Immune escape after adoptive T cell therapy for malignant ......2020/08/11 · Introduction Immunotherapy has revolutionized cancer care [1, 2]. However, tumor escape is common and

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Immune escape after adoptive T cell therapy for malignant gliomas Tyler J. Wildes1, Kyle A. Dyson1, Connor Francis1, Brandon Wummer1, Changlin Yang1, Oleg Yegorov1, David Shin1, Adam Grippin1, Bayli DiVita Dean1, Rebecca Abraham1, Christina Pham1, Ginger Moore1, Carmelle Kuizon1, Duane A. Mitchell1, Catherine T. Flores1* 1University of Florida Brain Tumor Immunotherapy Program, Preston A. Wells, Jr. Center for Brain Tumor Therapy, Lillian S. Wells Department of Neurosurgery, McKnight Brain Institute, University of Florida, Gainesville, Florida, USA. Running Title: Glioma immune escape after immunotherapy Keywords: Glioma, Immune escape, Immunotherapy, Adoptive cellular therapy (ACT), Immune Checkpoint Blockade, Tumor escape Acknowledgments: This research was supported by the University of Florida Health Cancer Center Predoctoral Award (T. Wildes); American Brain Tumor Association Research Collaboration Grant (C. Flores); Alex’s Lemonade Stand Young Investigator Grant (C. Flores); Florida Center for Brain Tumor Research Grant (C. Flores); Wells Foundation; and University of Florida Clinical and Translational Sciences Award (UL1TR001427). *Corresponding author: Catherine T. Flores, Ph.D., University of Florida Brain Tumor Immunotherapy Program, Preston A. Wells, Jr. Center for Brain Tumor Therapy, Lillian S. Wells Department of Neurosurgery, McKnight Brain Institute, PO Box 100265, Gainesville, FL 32610; (352) 294-5269; [email protected] Conflict of interest: CTF and DAM have patents related to material disclosed in this publication that have been licensed to iOncologi, Inc. CTF and DAM hold interests in iOncologi, Inc., a biotechnology company focused on immuno-oncology. TJW, KAD, CF, BW, CY, OY, DS, AG, BD, RA, CP, GM, and CK declare no conflicts of interest. Clinical Cancer Research Translational Relevance: 150. Current: 145 Abstract limit: 250. Current: 212 Text limit: 5000. Current: 5052 Figures limit: 6 main text figures, Current: 6+4+1

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Translational Relevance

Tumor escape from immunotherapy remains a problem. While research in

peripheral cancers has identified common mechanisms of escape, escape mechanisms

in brain tumors remain unclear. Herein, we investigated tumor escape after tumor-

specific adoptive T cell immunotherapy. We developed an immune-escaped tumor

model system to study escape mechanisms as well as secondary immunotherapy

treatment. These studies revealed multiple mechanisms of escape including a shift in

immunogenic tumor antigens, downregulation of MHC-I, and upregulation of checkpoint

molecules. Despite these changes, a new population of escape variant-specific

polyclonal T cells could be generated to target immune-escaped tumors through using

tumor escape variant RNA. These T cells were more specific for the escaped tumors

when compared to primary gliomas and were unique to each escape variant. When

applied in a treatment model with checkpoint blockade, tumor-specific adoptive T cell

therapy significantly prolonged survival of immune-escaped and primary glioma-bearing

mice.




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Abstract

Purpose: Immunotherapy has been demonstrably effective against multiple cancers,

yet tumor escape is common. It remains unclear how brain tumors escape

immunotherapy and how to overcome this immune escape.

Experimental Design: We studied KR158B-luc glioma-bearing mice during treatment

with adoptive cellular therapy (ACT) with polyclonal tumor-specific T cells. We tested

the immunogenicity of primary and escaped tumors using T cell restimulation assays.

We used flow cytometry and RNA profiling of whole tumors to further define escape

mechanisms. To treat immune-escaped tumors, we generated escape variant-specific T

cells through the use of escape variant total tumor RNA and administered these cells as

ACT. Additionally, PD-1 checkpoint blockade was studied in combination with ACT.

Results: Escape mechanisms included a shift in immunogenic tumor antigens,

downregulation of major histocompatibility complex (MHC) class I, and upregulation of

checkpoint molecules. Polyclonal T cells specific for escape variants displayed greater

recognition of escaped tumors than primary tumors. When administered as ACT, these

T cells prolonged median survival of escape variant-bearing mice by 60%. The rational

combination of ACT with PD-1 blockade prolonged median survival of escape variant

glioma-bearing mice by 110% and was dependent upon NK cells and T cells.

Conclusions: These findings suggest that the immune landscape of brain tumors are

markedly different post-immunotherapy yet can still be targeted with immunotherapy.




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Introduction

Immunotherapy has revolutionized cancer care [1, 2]. However, tumor escape is

common and poorly understood [2-4]. Herein, we studied tumor escape variants after

immunotherapy to draw meaningful insights about escape mechanisms. We then

applied that information to study secondary immunotherapy based on escape variant

total tumor RNA to treat tumor escape variants.

Gliomas are resistant to chemotherapy, radiation, surgical resection, and even

recent developments in immunotherapy, yet glioma escape mechanisms remain poorly

understood [5-14]. One of the hypothesized methods of brain tumor escape is

immunoediting, or the elimination of cells expressing targetable epitopes, the

equilibration of remaining tumor, and the outgrowth of tumor escape variants. In

peripheral tumors, immunoediting is amplified in the presence of IFN- and Fas-

mediated targeting of tumor, two primary components of T cell-mediated killing [15, 16].

Furthermore, it was also recently demonstrated that programmed cell death protein-1

(PD-1) checkpoint blockade can promote T-cell immunoediting of tumors in the

periphery [14, 17, 18]. The expectation is that once the immunogenic antigens are

deleted during immunoediting the optimal opportunity to target immunogenic tumor

antigens has largely passed [12, 17, 19].

Recent evidence in human trials has shown evidence of immunoediting including

widespread loss of single antigen targets in gliomas and other cancers after monoclonal

chimeric antigen receptor (CAR) T-cell therapy [4, 6-9]. While some preclinical studies

have indicated that this single antigen loss may not affect anti-tumor immunity [20],

conclusions from these recent human studies recommend employing cell therapies with




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multiple antigen targets and the use of combinatorial therapies to activate host immunity

and overcome the immunosuppressive tumor microenvironment [7, 21]. Given these

findings and similar evidence in the periphery, there is now an expectation that

treatments focused on single or limited antigen pools may have limited long-term

success and may even promote immunoediting and formation of tumor escape variants.

Additional tumor escape mechanisms implicated in peripheral tumors include

loss of major histocompatibility complex class I (MHC-I) and upregulation of immune

checkpoint molecules [3, 18]. MHC-I is required for CD8+ cytotoxic T-cell targeting and

killing of cells that present the T-cell’s cognate antigen. Tumor cells can evade T-cell

targeting by downregulation or deletion of MHC-I [18, 22]. In this setting, natural killer

(NK) cells possess cytotoxic capacity against MHC-Ilo tumors since MHC-I is a key

inhibitory ligand for NK immunoglobulin-like receptors (KIRs) [23]. While various

regimens of lymphokine-activated killer (LAK) cells have been investigated for the

treatment of brain tumors, convincing demonstrations of NK cell anti-tumor efficacy

remain elusive [24-28]. It also remains unclear if NK cells provide any role during

adoptive cellular therapy (ACT) for brain tumors.

Our group developed an ACT platform that targets multiple tumor antigens with

one infusion and is demonstrably efficacious in multiple murine models of brain

malignancies [29-31]. ACT employs bone marrow-derived DCs pulsed with total tumor

RNA to ex vivo activate a polyclonal population of tumor-specific T-cells [29, 31]. These

cells are adoptively transferred into tumor-bearing hosts following host conditioning and

hematopoietic stem and progenitor cell (HSPC) transplant and anti-tumor immunity is

maintained with weekly tumor RNA-pulsed DC vaccines (Fig. 1A). This combinatorial




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strategy strongly modulates the tumor microenvironment and promotes continued

intratumor T-cell activation [29-32].

ACT prolongs median survival and mediates ~30% long-term cures in malignant

brain tumor-bearing hosts. However, ~70% of treated animals succumb to disease for

unknown reasons [31]. We hypothesized, based on our previous data highlighting the

ACT-mediated upregulation of Ifng and Fasl, that brain tumors are primed for

immunoediting during ACT [15, 16, 31]. While investigation of viable human brain tumor

tissue after failure of immunotherapy is limited due to a small number of biopsies post-

progression, mouse brain tumor tissue is readily available after escape of

immunotherapy. Therefore, we isolated treatment-resistant tumor escape variants from

mice that succumbed to disease after initial suppression of tumor growth and eventual

escape from ACT (termed TOGA). We used these immune-escaped models and in vitro

T-cell functional assays, flow cytometry, and RNA analysis to investigate mechanisms

of immune escape. These studies revealed that escape mechanisms include a shift in

the immunogenic tumor antigens, downregulation of MHC-I on tumor, and upregulation

of checkpoint molecules on tumors, NK cells, and T-cells.

To evaluate the retreatment of escape variants with immunotherapy, we

developed escape variant-specific T-cells that were primed and expanded using DCs

pulsed with tumor escape variant total tumor RNA. This polyclonal population of escape

variant-specific T-cells demonstrated heightened ability to target TOGA tumors

compared to primary glioma-specific T-cells. When administered with DC vaccines, host

conditioning, and HSPC transplant to TOGA-bearing animals, TOGA-ACT prolonged

median survival by 60% compared to untreated animals. When we introduced PD-1




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blockade during ACT administration, TOGA-ACT+PD-1 blockade prolonged median

survival by 110% compared to untreated animals. When CD8+ T-cells or NK1.1+ NK

cells were depleted during TOGA-ACT+PD-1 therapy, the therapeutic benefit was

significantly ablated. With this flexible combinatorial approach, ACT+PD-1 blockade can

be employed to immunologically reject primary and recurrent gliomas.




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Materials and Methods

Mice

Female six- to eight-week-old C57BL/6 mice (Jackson Laboratories, 000664),

transgenic DsRed mice (Jackson Laboratories, 006051), transgenic GFP mice (Jackson

Laboratories, 004353) and GREAT mice (Jackson Laboratories, 017580) were used.

The facilities at the University of Florida Animal Care Services are fully accredited by

the American Association for Accreditation of Laboratory Animal Care, and all studies

were approved by the University of Florida Institutional Animal Care and Use

Committee.

Bioluminescent imaging

Imaging was performed as previously described using the IVIS system [29].

RNA isolation

Total tumor RNA was isolated from two sources: either in vitro cell lines or

directly post-excision. Qiagen RNAeasy kit (Qiagen, 74104) was utilized for all

extractions. Manufacturer’s guidelines were followed.

RNA-seq

Untreated KR158B and GL261 tumors were harvested 3 weeks post-implantation

and 6-week old C57BL/6 mouse brains were harvested for transcriptome analysis.

cDNA preparation and sequencing for these 9 samples were described previously [33].

TOGA1.1 tumor was harvested at humane endpoint at 63 days post-tumor implantation.

For this sample, RNA-Seq libraries were generated using the SMARTerTM Ultra Low

input RNA Kit and KAPA LTP Library Preparation Kit Illumina platforms following the

manufacturers recommended protocols (Clontech cat. #634935 and KAPA Biosystems




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cat. #KK8230). Analysis for all RNASeq samples was preformed on University of

Florida High Performance Cluster (HiPerGator). Briefly, low quality reads and adaptors

of fastq data were trimmed by trim_galore [34] then aligned to Ensembl 91 mouse

genome by RSEM to extract sample gene expression [35, 36]. This algorithm allows us

to align reads from different library preparation on transcript-level and normalized gene

expression by TPM (Transcripts Per Kilobase Million) which makes samples more

comparable among different groups. TPM were compared among the groups.

CancerSubtypes [37] and pheatmap [38] were used for data normalization and

visualization. The top 1000 most variable genes were extracted by CancerSubtypes and

clustered with pheatmap. Then, genes were extracted from each of 6 clusters (Fig. 1F).

In addition, gene networks were generated with stringdb's confidence mode. Principle

component analysis (PCA) were performed with pca3d and rgl [39]. (Supp. Fig.5).

Adoptive Cellular Therapy

Tumor-reactive T-cells were generated as previously described [29-31, 40]. For

TOGA T-cells used in TOGA-ACT, the same protocol was used with a different RNA

species that was isolated from immune-escaped TOGA lines. Briefly, total tumor RNA

was electroporated into DCs and tumor-specific DCs were then used to prime naïve

hosts. One week later, primed splenocytes were harvested and co-cultured with

additional tumor RNA-specific DCs and IL-2. After 5-7 days of co-culture and periodic

splitting, polyclonal, tumor-specific T-cells were harvested and utilized. Treatment of

tumor-bearing mice began with 5Gy lymphodepletion or 9Gy myeloablation on day 5

post-intracranial injection with X-ray irradiation (X-RAD 320). On day 6 post-intracranial

tumor injection, mice received a single intravenous injection with 107 autologous ex-vivo




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expanded TTRNA T-cells with either 5x104 lineage-depleted (lin-) hematopoietic stem

and progenitor cells (HSCs) (Miltenyi Biotec cat. 130-090-858). Beginning day 7 post-

tumor injection, 2.5x105 TTRNA-pulsed dendritic cells were injected intradermally

weekly for 3 weeks.

T-cell functional assays

In vitro experiments utilized IFN-γ release from T-cells in functional assays as a

measure of T-cell activity. Functional assays included effector T-cells and targets

(pulsed DCs or tumor cell lines) that are co-cultured in a 10:1 ratio in 96-well U-bottom

plates in triplicate. IFN-γ Platinum ELISAs (Affymetrix, #BMS606) were performed on

acellular media that was harvested and frozen from the supernatants after 48 hours.

The supernatant transfer system utilized the 10:1 ratio of T-cells and DCs to generate

supernatants.

Tumor models

KR158B-luc was murine glioma was used courtesy of Dr. Karlyne M. Reilly [29,

41] as previously described [31]. TOGA cell lines were isolated from excision of brain

tumors from mice after succumbing to KR158B-luc tumors post-ACT treatment

(including 9Gy irradiation, HSPC transplant, tumor-reactive T-cells, and 3 DC vaccines).

TOGA tumors were utilized identically to KR158B-luc tumors. Cell lines tested negative

for mycoplasma contamination (IDEXX, 9/26/2017).

In vivo antibodies

In vivo antibodies included anti-PD-1 monoclonal antibody (BioXcell, BE0146),

anti-CD8 depletion antibody (BioXcell, BE0223), and anti-NK1.1 depletion antibody

(BioXcell, BE0036) were administered as previously described [42]. Antibodies were




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administered according to treatment diagrams in figures.

Tissue processing

Tissue was processed as previously described [30, 31]. Brain tumor dissection

began posteriorly with a midline cut in the skull and rongeur removal of skull laterally.

Tumor resection extended to gross borders of tumor mass near the site of injection.

Tumors were dissociated mechanically with a sterilized razor blade and chemically with

papain (Worthington cat. NC9809987) for 30 minutes. Tumors were filtered with a 70μm

cell strainer (BD Biosciences cat. 08-771-2) prior to antibody incubation

RT-qPCR

Quantity of RNA was measured using NanoDrop 2000. Each reverse

transcription reaction was performed us

libraries were generated using the SMARTscribe reverse transcriptase kit from total

tumor RNA per the manufacturer’s instructions (Clontech, cat. 639537). Samples were

stored at -80°C for subsequent qPCR analyses. The CFX Connect™ Real-Time PCR

Detection System (BioRad Laboratories, 1855201), TaqMan® Universal PCR Master

Mix (Applied Biosystems, 4324018), and validated TaqMan® probes were used for

qPCR analyses. Transcriptional expression of H2k1 (cat. 4331182, Mm01612247_mH)

and Pdl1 (cat. 4331182, Mm00452054_m1) were normalized to Hprt (cat. 4331182,

Mm03024075_m1) per sample and expressed as fold-change versus untreated tumors.

well. Reagent preparation and thermal cycling parameters were followed per

manufacturer instruction. No template controls and reverse transcriptase negative

samples were included to ensure the absence of contamination and genomic DNA.




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Flow cytometry

Flow cytometry was done with the BD FACS Canto-II machine under the

management of the University of Florida Cancer Center. Cell-sorting was performed

using the BD FACS Aria-II. DsRed+ mouse-derived cells were detected in FL-2.

Analysis was performed with FlowJo version 10 (Tree Star). Results were analyzed

using isotype controls after debris and doublets were excluded and target populations

were gated on size and granularity. FACS antibodies are listed in Supplemental Table

1.

Statistical analysis

Statistical tests were performed using GraphPad Prism 8. For in vitro

experiments we utilized the unpaired Student’s t-test and for in vivo experiments we

utilized the Mann-Whitney rank sum test. Correlation studies employed Pearson’s two-

tail test for correlation. Experiments are powered to include at least 5 randomized

animals per group. For survival experiments we utilized 7 or more animals to attain

enough power to distinguish groups after analysis by Mantel-Cox log-rank test. Median

survival for KR158B-luc is 42 days and for TOGA1.1 is about 21 days. Significance was

determined as P<.05.




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Results

Escape from adoptive cellular therapy

ACT leads to approximately 30% long-term cures in preclinical murine models

including NSC medulloblastoma, K2 brainstem glioma, and KR158B-luc glioma [31].

Here, C57BL/6 mice received orthotopic injection of syngeneic KR158B-luc high grade

glioma and were treated with ACT as previously described [29-31]. Despite a significant

increase in median and overall survival, a proportion of glioma bearing mice treated with

ACT succumb to disease [29-31]. Bioluminescent imaging revealed that all ACT treated

mice maintain control of tumor growth up to 21 days post-ACT (p<0.0001) with a

fraction demonstrating long-term survival (Fig.1A-C).

Tumors from this experiment that escaped ACT treatment after a period of

immunological control were referred to as TOGA1.1 and TOGA1.2. When orthotopically

injected into naïve C57BL/6 mice, TOGA1.1 tumor was demonstrably more aggressive

than its primary counterpart KR158B-luc (median survival, 21.5 days vs. 41 days;

p<.0001; Fig. 1D). RNA-Seq revealed global genetic differences between the primary

KR158B-luc glioma and the escaped tumor TOGA1.1 (Fig. 1E). Gene expression of

TOGA1.1 was also compared to global gene expression of primary murine glioma cell

lines KR158B-luc, GL261 glioma, and normal brain tissue (Fig. 1E). We found that

between TOGA1.1 and primary KR158B-luc 8,487 genes are differentially expressed by

at least 2-fold, indicating that the selection process of ACT led to genetically distinct

gliomas.

Brain tumor escape variants are immunologically distinct from primary tumors




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After observing genetic differences between primary KR158B-luc and escaped

TOGA1.1, we next sought to determine if the tumors were immunologically discrete. We

hypothesized that if the escape variants were immunologically distinct, T-cells

generated from the primary tumor would no longer provide a remarkable treatment

effect. To test this, we administered serial adoptive T-cell transplants specific for the

primary KR158B-luc to KR158B-luc-bearing animals. While there was not a significant

difference in median survival, giving two serial T-cell transplants did induce a shift from

30% to 40% long-term cures (p=.2503, p=.5427; Fig. 1G, Supp. Fig. S1A). Regardless,

tumor escape persisted.

We next asked if T-cells generated against the primary KR158B-luc glioma

maintain immunological recognition of the escaped tumor TOGA1.1. To determine if

KR158B-luc-specific T-cells recognize cognate antigen on TOGA1.1, KR158B-luc-

specific T-cells were generated then used as effector cells against either KR158B-luc,

TOGA1.1, or B16-F10-OVA melanoma tumor target cells in a functionality assay (Fig.

2A). Supernatant IFN- secretion was measured as an indication of anti-tumor T-cell

reactivity. KR158B-luc T-cells secreted IFN- upon recognition of KR158B-luc but had

markedly diminished recognition of TOGA1.1 or B16-F10-OVA melanoma cells

(KR158B-luc: 2911pg/mL, TOGA1.1: 448pg/mL, p=.0767, Fig. 2A). This strongly

suggests that T-cells specific for primary tumor provide very little immune function

against escape variant tumor cells.

We next investigated whether we could regenerate a secondary T-cell therapy

that was more specific for the TOGA1.1 escape variant and could mediate significant

anti-tumor function. To do this, we generated TOGA1.1-specific T-cells by using total




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tumor RNA isolated from TOGA1.1 cells. TOGA1.1-specific T-cells were then tested in a

functionality assay to target either KR158B-luc, TOGA1.1, or B16-F10-OVA tumor cells.

TOGA1.1-specific T-cells secreted significantly more IFN- upon co-culture against

TOGA1.1 over primary KR158B-luc tumor (TOGA1.1: 3782pg/mL, KR158B-luc: 1200

pg/mL, p=.0128; Fig. 2B). Importantly, this demonstrates that the TOGA1.1 tumor,

which was outgrown from a primary KR158B-luc glioma that escaped ACT, is

immunologically distinct from its primary counterpart.

Individual tumor escape variants are immunologically distinct from each other

Our data thus far demonstrates that escape from ACT results in immunologically

distinct tumors from the primary tumor. We next sought to determine if the escaped

tumors after treatment are immunologically distinct from other escape variants

originating from the same tumor that received the same ACT treatment.

Since T-cells generated against the primary KR158B-luc glioma no longer

recognize escaped TOGA1.1 glioma, we then specifically asked if T-cells generated

against tumors that have escaped ACT recognize and target each other. Here we used

TOGA1.1 and TOGA1.2 which are escaped tumors that both originated from the same

KR158B-luc tumor which escaped the same T-cell treatment (Fig. 1D). We generated

antigen-specific T-cells against TOGA1.1 and used those to target either TOGA1.1

tumor cells or TOGA1.2 tumor cells and supernatant IFN- was measured as an

indicator of T-cell recognition of cognate tumor antigen. While IFN- was released when

TOGA1.1 T-cells were cultured with TOGA1.1, minimal IFN- was detected when

TOGA1.1 T-cells were cultured with TOGA1.2 tumor cells (TOGA1.1 vs TOGA1.2,




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p=.0224, Fig. 2C). The converse experiment was conducted when TOGA1.2-specific T-

cells were generated and used to target either TOGA1.1 or TOGA1.2 glioma cells. The

TOGA1.2 T-cells failed to recognize TOGA1.1 escaped tumor cells (TOGA1.1 vs.

TOGA1.2, p=.0313, Fig. 2D). Therefore, there was successful recognition of cognate

tumors, yet minimal cross-reactivity between T-cells and the converse tumor target (Fig.

2, C-D). This indicates that tumor escape variants were at least partly unique.

It has been previously reported that spectratyping using fluorescence-activated

cell sorting (FACS) analysis of TCR V may be used to track the reactivity of specific

TCR V families towards target cells [43-45]. For validation of this method, we verified

the reactivity of TCR V 5.1, 5.2 T-cells for OVA-expressing target cells [46] (Supp. Fig.

S1, B-C). We next evaluated the proportion of TCR V families within the polyclonal

pools of KR158B-luc T-cells or TOGA1.1 T-cells belong after ex vivo expansion and

determined they were largely comparable (Supp. Fig. S1D). In a recent manuscript, we

discovered that the primary TCR V family that drives ACT response to KR158B-luc in

vivo is TCR V6 [45]. We therefore FACS-sorted TCR V6+ T-cells from the polyclonal

KR158B-luc T-cell pool. After sorting, we performed T-cell functional assays against

KR158B-luc or TOGA1.1 tumor cells. TCR V6+ KR158B-luc-specific T-cells were 8-fold

more reactive towards KR158B-luc when compared with TOGA1.1 tumor cells

(KR158B-luc: 771pg/ml, TOGA1.1: 94pg/ml, p=.0033, Fig. 3A). This indicates that TCR

V can be used to broadly demarcate specificity of T-cells between KR158B-luc T-cells

and TOGA1.1 T-cells and that TCR V6+ KR158B-luc T-cells were not as capable of

targeting epitopes that were present on TOGA1.1. We therefore tested the reactivity of

individual FACS-sorted TCRV families in vitro against their cognate tumor. This




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revealed that the T-cell TCRV families that are reactive for TOGA1.1 tumors are

largely different than those that are reactive for KR158B-luc and TOGA1.2 tumors (Fig.

3, B-C).

MHC class I is downregulated on a subset of tumor escape variants

We next investigated alternative mechanisms that could be responsible for

escape following ACT. Given the dependence of CD8+ T-cells on tumor MHC-I

expression, we investigated the potential for MHC downregulation on ACT-treated

tumors. We identified a downregulation of MHC-I by percent and MFI on the TOGA1.1

tumor compared to KR158B-luc (Fig. 4, A-B). We then investigated MHC-I expression

on tumors of a cohort of animals treated with ACT in a separate experiment (Fig. 4C). In

the KR158B-luc-bearing group, 4/7 tumors maintain expression of MHC-I after ACT, 3/7

tumors displayed a marked decrease in expression of MHC-I (p=.0289, Fig. 4, D-F).

This bifurcation in response was verified by percent expression, MFI, and PCR (Fig. 4,

D-F).

We additionally tested the impact of ACT on MHC-I expression in TOGA1.1-

bearing animals. This revealed that MHC-I was ubiquitously downregulated on

TOGA1.1 tumors after TOGA-ACT by percent expression and MFI (MHC-I+, 22.59% to

10.12%, p=.0033, Fig. 4, D, G-H). It should be noted that while only a fraction of ACT-

treated KR158B-luc-bearing hosts demonstrated downregulation of MHC-I, all ACT-

treated TOGA-bearing animals demonstrated a uniform downregulation of MHC-I by

flow cytometry. We then generated TOGA-ACT T-cells from DsRed transgenic animals

and tracked T-cells in tumors of ACT-treated hosts. At endpoint we excised tumors and




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analyzed tumor-infiltrating lymphocyte (TIL) TCR V families. The V families driving at

least partial in vitro reactivity against TOGA1.1 tumors (4, 5.1/5.2, 6, 8.1/8.2, and 8.3)

also comprised the majority of the TIL fraction of the families tested at endpoint (Supp.

Fig. S1E).

PD-L1 is upregulated on a subset of escape variant tumors

Given recent reports detailing the importance of checkpoint molecules and MHC-

I expression in determining the cytotoxic response in peripheral tumors [42], we next

investigated PD-L1 expression on tumors. We determined that ACT induces an

upregulation of PD-L1 by PCR and flow cytometry (p=.0466, p=.001; Fig. 5, A-B). PD-L1

expression was then compared with MHC-I at the gene level (Fig. 5C) and the protein

level (Fig. 5D) within each sample. Within-sample analysis revealed a strong positive

correlation between the two molecules (r=.9953, p<.0001), indicating that tumors that

escape by downregulating MHC-I are largely PD-L1lo. Additionally, in concordance with

this correlative data, TOGA-ACT treated hosts, which are low for MHC-I, remained low

for Pdl1 even after ACT (Fig. 5E). This suggests that TOGA1.1 is a variant that primarily

escapes by MHC-I downregulation and not necessarily by PD-L1 upregulation. This

may suggest distinct mechanisms of escape whereby brain tumors primarily either

upregulate PD-L1 or downregulate MHC-I in response to escape T-cell pressure.

ACT promotes activation and PD-1 expression on T-cells and NK cells

To determine if ACT induced a cellular immune response, we investigated

cytotoxic immune cell infiltration in the tumor-draining cervical lymph nodes (TDLN) and




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tumor microenvironment during ACT. For these analyses, we specifically investigated

the presence and activation of CD8+ T-cells and CD3-CD19-F4/80-Ter-119-

NK1.1+NKp46+ NK cells. We studied expression of CD107a as a marker of

degranulation of activated cytotoxic cells as well as PD-1, a marker of T-cell activation

that functions as a regulatory receptor [47-49]. These analyses revealed that ACT

induced a 22-fold increase in CD8+ T-cells and a 3-fold increase in NK cells in TDLN by

percent and absolute counts (p=.001, p=.001, Fig. 5, F-G). Importantly, NK cells are not

derived from the adoptive T-cell transfer (Supp. Fig. S2). This demonstrates an

important link between T-cell therapy and NK cell engraftment in TDLN. A concomitant

increase of both absolute number and percent of CD107a+ and PD-1+ T-cells and NK

cells were also seen with ACT (Fig. 5, H-I). ACT induced a 2.5-fold greater expression

of CD107a on T-cells and a 4.5-fold greater expression on NK cells (p=.001, p=.001,

Fig. 5, H-I). Additionally, ACT induces a 6-fold greater expression of PD-1 on T-cells

and a 5-fold greater expression on NK cells (p=.001, p=.001, Fig. 5, H-I). When we

investigated MFI expression on tumor-infiltrating T-cells and NK cells, we determined

that ACT induced greater expression of PD-1 on T-cells and NK cells (p=.001, p=.042,

Supp. Fig. S2-4) while inducing greater expression of CD107a on NK cells (p=.001,

Supp. Fig. S2-4). This data demonstrates that ACT mediates T-cell and NK cell

engraftment and activation.

ACT and ACT+PD-1 blockade prolongs survival of escape variant-bearing hosts

We next performed a series of survival experiments to test the capacity of ACT to

overcome the three described immune escape mechanisms: tumor antigen changes,




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MHC-I downregulation, and checkpoint molecule upregulation on immune cells and

tumor cells. Based on the in vitro data, we anticipated that tumor-specific ACT would

overcome the shift in immunogenic tumor antigens and allow for tumor-specific

targeting. We also anticipated MHC-I downregulation could be overcome because

tumor-specific T-cells still targeted escape variants in vitro and simultaneously

enhanced infiltration and activation of NK cells and T-cells in vivo. Lastly, we anticipated

combinatorial addition of PD-1 checkpoint blockade could prevent immune checkpoint-

mediated escape. Administration of TOGA1.1-specific ACT prolonged median survival

of TOGA1.1-bearing hosts by 60% (median survival, 24 to 33 days, p=.0003; Fig. 6, A-

B). Since we identified upregulated PD-1 on NK cells and T-cells previously during ACT,

we tested the ability of PD-1 to enhance the anti-tumor benefit of TOGA-ACT (Fig. 6C).

When we administered TOGA-ACT+PD-1 to TOGA-bearing animals, this yielded 110%

prolongation of median survival (UnTx vs. ACT+PD-1, 20 to 42 days, p=.0002, ACT vs.

ACT+PD-1, 32 to 42 days, p=.1522, Fig. 6D).

We previously demonstrated an ACT-induced engraftment and activation of NK

cells and T-cells in TDLN and tumors but had not yet investigated their impact on anti-

tumor efficacy. To test the functional impact of NK cells and T-cells during therapy, we

utilized depleting antibodies before and during TOGA-ACT+PD-1. When depleting

antibodies were administered to deplete NK1.1+ NK cells or CD8+ T-cells from animals

during ACT+PD-1 therapy, the survival benefit was significantly diminished (anti-NK1.1,

42 to 31 days, p=.0122; anti-CD8, 42 to 27 days, p=.0038, Fig. 6E). Therefore, both cell

types are required for optimal efficacy.




21

In summary, brain tumor escape from ACT occurs through at least three

mechanisms including a shift in immunogenic tumor antigens, MHC-I downregulation,

and upregulation of checkpoint molecules. However, ACT promotes the infiltration of

both NK cells and T-cells, two populations that can cytotoxically target tumors

regardless of MHC-I status. When we regenerated T-cells specific for tumor escape

variants, they were more specific for their cognate escape variant tumor cells when

compared to primary glioma cells. When this escape variant-specific T-cell approach

was applied in vivo with an ACT regimen, it significantly prolonged median survival. PD-

1 blockade during ACT enhanced this benefit and depletion of NK cells and CD8+ T-

cells highlighted the requirement for both cell types for optimal efficacy.




22

Discussion

This report highlights multiple mechanisms of escape during immunotherapy for

malignant gliomas including a shift in immunogenic tumor antigens, downregulation of

MHC-I, and upregulation of checkpoint molecules. This study also demonstrates a

translatable method of analyzing tumor that has escaped immunotherapy in situations

where re-biopsy is feasible. This mechanistic investigation which was done in

recalcitrant primary and recurrent murine gliomas demonstrates that tumor immunity is

complex and that single pathways are not solely responsible for escape, which is highly

relevant to cancer immunology. For instance, when tumors are targeted with antigen-

specific CAR-T cells or other single-antigen targeting modalities, antigen loss provides a

tumor escape route [4, 7, 8, 20, 50]. Alternatively, checkpoint blockade strategies may

promote anti-tumor immunity, but can encourage escape from immune surveillance [17,

51-55]. In another pathway altogether, the most advanced adaptive immunotherapy

strategy can be immobilized or stripped of activation by the network of pro-tumor

myeloid cells that are endemic to all anti-tumor immune responses [56, 57]. These are

all part of a larger coordinated immune system that self-regulates to the extent that

opportunistic tumor cells can benefit in the fray.

The role of CD8+ T-cells in anti-tumor immunity is well-established and was been

recapitulated in this report. They were required for optimal anti-tumor immunity even

against TOGA tumors, which express relatively lower MHC-I. However, the role of NK

cells in brain tumor immunity is not as well-appreciated. While much of the

immunotherapy field has focused on checkpoint molecules on T-cells, recent evidence

has also implicated checkpoint molecules on NK cells. In lymphoma models, PD-1




23

checkpoint blockade can promote activation of natural killer (NK) cell-mediated

immunity, opening the possibility for simultaneous activation of T and NK cells [42].

However, PD-1 blockade alone does not mediate remarkable efficacy in primary glioma

tumor models [30]. What we demonstrated here is that ACT+PD-1 blockade can

activate T-cells and NK cells and promote anti-tumor efficacy. We anticipate that NK

cells may follow parallel pathways of cytokine and chemokine-mediated migration and

activation that promote T-cell immunity. We previously demonstrated that HSPCs in the

tumor microenvironment release MIP-1 which promotes T-cell migration to tumors in

coordination with ACT that induces upregulation of Ccl5, Ifng, and other T-cell cytokines

and chemokines [10, 29, 31]. There are multiple reports that NK cells can migrate and

become activated through the same molecules and future investigation will explore this

further [58, 59]. Regardless, through this combined engagement of T-cells and NK cells

during ACT+PD-1, CD8+ T-cells can kill any tumor cell that has MHC-I while NK cells

can kill MHClo tumors that may appear in response to T-cell pressure. Perhaps this

encourages tumors into “escaping” into the cytotoxic snare of either NK cells or T-cells

in MHC-pliable tumors.

After ACT, some tumors displayed preferential escape through PD-L1

upregulation or MHC-I downregulation. In the within-sample analysis, there were largely

no tumor samples that expressed low MHC-I and high PD-L1 or high MHC-I and low

PD-L1. Previous experiments that demonstrated the impact of NK cells during PD-1/PD-

L1 blockade used a cell line that was MHClo and transduced with Pdl1 [42]. Then with

increased tumor Pdl1, NK cells were engaged by PD-1 or PD-L1 blockade to generate

anti-tumor efficacy against MHC-Ilo tumors. In our study, PD-L1 was only highly




24

expressed when MHC-I was highly expressed. Since CD8+ T-cells rely on MHC-I and

NK cells depend on its absence, our study suggests that the PD-1/PD-L1 axis is more

relevant in the function of CD8+ T-cell immunity during ACT. This may partially explain

why PD-1 blockade did not induce significant activation of NK cells in TDLN, whereas

PD-1 blockade did induce CD8+ T-cell activation. However, NK cells and CD8+ T-cells

were both required for optimal efficacy during combined ACT+PD-1. In total, these data

indicate a potential bifurcation in the escape mechanisms of brain tumors that is

reminiscent of a mechanistic model that has been described in other tumors as

escaping by “natural selection” or “acquired resistance” [22, 60]. Future studies in brain

tumors should perform single cell analysis to longitudinally track single cells and their

pre-determined or acquired resistances.

In the cohort of tumors that escape by PD-L1 upregulation and we anticipate that

TOGA-ACT+PD-1 may provide considerable benefit and perhaps long-term tumor

control. Given recent articles on neoadjuvant PD-1 blockade in glioblastoma [61, 62],

we anticipate robust development in neoadjuvant or adjuvant ACT+PD-1 combinations

in primary and resistant tumors. With ACT inducing a subset of tumors to upregulate

PD-L1, perhaps some escaped tumors are more readily treatable with PD-1 blockade

after stratification in the PD-L1hi escape variant subtype. On the contrary, the addition of

PD-1 to TOGA-ACT in the TOGA1.1 model was beneficial but limited. We anticipate this

may be due to TOGA1.1 demonstrating a preference for MHC-I downregulation (a

potential form of “natural selection”), not PD-L1 upregulation. Even though ACT induces

considerable T-cell engraftment (22-fold increase) and significant but limited NK cell

engraftment (3-fold increase), we generated a significant survival benefit. We anticipate




25

that for tumors that escape by MHC-I downregulation, additional combinatorial NK cell

activation strategies in addition to ACT+PD-1 may be beneficial. MHC-I was

downregulated on TOGA1.1, but it is important to recognize that MHC-I does not appear

completely lost on TOGA1.1. It was detectable by PCR and flow cytometry and our

functional assays indicate T-cell targeting despite lower MHC-I. However, future studies

should investigate the single cell-level kinetics of MHC-I changes and antigen

expression levels in escape variants and determine the relative requirement of MHC-I

and antigen presence for adequate T and NK cell function.

The strengths of this study include the use of multiple therapeutic brain tumor

models including the generated escape variant models. Here we laid the groundwork for

three primary mechanisms of escape in malignant gliomas. Even after escape, we

generated a significant benefit through novel generation of escape variant-specific ACT.

Future directions of these studies include the stratification and early detection of escape

variant subtypes. With that classification, future cancer regimens can be diversified and

tailored with attention to how and when specific tumors escape.




26

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Author Contributions: Conception or design of the work: TJW, DAM, CTF, KAD, CF, AG, and CP. The acquisition, analysis, or interpretation of data: TJW, DAM, CTF, KAD, CF, BW, CY, OY, DS, BD, RA, CP, GM, and CK. Drafted the work or substantively revised it: TJW, DAM, CTF, KAD, BW, AG, and BD. The creation of new software used in the work: n/a TJW, KAD, CF, BW, CY, OY, DS, AG, BD, RA, CP, GM, CK, DAM, and CTF approved the submitted version (and any substantially modified version that involves the author's contribution to the study). TJW, KAD, CF, BW, CY, OY, DS, AG, BD, RA, CP, GM, CK, DAM, and CTF agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.




30

Figure Legends Figure 1. Adoptive cellular therapy prevents early tumor growth and promotes

long-term survival in malignant primary glioma-bearing mice. A). Treatment platform for tumor-bearing mice. B-C). Bioluminescent imaging of KR158B-luc glioma-bearing mice untreated or treated with ACT. 8/10 animals escaped ACT while 2/10 were long-term cures. D). Survival of KR158B-luc glioma-bearing mice untreated or treated with ACT. Experiment performed at least five times. E). Tumorigenicity of TOGA1.1 tumor after re-implantation into naïve hosts. Passaging of TOGA1.1 cell line remained below 5 passages. Experiment performed two times. F). Heatmap of RNA-seq of primary tumors (KR158B-luc or GL261) and recurrent immune-escaped tumor (TOGA1.1) compared to normal brain. G). Survival of KR158B-luc glioma-bearing mice treated with ACT including 1 injection of T cells or serial ACT with 2 injections of T cells (treatment platform Supp. Fig. S1). *P<.05, **P<.01, ***P<.001, ****P<.0001, by Mantel-Cox Log Rank Test for survival experiments (n≥7).

Figure 2. Reactivity of tumor escape variant-specific T cells for primary and

recurrent tumors. A-B). IFN- ELISA of restimulation assay between B). KR158B-luc-T cells and KR158B-luc or TOGA1.1 tumor cells or C). TOGA1.1-T cells and KR158B-luc or TOGA1.1 tumor cells. KR158B-luc-primary glioma, TOGA1.1-immune-escaped glioma, B16-melanoma negative control. Experiment

performed twice. C-D). IFN- ELISA of restimulation assay between TOGA1.1-T

cells and TOGA1.1 or TOGA1.2 tumor cells. G). IFN- ELISA of restimulation assay between TOGA1.2-T cells and TOGA1.1 or TOGA1.2 tumor cells. All data represent the mean +/-SEM. *P<.05, **P<.01, ***P<.001, ****P<.0001, by

unpaired students t test for in vitro studies (n3).

Figure 3. TCR V families of TOGA1.1-T cells and TOGA1.2-T cells. A). IFN-

ELISA of restimulation assay performed after FACS-sorting for TCR V6+

KR158B-luc-T cells. Restimulation assay contained unsorted or V6+-sorted KR158B-luc-T cells cultured with KR158B-luc or TOGA1.1 tumor cells. B-C). IFN-

ELISA of restimulation assay performed after FACS-sorting for TCR V

families. Restimulation assay contained either B). unsorted or sorted TCR V-specific TOGA1.1-T cells with TOGA1.1 tumor cells or C). unsorted or sorted

TCR V-specific TOGA1.2-specific T cells with TOGA1.2 tumor cells. All data represent the mean +/-SEM. *P<.05, **P<.01, ***P<.001, ****P<.0001, by

unpaired students t test for in vitro studies (n3). Figure 4. MHC class I downregulation during adoptive cellular therapy. A-B). Flow

cytometry of MHC-I expression on KR158B-luc primary glioma or the isolated tumor escape variant TOGA1.1 C). Treatment plan for D-H. D). Flow cytometry MFI of MHC class I on brain tumors of mice treated untreated or treated with ACT at humane endpoint. E) Quantification of MFI and percent MHC-I expression in KR158B-luc-bearing animals. F). Correlation between % MHC-I positivity by flow cytometry and PCR expression of H2k1 for KR158B-luc-bearing animals. G). Quantification of MFI and percent MHC-I expression in TOGA1.1-bearing animals. H). Correlation between % MHC-I positivity by flow cytometry and PCR expression of H2k1 for TOGA1.1-bearing animals. All experiments performed twice and data represent the mean +/-SEM. *P<.05, **P<.01,




31

***P<.001, ****P<.0001, by Mann-Whitney t test for in vivo studies (n5) and and

Pearson’s two tail test for correlation (n5). Figure 5. PD-L1 upregulation on tumors and PD-1 upregulation on T cells and NK

cells during adoptive cellular therapy. A). PCR for Pdl1 (Cd274), the gene for PD-L1 in mice, at humane endpoint in KR158B-luc-bearing hosts. B). Flow cytometry of PD-L1 of brain tumors of mice at day 23 post-T cell transplant. Experiment performed twice. C). Correlation between Pdl1 and H2k1 determined by PCR in tumors from untreated or ACT-treated animals at humane endpoint. Statistics represent Pearson’s test for ACT-treated group. D). Correlation between PD-L1 and MHC-I determined by flow cytometry in tumors from untreated or ACT-treated animals at day 23 post-T cell transplant. Experiment performed twice. Statistics represent Pearson’s test for ACT-treated group. E). PCR for Pdl1 (Cd274) at humane endpoint in TOGA1.1-bearing hosts. F). Flow cytometry of CD8+ T cells in TDLN untreated or treated with ACT at day 23 post-T cell transplant. G). Flow cytometry of NK cells in TDLN untreated or treated with ACT at day 23 post-T cell transplant. NK cell phenotype is CD3-CD19-F4/80-

Ter-119-NK1.1+NKp46+. H). Flow cytometry of markers CD107a and PD-1 on T cells in TDLN. I). Flow cytometry of markers CD107a and PD-1 on NK cells in TDLN. Data represent the mean +/-SEM. *P<.05, **P<.01, ***P<.001,

****P<.0001, by Mann-Whitney t test for in vivo studies (n5) and Pearson’s two

tail test for correlation (n5). Figure 6. Anti-tumor efficacy of ACT alone or in combination with PD-1 blockade

for immune-escaped and primary brain tumors. A). Treatment outline for Fig. 6B. B). Survival curve of TOGA-bearing animals treated with TOGA-specific ACT. Experiment performed twice. C). Treatment outline for Figure 6, D-E. D). Survival curve of TOGA-bearing animals treated with TOGA-ACT+PD-1 blockade. PD-1 blockade was administered on days 6, 11, 16, and 21 as depicted by ticks on the graph. E). Survival curve of TOGA-bearing animals depleted of NK1.1+ NK cells or CD8+ T cells during ACT+PD-1 blockade. PD-1 blockade was administered on days 6, 11, 16, and 21 as depicted by ticks on the graph. NK1.1+ NK cells or CD8+ T cells were depleted on days -2, -1, 5, 10, 15, 20, 25, and 30 as depicted by ticks on the graph. *P<.05, **P<.01, ***P<.001, ****P<.0001, by Mantel-Cox Log Rank Test for survival experiments (n≥7).













































































































Published OnlineFirst August 11, 2020.Clin Cancer Res Tyler J Wildes, Kyle A Dyson, Connor P Francis, et al. gliomasImmune escape after adoptive T cell therapy for malignant

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