCR avidity, and CTL activation status.

However, methodologies to engineer and expand CTLs are oftentimes

hindered by the inadequate quantity of resulting effector cells and by the

scant tumor infiltration of the adoptively transferred CTLs (45,48).

Furthermore, methodologies that drive T cells to acquire potent cytotoxic

functions in vitro can lead to the downregulation of lymphoid homing

molecules (52). Our approach, therefore, is highly clinically relevant as it

allows for the generation and infusion of fewer antigen-specific CTLs with

improved homing capabilities. Additionally, our simple fucosylation strategy

involves a short incubation of effector T cells with an enzyme/substrate

cocktail, which can be applied rapidly to expanded/engineered cells. This

approach was shown to be safe in the setting of SCT and therefore can be

rapidly translated to the clinical setting for the treatment of hematologic as

well as solid tumor malignancies, and possibly to CAR-T cell therapies.




26

Acknowledgements

This work was supported by a grant from the Leukemia Research Foundation

(G.A.); Leukemia & Lymphoma Society (G.A.); Nancy Owens Memorial

Foundation and Pink Ribbons Project (EAM) and Cancer Center Support Grant

NCI #CA16672. E.A. Mittendorf is an R. Lee Clark Fellow at The University of

Texas MD Anderson Cancer Center, supported by the Jeanne F. Shelby

Scholarship Fund.




27

Author Contributions

G.A. and E.A.M. proposed the study given their observation that ex vivo

fucosylation enhances hematopoietic stem cell engraftment and homing of

regulatory T-cells. G.A. and E.A.M designed the experiments with the help of

N.Q., M.Z., A.P., and P.S. N.Q., M.Z, and C.K. conducted the in vivo and in vitro

migration and cytotoxicity studies. A.P., P.S., and M.Z. conducted the phenotypic

and CFU studies. J.J.M., E.J.S. providing guidance with the experimental design.

L.M., S.W. provided expert consultation on the use of Targazyme products in the

project. A.P., H.G., K.C.D. and L.S.J. helped with the analysis of flow cytometry

data. Q.M. provided consultation for planning in vivo studies. H.K. and W.O.

provided the pmel-1 mouse model and assisted with the design and

implementation of the related studies. M.Z., M.K., E.A.M. and G.A. drafted the

manuscript; all authors provided feedback on the manuscript. G.A. and E.A.M.

supervised and secured funding for the project.




28

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33

Figure Legends.

Figure 1. Fucosylation enhances CTL migration. CD8 enriched antigen

specific CTL were fucosylated with FT-VII. Results show representative HECA-

452 staining by flow cytometry of A, leukemia specific CTL and B, breast cancer

specific CTL. C, Fucosylated or non-fucosylated antigen specific CTL were

passed through a HUVEC barrier to assess functional alterations to

transmigration. LeukoTracker fluorescence was measured in the post-barrier

well. Relative fluorescence units (RFU) are indicative of three independent

experiments. Statistical testing was performed using unpaired t-test (*p < 0.05).

Figure 2. Fucosylation alters expression of select CTL surface markers.

Fucosylated antigen specific CTL were assessed for known trafficking and

regulatory cell surface molecules using flow cytometry. Fucosylation of antigen-

specific CTL increased the cell surface expression of a number of trafficking

molecules. Cell surface expression of CD162 (PSGL-1) (1.7-fold) and CD137

(41BB) (3-fold) was significantly increased in comparison to non-fucosylated

CTL. Results are representative of 6 independent experiments. Statistical testing

was performed using ANOVA (*p < 0.05; ****p < 0.0001).

Figure 3. Fucosylation enhances targeted cytotoxicity in vitro. Antigen-

specific CTL were co-cultured with cognate peptide-MHC expressing target cells

at varying E (effector):T (target) ratios in a calcein-AM release cytotoxicity assay.

Results are depicted as % specific lysis of target cells. A, CG1-CTL and B, PR1-

CTL were co-cultured with T2 cells pulsed with corresponding peptide, patient

AML (UPN#2) or U937-A2 cells. Similarly, C, E75-CTL were co-cultured with E75

peptide-pulsed T2 cells or SKBR3-A2 cells at varying E:T ratios. Results are

representative of 3 independent experiments. Statistics were obtained using

unpaired t-test (*p < 0.05; **p < 0.005; ***p < 0.001).




34

Figure 4. Fucosylation enhances the efficacy of CG1-, PR1- and E75-CTL in

vivo. Irradiated NSG mice were engrafted with primary patient AML UPN#1 (1 x

106 to 1 x 107 cells) on day 0. Fucosylated or non-fucosylated A, CG1- or B,

PR1- CTL were injected on day 2 (0.5 x 106). Mice were sacrificed and bone

marrow was harvested on week 2 and analyzed by flow cytometry. Results are

expressed as percent hCD33+/hCD45+/mCD45- cells of total live cells in bone

marrow. C, For the breast cancer model, irradiated NSG mice were engrafted

with SKBR3-A2 (1.5 x 107 cells) in the mammary fat pad on day 0. Fucosylated

or non-fucosylated E75-CTL (2 x 106 cells) were injected IV on days 7 and 10.

Mice were sacrificed at week 5 and tumor sizes were measured using

bioluminescence imaging. Results are expressed as percent tumor size following

treatment with CTL in comparison with pre-treatment tumor size. D, C57BL/6

mice were inoculated with B16-F10 melanoma cells (0.3 x 106). Fucosylated or

non-fucosylated expanded pmel-1 CD8+ T cells (5 x 106) were injected IV on day

0. Mice tumor volumes were measured daily for 9 days. Results are expressed

as mean tumor size on day 9 from two separate experiments. Statistical testing

was performed using ANOVA (*p < 0.05; **p < 0.005).

Figure 5. Fucosylation enhances CTL homing in vivo. Irradiated tumor-

bearing NSG mice treated with fucosylated or non-fucosylated CTL were

analyzed for CTL homing into tumor using flow cytometry. A, Results show

percent of CG1-CTL (hCD33-/hCD45+/mCD45-/hCD13-/hCD3+/hCD8+) of total

live cells in bone marrow harvested from leukemia-engrafted mice. B, Breast

cancer engrafted mice were sacrificed at week 5, and TILs were enriched from

the tumors. Results show percentage of E75-CTL (hCD45+/ mCD45-/hCD3+/

hCD8+) of total live cells from TIL extraction. C, Melanoma-engrafted C57BL/6

mice were sacrificed on Day 9, and TILs were extracted from the primary tumor.

Results show percent of pmel-1 T cells (mCD45+/mCD3+/mCD8+/CD90.1+) of

total live cells. Statistical testing was performed using unpaired t-test (*p < 0.05;

***p < 0.001).




35

Figure 6. Fucosylation does not alter CTL specificity or homing to normal

tissue. A, Healthy donor bone marrow was co-cultured with fucosylated or non-

fucosylated CG1-CTL at a 1:5 ratio of BM:CTL. Cultures were grown in

Mammocult medium in a 6-well plate for 14 days before CFU counts were taken.

Results show CFU on day 14 for CG1-CTL. BM co-cultured with cytarabine (1

mM) served as a positive killing control. Data is representative of 3 independent

experiments. B, Fucosylated (fuco) and non-fucosylated (non-fuco) antigen-

specific CTL were administered to mice twice weekly IV via tail vein. After 4

weeks, mice were sacrificed and tissues were fixed, paraffin-embedded,

sectioned, and stained with hematoxylin and eosin prior to histological

examination. Data show equal lymphocytic infiltration in normal mouse tissue

between fuco CTL and non-fuco CTL treated groups. Statistical testing was

performed using ANOVA. BM, bone marrow; NS, not significant.




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Published OnlineFirst January 15, 2019.Clin Cancer Res Gheath Alatrash, Na Qiao, Mao Zhang, et al. antigen-specific cytotoxic T lymphocytesFucosylation enhances the efficacy of adoptively transferred

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cytotoxic t lymphocytes , na qiao , mao zhang , madhushree ... · fucosylation of selectin ligands...

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