Restoring Innate NK-Cell Immunity with Antibody Therapeutics in CLL B-Cell Malignancy

Restoring Innate NK-cell Immunity with Antibody Therapeutics in CLL B-Cell Malignancy

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Emily Mary McWilliams, B.S.

The Biomedical Sciences Graduate Program

The Ohio State University

2016

Dissertation Committee:

Dr. John C. Byrd, Co-Advisor

Dr. Natarajan Muthusamy, Co-Advisor

Dr. Susheela Tridandapani

Dr. Virginia Sanders

Emily Mary McWilliams

2016

Abstract

Chronic Lymphocytic Leukemia (CLL) is the most prevalent adult leukemia with

estimations in the US of over 18, 900 newly diagnosed cases and 4,600 deaths for 2016.

Patients suffer from profound defects in immune function, with secondary infection a

leading cause of morbidity and mortality that is further exacerbated by frontline therapy.

A deeper understanding of CLL-associated immunosuppression is needed in order to overcome immune compromise. Enhancing Natural Killer (NK) effector functions has emerged as a promising immunotherapy. NK cells are innate immune effectors that survey and kill pathogen-infected or malignant cells. These cells are a major component of anti- tumor response especially with monoclonal antibody therapeutics (mAbs) that engage NKs and mediate tumor killing. Activating NK anti-tumor functions depends on a tightly regulated network of inhibitory and activating receptors that detect “self” antigens, which are severely deregulated in CLL leading to tumor escape. MAbs that activate NK anti- tumor immunity represent a promising chemotherapy-free therapeutic option that would not only spare patients’ immune systems but even enhance the anti-tumor response.

Antibodies generated by glyco-engineering have improved capacity to recruit and activate NK anti-tumor response. Defucosylation is a form of glyco-engineering that

removes a fucose moiety at the CH2 locus within the antibody constant region (Fc) and

raises the affinity between antibody and NK resulting in more effective NK-mediated

ii killing of targeted cancer cells. B-cell activation factor (BAFF) ligating to BAFF receptor

(BAFF-R) triggers critical pro-survival signals in B cells and blocking this interaction represents a novel target for immunotherapy. Glyco-engineered anti-BAFF-R activated superior NK antibody-dependent cellular cytotoxicity (ADCC) over CD20 antibodies including glyco-engineered obinutuzumab, and activated additional innate immune response as demonstrated by TNFα release by monocytes and macrophages, and induction of antibody cellular phagocytosis (ADCP). Anti-BAFF-R antagonizes BAFF-mediated protection of CLL cells from apoptosis and blocks NF-κB signaling, as shown both in bulk protein analysis and at the single-cell level. Similarly, BAFF signaling was observed in

CLL B-cells treated with ibrutinib, which blocks Bruton’s tyrosine kinase (Btk), a mediator of B-cell receptor (BCR) signaling cascades, and this was antagonized by anti-BAFF-R pretreatment. In vivo, anti-BAFF-R treatment rapidly cleared peripheral blood and synergistically combined with ibrutinib to provide survival advantage in a murine CLL model.

CLL patient NK-cell counts correlates with stage of disease and time to treatment, and are severely dysfunctional. CLL B-cells overexpress HLA-E, a MHC class I molecule that differentiates “self” from “non-self,” and leads to NK cell inhibition by binding

NKG2A. NKG2A, the primary inhibitory receptor on NKs, has garnered much attention as a promising target of antibody therapy to alleviate NK cell suppression, especially as anti-

NKG2A, monalizumab, is entering phase II clinical trials in various cancers. NKG2A is expressed on CD56+ CD16+ NKs, and blocking NKG2A with monalizumab is sufficient

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to restore NK-killing capacity indicating the significance of HLA-E/NKG2A driving tumor escape in CLL.

These findings demonstrate the impact of NK-cell dysfunction in CLL, and the novel therapeutic approaches of glyco-engineering and antigen selection to generate effective antibody-therapy. Together, these studies deepen our understanding of NK cell

immunosuppression in CLL and contributes to the development of rational combination

therapies to eradicate tumor and enhance anti-tumor response.

Dedication

This document is dedicated to my family.

Acknowledgments

I would like to thank my mentors Dr. John C. Byrd and Dr. Natarajan (Raj)

Muthusamy for their continued support and guidance through my graduate education. They created an outstanding training environment that fosters scientific excellence and their constant enthusiasm to continue asking the hard questions and appreciate that “data are data.” I entered my graduate program with minimal biology research experience and I am grateful for the opportunities to grow as a scientist and their patience along the way. I would also like to thank my committee members Dr. Susheela Tridandapani and Dr.

Virginia Sanders for serving on my committee as well as for their encouragement and always challenging me to perform at my best. I am humbled to have been trained by such talented scientists and caring mentors.

Additionally, I would like to thank all the past and present lab members who provided a rich learning environment and supported my training in an exciting work environment. Especially, I would like to thank Carolyn Cheney, Lisa Smith, Jennifer Mele for their technical assistance and support through my training as well as Xiaokui “Molly”

Mo for biostatistics work. I am thankful for the assistance of Michelle Grindley, Sue Scott, and Laura Handley for keeping our meetings and weeks organized. I am grateful for the support and friendship of my colleagues Drs. Amy Johnson, Kerry Rodgers, and Ema

Coccuci, as well as my peers J.T. Greene, Dr. Rebekah Browning, Eileen Hu, Shaneice

Mitchell, Sean Reiff, and Emily Smith. I would also like to acknowledge the invaluable contributions and mentorship from Dr. Chris Lucas.

I would like to extend my gratitude to my family for their continued support. My parents, my grandparents, and my brother that encourage and inspire me, and, I am thankful for my partner, Jenna Thrash, for her continued support of pursuing my goals within biomedical research.

vii

Vita

1989 ...... Born – Elgin, IL

2011 ...... B.S. Chemistry, North Park University

2011 to present ...... Graduate Research Associate, The Ohio

State University

Publications

E.M. McWilliams, J.M. Mele, C. Cheney, B.A. Timmerman, E.J. Strattan, X. Mo, F.

Fiazuddin, J.C. Byrd, N. Muthusamy, F.T. Awan. “Therapeutic CD94/NKG2A Blockade

Improves Natural Killer Dysfunction in Chronic Lymphocytic Leukemia.”

OncoImmunology. Accepted, July 2016.

P.D. Halley*, C.R. Lucas*, E.M. McWilliams, M.J. Webber, R.A. Patton, C. Kural,

D.M. Lucas, J.C. Byrd, C.E. Castro. “Daunorubicin-Loaded DNA Origami

Nanostructures Circumvent Drug Resistance Mechanisms in a Leukemia Model.” Small.

Epub date, November 2015

viii

Fields of Study

Major Field: Biomedical Sciences Graduate Program

Specialization: Immunology

Table of Contents

Abstract ...... ii

Dedication ...... v

Acknowledgments...... vi

Vita ...... viii

Table of Contents ...... x

List of Figures ...... xv

List of Abbreviations ...... xvi

Chapter 1: Background and Introduction ...... 1

1.1 Chronic Lymphocytic Leukemia, Overview ...... 1

1.1.2 Genomic Risk Factors ...... 1

1.1.3 Therapies approved for CLL ...... 2

1.2 CLL B-cell Survival ...... 4

1.2.3 Tumor Microenvironment in CLL ...... 8

1.2.4 Innate Immunosuppression by CLL B-cells ...... 13

1.2.5 Adaptive Immunosuppression by CLL B-cells ...... 15

1.2.6 Mouse models to study CLL ...... 17

1.3 Antibody Therapeutics ...... 19

1.3.2 Currently approved mAb for CLL ...... 19

1.3.3 Anti-tumor mechanisms of mAb ...... 22

1.4 Conclusion and Significance ...... 24

1.5 References ...... 25

1.6 Figures ...... 38

Chapter 2: Glyco-Engineered anti-BAFF-R, B-1239, Increases NK-Cell Activation and

Combines with Ibrutinib to Block Multiple Pro-Survival Signals and Eradicate CLL B- cells ...... 41

2.1 Introduction ...... 41

2.2 Materials and Methods ...... 44

2.2.1 Human sample preparation and cell culture ...... 44

2.2.2 Reagents...... 46

2.2.3 Flow cytometry and reagents ...... 46

2.2.4 Assessment of apoptosis ...... 47

2.2.5 Antibody Dependent Cellular Cytotoxicity assay ...... 48

2.2.6 Complement Dependent Cytotoxicity assay...... 49

2.2.7 Antibody Dependent Cellular Phagocytosis assay ...... 49

2.2.8 Cytokine assays ...... 50

2.2.9 Single cell fluorescent microscopy ...... 51

2.2.9 Lysates preparation and immunoblot ...... 53

2.2.10 Animal Studies ...... 53

2.2.11 Histopathological Analysis ...... 55

2.2.12 Statistical Considerations ...... 55

2.3 Results ...... 56

2.3.1 Anti-BAFF-R enhanced NK-cell ADCC and cytokine production ...... 56

2.3.2 Anti-BAFF-R activates innate immunity ...... 58

2.3.3 Specificity of anti-BAFF-R on B cells and FcγR on NK cells ...... 59

2.3.4 B-1239 obstructed alternative NF-κB and decreased cell viability ...... 61

2.3.5 Murine activity of anti-BAFF-R and survival advantage, in vivo ...... 64

2.3.6 Conserved BAFF-mediated NF-κB activity in ibrutinib treated CLL patient

cells ...... 66

2.3.7 Anti-BAFF-R and ibrutinib combination enhances efficacy and survival

advantage, in vivo ...... 68

2.4 Conclusions and Discussion ...... 70

2.5 References ...... 74

2.6 Figures ...... 80

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Chapter 3: Therapeutic CD94/NKG2A Blockade Improves Natural Killer Cell

Dysfunction in Chronic Lymphocytic Leukemia ...... 95

3.1 Introduction ...... 95

3.1 Materials and Methods ...... 97

3.1.1 Cells and Culture ...... 97

3.1.2 Flow Cytometry, HLA-E & NKG2a surface expression ...... 98

3.3.3 Real-time PCR ...... 99

3.3.4 NK cell killing assays (Direct Cytotoxicity and ADCC) ...... 100

3.3 Results ...... 101

3.3.1 HLA-E and NKG2A landscape in CLL ...... 101

3.3.2 Monalizumab, blocks NKG2A and enhances CLL NK-cell activity ...... 103

3.4 Conclusion and Discussion ...... 105

3.5 References ...... 109

3.6 Figures ...... 112

Chapter 4: Conclusions and Future Perspectives ...... 121

4.1 Conclusions ...... 121

4.2 Future Perspectives ...... 130

4.3 References ...... 134

4.4 Figures ...... 137

xiii

Works Cited ...... 139

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List of Figures

Figure 1. 1 Proximal B-cell receptor signaling pathway ...... 38

Figure 1. 2 BAFF/BAFF-R ligation activates alternative NF-κB ...... 39

Figure 1. 3 Mechanisms of antibody mechanisms to target and clear malignant cells ..... 40

Figure 2. 1 B-1239 enhances ADCC and NK cell activation ...... 80

Figure 2. 2 B-1239 activates innate immunity of monocytes and macrophages ...... 82

Figure 2. 3 B-1239 engages surface molecules on CLL B and NK -cells ...... 83

Figure 2. 4 BAFF-mediated survival is blocked by B-1239 pretreatment of CLL B-cells

...... 85

Figure 2. 5 In vivo efficacy of B-1239 in the TCL-1 mouse model of CLL ...... 89

Figure 2. 6 BAFF induced NF-κB signaling in CLL treated with ibrutinib ...... 91

Figure 2. 7 B-1239 combines effectively with ibrutinib in vivo ...... 93

Figure 3. 1 HLA-E and NKG2A landscape in CLL ...... 112

Figure 3. 2 Monalizumab blocks NKG2A and enhances CLL NK-cell mediated cytotoxicity ...... 117

Figure 4. 1 A model of overcoming CLL NK-cell immunosuppressive mechanisms ... 137

List of Abbreviations

Abs:……………………………………………………………………………..Antibodies

ADC:……………………………..………………………………Antibody drug conjugate

ADCC:.…………….……………………..Antibody-dependent cell-mediated cytotoxicity

ADCP:……………………...………….. Antibody-dependent cell-mediated phagocytosis

AIHA:……………………..…………………………….Autoimmune hematolytic anemia

ALL:……………………………………….……………… Acute lymphoblastic leukemia

ALE:…………………………………………………………………………Alemtuzumab

AML:……………………………………………………….…….Acute myeloid leukemia

APRIL:…………………………………………………… A proliferation-inducing ligand

Asp:………………………………..……………………………………………Asparagine

BAFF:…………………………………………………………...… B-cell activating factor

BAFF-R:…………………………………………..…….. B-cell activating factor receptor

BCMA:……………………………………………………...….. B-cell maturation antigen

BCR:……………………………………………………………...…………B-cell receptor

BCL10:……………………………………………………….…B-cell CLL/lymphoma 10

BLNK:……………………………………………………………..………….B-cell linker

BLK:……………………………………………….……………B-cell lymphocyte kinase

BM:…………………………………………………………………………. Bone marrow

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BMSC:……………….…………………………………………Bone marrow stromal cell

Btk:………………………………………………………………. Bruton’s tyrosine kinase

Breg:………………………………………………………………………B regulatory cell

CAR:……………………………………………………………Chimeric antigen receptor

CARD11:………..……………………….Caspase recruitment domain family member 11

CBM:……………………..…………………………..CARD11/BCL10/MALT1 complex

CDC:………………………………….Complement-dependent cell-mediated cytotoxicity

CHO:…………………………………..…………………………..Chinese Hamster ovary

CLL:………………………………………………...……..Chronic lymphocytic leukemia

CRA:…………………………………………………………..Chromium-51 release assay

CTL:……………………………………..………………………Cytotoxic T-lymphocytes

DC:……………………………………...……………………………………Dendritic cell

DLBCL:…………………………………………………..Diffuse Large B-cell lymphoma

ELISA:…………………………………………... Enzyme-linked immuno-sorbent assays

ERK:……………………………………………………….. Extracellular regulated kinase

E: T:……...……………………………………………………….....Effector to target ratio

Fab:……………….…………………………………………..Heavy chain variable region

FBS:………………………………………………..…………………...Fetal bovine serum

Fc:………………………………………………………………..Antibody constant region

FCR:…………...... ………………….Fludarabine, cyclophosphamide, rituximab therapy

FcγR:……………………………………………………………….…..Fc gamma receptor

FITC:…………………………………………………….……..Fluorescein isothiocyanate

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FMO:…………………………………………………………...…Fluorescence minus one

GC/MS:……………………….………………….Gas chromatography/mass spectrometry

G-CSF:…………………………..……………….. Granulocyte-colony stimulating factor

GEP:……………………………….……………………………Gene expression profiling

HLA-E:…………….....………….. HLA class I histocompatibility antigen, alpha chain E

IACUC:………...………………………….Institutional Animal Care and Use Committee

IFNγ:……..…………………………………………………………….. Interferon gamma

IgG:……………………………………………………………………… Immunoglobulin

IGH:…………………………………………………………Immunoglobulin heavy chain

IGVH:………………...…………………… Immunoglobulin heavy chain variable region

ILT2:……………………………………………………………………Ig-like transcript 2

IRB:……………………………………………………………..Institutional review board

ITAM:………………….………………. Immunoreceptor tyrosine-based activation motif

ITK:……………………………………………….……………….. IL-2 –inducible kinase

JAK3:……………………………………………….………….Janus family of kinases - 3

JNK:………………………………………………………………C-jun N-terminal kinase

KIR:…………………………………………………………………Killer Ig-like receptor

LN:…………………….………………………………………………………Lymph node

MAb:….…………………………………………………………..… Monoclonal antibody

MALT1:....………….Mucosa-associated lymphoid tissue lymphoma translocation gene 1

MCL:……………………………….……………………………….Mantle cell lymphoma

M-CSF:…………………………….…………………Monocyte-colony stimulating factor

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MDM:……………………………………………………...Monocyte derived macrophage

MDS:……………………………….…………………………. Myelodysplastic syndrome

MFI:……..……………………………………………………Mean fluorescence intensity

MHC:…………………...………………………………Major histocompatibility complex

MM:………………………………..……………………………………Multiple myeloma

MRD:…………………………………………………………….Minimal residual disease

MYD88:…….……………………………….Myeloid differentiation primary response 88

NAMPT:…………………………………………Nicotinamide phosphoribosyltransferase

ND:…………………………………………………………………………..Normal donor

NF-κB:…………………………………………………………… Nuclear factor kappa- B

NHL:………………………………………….………………..Non-Hodgkin’s lymphoma

NIK:……………………………………………………………….NF-κB inducible kinase

NK:…………………………………………………………..…………. Natural killer cell

NLC:……………………………………………………………………… Nurse-like cells

NOAEL:..……………………………………………..No observable adverse effects level

NZB:…….…………………….…………………………New Zealand black mouse strain

OBN:……………………………………………………………………..…Obinutuzumab

OFA:………………………………………………………………………….Ofatumumab

OR:……………….………………………………………………………Overall response

OS:……….………………………………………………………………..Overall survival

PALS:……………………………………………………. Periarteriolar lymphoid sheaths

PB:……………………………………………………………….………..Peripheral blood

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PBMC:……………………………………………….. Peripheral blood mononuclear cells

PBS:…………………………………………………….……….Phosphate-buffered saline

PC:………………………………………..……………………………Proliferation center

PFS:…………...………………………………………………….Progression free survival

PH:…………………………………….………………………...……Pleckstrin homology

PI:……………………………………………………………..………….Propidium iodide

PI3K:………………………………………………………….. Phosphoinositude 3 kinase

PKCβ:…………………………………………………………..……Protein kinase C-beta

PLCγ2:…………... 1-Phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2

PLL:………………………………….………………………….Prolymphocytic leukemia

RFU:……………………………………………………………..Relative fluorescent units

RLK:………………………………………………………….. Resting lymphocyte kinase

ROS:……………………………………………………….……..Reactive oxygen species

RTX:……………………………....……………………………………………..Rituximab

RT-PCR:……………………...... ………………Real time- Polymerase chain reaction

SCID:………………………………………..………Severe combined immunodeficiency

SLE:……………………………………….……………….Systemic lupus erythematosus

SLL:…………………….…………………………………….Small lymphocyte leukemia

SNP:……………………….……………………………..Single nucleotide polymorphism

STAT3:……………..…………………..Signal transducer and activator of transcription 3

SYK:……………………………………………………………..... Spleen tyrosine kinase

TACI:………………...…………...…… Trans membrane activator and CAML interactor

TCL-1:………………………...……………………………..T-cell leukemia/lymphoma 1

TCR:…………………………..…………………………………………….T-cell receptor

Tec:……………………………….…………………….………….Tyrosine protein kinase

Tg:………………………………………………………………………………Transgenic

TH:……………………………….……………………………….…………. T- helper cell

TNF:………………………...….……………………………………Tumor necrosis factor

TRA:…………………………………………………………………………..Trastuzumab

TRAF:………………………………………….………………..TNF-R associated factors

Treg:………………………………………….…………………………Regulatory T-celll

TRPC1:……………………………………………..Transient receptor potential channel 1

VLA-4:………………...……………………………………………….Very late antigen 4

WBC:……………………………………..……………………………….White blood cell

XID:…………………………………………………………...… X-linked immune defect

XLA:……………………………….……………….……. X-linked agammaglobulinemia

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Chapter 1: Background and Introduction

1.1 Chronic Lymphocytic Leukemia, Overview

Chronic Lymphocytic Leukemia (CLL) is the most prevalent adult leukemia with estimations of 18,900 newly diagnosed cases and 4,600 deaths in the US for2016. This disease is characterized by the clonal proliferation and accumulation of morphologically mature, malignant B cells that express CD19+, CD20+, CD23+, and CD5+ [1]. The malignant cells reside in the spleen, bone marrow (BM), and lymphoid tissues and circulate through the peripheral blood (PB). The malignant clone migrates to microenvironmental niches within lymphoid tissues such as bone marrow, lymph nodes, and spleen where the cells receive pro-survival signals. The exact cause of CLL is unknown, though publications suggest that CLL arises from a pre-leukemic multipotent hematopoietic cell harboring CLL associated mutations [2, 3]. CLL predominantly affects an older population with a median age of diagnosis at 72 years, though 10-20% of patients are younger than 55 years [4-7].

Considering that age is linked to incidence of disease, prevalence and mortality are expected to rise with society’s aging population.

1.1.2 Genomic Risk Factors

Disease course is highly variable and patients’ clinical course varies depending on

the stage of disease diagnosed at onset, disease progression, therapeutic response, and

outcome. Cytogenetic statuses factor heavily into disease heterogeneity and are detected in

over 80% of CLL cases with associative prognostic value. Among these, mutational status

of Ig heavy chain V region (IGHV), deletions or translocations in 11q, 13q, and 17p,

trisomy 12, and complex karyotype are used to risk stratify patients to an aggressive disease

course and determine patient response to therapy [8-11].

1.1.3 Therapies approved for CLL

The last decade has seen great advancements that broaden the treatment armamentarium for CLL. Clinically approved treatment options for CLL patients include chemotherapy, monoclonal antibodies (mAbs), or small molecule inhibitors targeting specific signaling pathways. Currently, how clinicians approach treatment naïve patients is in flux due to disease heterogeneity and the arrival of new-targeted therapies. An indolent clinical course is seen in a subset of patients with prolonged survival that do not need treatment intervention while aggressive disease is found in some patients and requires early treatment along with frequent relapses.

Chemotherapeutics such as fludarabine, chlorambucil, or bendamustine are highly effective at eradicating tumor load and patients can live decades without relapse; however this class of drugs is severely immunosuppressive and some patients develop therapy-

related myelodysplastic syndrome (MDS) or myeloid leukemias [12, 13]. Estimations up

to 10% of non-Hodgkin’s lymphoma (NHL) patients treated with conventional- or high-

dose chemotherapy may develop these complications within 10 years of initial therapy

[14]. Many patients cannot tolerate high-dose chemotherapy due to comorbidities like

multiple strokes, and patients with the genetic abnormality deletion of chromosome 17p do not respond to chemotherapy and fail to qualify for this treatment modality [15].

Treatment-related toxicities as well as comorbidities limit therapeutic options for older patients and patients often die from recurrent infections. Unfortunately, many therapies exacerbate this defective immunity. The combination of fludarabine, cyclophosphamide, and anti-CD20 rituximab (FCR) associates with persistent myelosuppression and cytopenias like neutropenia; additionally major infections are reported in up to 25% of patients [16, 17]. Despite these complications, chemotherapy and combination therapy with anti-CD20 mAbs has improved patient outcomes including complete remissions, progression-free survival, and overall survival for both young and old CLL patients [18,

19].

Inhibitors of BCR signaling molecules are an important new class of targeted therapeutics to treat CLL as well as other lymphoid malignancies. Ibrutinib is a first-in- class small molecule inhibitor that irreversibly targets Bruton’s tyrosine kinase (Btk) and is FDA approved for patients with 17p deletion, for frontline therapy, and for relapsed patients [20-22]. Additional BTK inhibitors are in development, as are other small molecule inhibitors: idelalisib targets phosphoinositude 3-kinase (PI3K) isoform P110

[23], and entosplentinib targets Spleen tyrosine kinase (SYK) [24], all of which inhibitδ molecules demonstrated to be critical for CLL cell homing to microenvironments and survival. The aforementioned inhibitors are lifesaving therapeutic options for leukemic

patients, though current therapy follow-up on is limited and long-term effects are unknown.

Additionally, not all patients respond to the inhibitors; relapse can occur with development of a more aggressive disease and acquired mutations while on therapy drive drug resistance as has been shown with ibrutinib [25] and idelalisib [26].

MAb therapeutics are designed against a surface protein expressed by cancer cells and activate the patient’s immune system to kill the mAb-coated tumor cell. Also, this class of therapeutics are generally well tolerated. Many antibodies for B cell malignancies target

CD20 and activate Natural Killer (NK) cells to lyse the tumor [27]. Examples include rituximab, alemtuzumab, obinutuzumab, and ofatumumab that all demonstrate efficient tumor cell clearance though patient response is variable, patients have relapsed, and developed resistance. Also, mAb therapy leads to profound immune suppression in a patient population with an already ailing immunity [28]. Indeed, there is a window of opportunity for new treatment modalities in CLL. Deep remission is achievable with combination of the various treatments, but CLL generally remains incurable with current standard approaches.

1.2 CLL B-cell Survival

CLL cells receive proliferative and survival signals via cytokines and cell-to-cell contact in microenvironment niches of the bone marrow, spleen and lymph nodes.

Additionally, microenvironment signals promote disease progression and induce drug

resistance. Among the accessory cells residing in these microenvironments are monocyte-

derived nurse-like cells (NLCs), T cells, and mesenchymal stromal cells that produce

factors and activate pro-survival signaling pathways to support the leukemic clone.

1.2.2 B-cell Receptor Pathway

CLL patient B-cells from infiltrated tissues show up-regulated gene signatures

characteristic of B-cell receptor (BCR) activation [29]. The BCR complex regulates

cellular processes like proliferation, differentiation, cell migration, and apoptosis by

activating kinases and triggering signaling cascades. This network is tightly regulated and

constitutively active in select B-cell malignancies, including CLL and non-Hodgkin

lymphoma (NHL), which have tonic signaling through ligand-independent autonomous

mechanisms or are antigen-dependent [30, 31]. As depicted in figure 1.6.1, the BCR is

comprised of an antigen-specific IgM surface membrane immunoglobulin that is

noncovalently bound to signal transduction Ig- / Ig- heterodimers (CD79a, CD79b).

Upon BCR engagement, cytoplasmic immunoreceptorα β tyrosine-based activation-motifs

(ITAMs) within CD79a/b are phosphorylated by the Src family kinase Lyn and tyrosine

kinase Syk [32, 33] resulting in recruitment of kinases and adaptor proteins Btk, Vav

proteins, adaptor Grb-2 proteins, and B-cell linker (BLNK) thereby forming the

signalosome [34, 35]. CD19 promotes aggregation of BCR complexes and their

redistribution to lipid rafts furthers signal amplification and reduces the threshold of activation [36]. Calcium mobilization, MAP kinase and RAS activation, activation of phospholipase Cγ (PLCγ), protein kinase C-β (PKCβ), and initiation of NF-κB cascades

are hallmarks of BCR signalosome activation and together, promote survival of malignant

CLL B-cells [29]. The identification of critical proteins and kinases involved in BCR led to the development of small molecule inhibitors against this pathway, many of which have shown promising clinical outcomes.

Ibrutinib is a first-in-class orally bioavailable, small molecule covalent inhibitor of

BTK that was approved in 2016 for the treatment of CLL as front-line therapy. By

irreversibly binding the cysteine 481 (C481) phosphorylation-binding pocket, ibrutinib

inhibits BTK enzymatic activity and subsequent phosphorylation events downstream of

BTK including phospholipase Cγ2 (PLCγ2), AKT, and extracellular signal-regulated

kinase (ERK). This inhibition abolishes BCR signaling, which has been demonstrated in

vitro and in vivo [37, 38]. BTK is a member of the tyrosine protein kinase (Tec) family and

is indispensable for proper B cell development and activation. Point mutations in the

pleckstrin homology (PH) domain of BTK leads to the absence of peripheral blood mature

B-cells and circulating Igs in patients with X-linked agammaglobulinema (XLA) [39, 40]

and low B-cell numbers in murine X-linked immunodeficiency (XID) [41, 42]. Compared

to normal B-cells, Btk expression is upregulated in CLL cells and inhibition by ibrutinib

leads to induction of apoptosis in a caspase-dependent manner as well as decreased

migration and adhesion to the tumor microenvironment [37]. Ibrutinib binds other kinases that are expressed throughout the lymphocyte lineage such as interleukin-2-inducible T-

cell kinase (ITK), B-cell lymphocyte kinase (BLK), BMX, Tec, resting lymphocyte kinase

(RLK), and Janus family of kinases – 3 (JAK3) [43, 44] leading to further immune modulation including T-cell polarization towards T helper – 1 (Th1) [45], DC maturation

and activation [46], and antagonized NK-cell anti-tumor activity [47]. Further investigations into ibrutinib binding ITK confirmed that treatment decreased serum Th2-

type cytokines like IL-10, IL-4, and IL-13 in patients on therapy for at least 28 days [45,

48]. The therapeutic on-target, off-tumor effects of ibrutinib have focused on hematopoietic lineage cells and demonstrate a strong influence on the immune response and deepening the anti-tumor response against leukemias and lymphomas.

Idelalisib is another promising oral small molecule inhibitor targeting the hyper

active BCR for treating CLL. Specifically, idelalisib binds and inhibits PI3Kδ and

promotes apoptosis ex vivo independent of mutational prognostic markers of patients.

Similarly to ibrutinib, idelalisib inhibits CLL cell homing, migration, and adhesion to

microenvironment niches [49, 50] and PI3Kδ activity is increased in CLL B-cells

compared to normal [51]. In the clinic, idelalisib has demonstrated effectiveness as a

monotherapy, in combination therapy, and is approved for patients with relapsed CLL in

combination with rituximab.

Clinical long-term follow-up data on ibrutinib and idelalisib treated patients is

currently limited with the longest follow-up approaching five years. Three-year follow-up

of ibrutinib demonstrated continued activity with durable response, and treatment-related

toxicities diminished over time in CLL [52]; however patients must remain on therapy

indefinitely. Though promising over other therapies as a single agent, complete response

measures at 23% for previously untreated patients receiving ibrutinib for CLL [52]. When

patients discontinue ibrutinib therapy, disease progresses rapidly and there is high morbidity; the median overall survival is 3.1 months after discontinuation [53]. In

combination, ibrutinib plus rituximab improves quality of life and overall response rate

reached 95% at a median follow-up of 17 months that was 20% higher than single agent

ibrutinib [54].

The promising results from clinical trials with these tyrosine kinase inhibitors has

revolutionized treatment towards a non-chemotherapy era for patients with B-cell

malignancies with strong in vitro data demonstrating the impact of inhibiting BCR

signaling, and preventing proliferation of the leukemic B-cell in response to external or

microenvironmental stimuli.

1.2.3 Tumor Microenvironment in CLL

Tumor microenvironments existing within secondary lymphoid organs provide

essential pro-survival signals to CLL cells that facilitate progression into the cell cycle,

circumvention from apoptosis, and accumulation of the tumor clone. Scattered throughout

the leukemia-infiltrated tissues are pseudofollicular proliferation centers (PCs) where

leukemic cells are generated through a highly dynamic process with a daily birth rate of 1-

2% as determined with deuterium (2H) DNA incorporation quantification by gas

chromatography/mass spectrometry (GC/MS) [55, 56]. Clinical correlations were found

between higher birth rates with progressive disease and unmutated IGHV status [57]. CLL

B-cells die rapidly in vitro without the addition of cytokines or co-culture conditions

implying that these factors are not intrinsic to the leukemic cell but rather secreted by

supportive tissues. Additionally, microenvironment signals promote disease progression and drug resistance, which could support minimal residual disease (MRD) after

8 conventional therapy. This warrants further investigation to understand the role the microenvironment has in driving leukemogenesis and to improve therapies to target the pro-leukemic niches with the goal to eliminate all malignant clones.

CLL cells recirculate between peripheral blood and secondary lymphoid tissues.

Homing to these tissues depends on chemokine gradients secreted by stromal cells in cooperation with corresponding chemokine surface receptors expressed on the CLL B- cells. CXCL12 binds CXCR4, CXCL13 binds CXCR5, and CCL19/21 binds CCR7 receptors leading to transmigration into the lymph nodes and bone marrow niches ultimately protecting the tumor cells through various over-lapping signaling cascades.

Engagement of CXCR4 with CXCL12 mediates pleiotropic effects on malignant B-cells including activation of PI3K, serine phosphorylation of signal transducer and activator of transcription 3 (STAT3), and p44/42 MAPK (Erk1/2), as well as calcium mobilization.

CXCR5 receptor stimulation by CXCL13 fosters follicular architecture of B cells within lymph nodes thereby aiding in the development of proliferation centers [58] mediating actin polymerization and p44/42 MAPK ERK1/2 signal cascades within the CLL cell. Both

CCL19 and CCL21 function as ligands for CCR7, a receptor highly expressed by CLL B- cells, and stimulates transmigration of CLL cells across the vascular endothelium [59].

While chemokines are major components to sequester leukemic cells to microenvironment niches, they also drive transformation of these tissues to support the tumor. Chemokines such as CCL3, CCL4, and CCL22 are secreted by the CLL B-cells and shape the tumor microenvironment by attracting and supporting accessory cells like monocyte-derived

NLCs, T cells, and mesenchymal stromal cells. Data show that CCL3/4 gene transcription

is upregulated in CLL cells isolated from the microenvironment [60] and it has been

demonstrated that CCL3/4 recruit regulatory T-cells (Treg) into closer proximity with B

cells in the lymph node and spleen [61].

Integrins expressed on the leukemic B-cells’ surface and selectins expressed by the stromal tissues are part of a diverse family of cell adhesion molecules that regulate cell growth and are critical to retaining CLL cells in the microenvironments while facilitating additional cytokine release and directing survival signals. Very late antigen 4 (VLA-4,

CD49d) is an essential integrin expressed by CLL cells and blocking VLA-4 inhibited

homing of the leukemia to bone marrow. Higher VLA-4 expression was associated with

poor prognosis in CLL patients [62].

Critical molecules are at the intersection of synapse formation that direct communication between CLL cells and supportive accessory cells within the microenvironment. These include tumor necrosis factor (TNF) superfamily members B- cell activation factor of the TNF family (BAFF) and a proliferation-inducing ligand

(APRIL). BAFF and APRIL are both homotrimeric type II transmembrane proteins, which can also exist as soluble molecules after cleavage by furin-like convertase [63, 64]. High affinity is demonstrated between these molecules to B-cell maturation antigen (BCMA) and transmembrane activator and calcium modulator and cycolphilin ligand interactor

(TACI). BAFF binds a third TNF receptor, BAFF-R, which has exclusive binding to BAFF

[65], demonstrates 100-fold selectivity for BAFF over BCMA [66], and primarily activates alternative NF-κB as depicted in figure 1.6.2. BCMA is normally expressed on mature B and T lymphocytes [67], TACI is detected on activated T-cells and subpopulations of B-

cells [68], and BAFF-R is expressed on B lymphocytes and a small subset of activated T- cells [65]. Ligation to these receptors activates lymphocyte differentiation, anti-apoptotic, or pro-survival signals like NF- B, c-jun N-terminal kinase (JNK), and p38, which is mediated by TNF-R-associated κfactors (TRAF) proteins that directly interact with the receptors [69-71]. BAFF-R was first identified in the A/WySnJ mouse strain harboring a mutation in Bcmd, the gene that encodes BAFF-R. Compared to parental A/J mice,

A/WySnJ mice have severe B-cell lyphopenia and fail to respond to BAFF-R stimulation

[72]. Monocytes, macrophages, dendritic cells, T cells, and granulocyte-colony stimulating factor (G-CSF)-stimulated neutrophils produce and excrete BAFF [73, 74]. In CLL, NLCs express higher levels of BAFF and APRIL than leukemic B-cells in the microenvironments promoting survival of the malignant cells by inducing anti-apoptotic Mcl-1 expression and activating NF- B [75]. Further implications of the TNF superfamily members in cancers include the B cellκ expression of BAFF-R in CLL and NHL, BCMA expression in multiple myeloma (MM) and NHL, and TACI expression on MM, NHL, and CLL, though BAFF-

R is uniformly found on all CLL B-cells and TACI only on some patients [76]. Similar expression levels of the three BAFF receptors were found comparing normal individuals to patients with diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and marginal zone lymphoma, whereas CLL and follicular lymphoma patients displayed lower expression [77]. The addition of exogenous BAFF and APRIL to CLL B-cells stimulates NF- B leading to protection of these cells from spontaneous or flavopiridol- induced apoptosisκ [78]. BAFF levels increase as tumors transform to more aggressive phenotypes of DLBCL and MCL, and serum BAFF correlates with poor median overall

survival and therapeutic response [79]. BAFF exerts its anti-apoptotic protection over CLL

B-cells in part through increasing expression of Bcl-2 family members. B cells isolated

BAFF transgenic mice express elevated levels of Bcl-2 protein along with increased numbers of mature B-cells and effector T-cells [80]. Single nucleotide polymorphisms

(SNPs) in BAFF (TNFSF13B) and BAFF-R (TNFRSF13C) genes have been documented in CLL patients and suggest an association of disease risk and need for treatment [81].

CLL cells rely on multiple pathways to evade apoptosis and proliferate including intrinsic autocrine signaling, constitutive activation of BCR, and cell-to-cell contact within tumor microenvironment niches. Many of these pathways overlap and therapeutic targeting the CLL pro-survival milieu is complex and has yet to be fully understood. CLL sensitivity to BAFF can by modulated by CD40L and IL4R stimulation [82], while release of soluble

BAFF and APRIL by stroma increases upon CD40 activation, and BAFF and APRIL act on the CLL cell to induce surface expression of CD40L [83]. Gene expression profiling

(GEP) of CLL cells from various lymphoid compartments such as peripheral blood, bone marrow, and lymph node has given rise to gene signatures specific to the tissue sample.

BCR, BAFF/APRIL, NF- B, and immune suppression related signatures were found upregulated in lymph nodesκ indicating this as a primary site of tumor growth. Indeed, peripheral blood CLL cells overexpress molecules associated with adhesion and migration confirming that a chemokine gradient is constantly attracting these cells to lymphoid niches

[84]. The interactions and exchanges in the microenvironment activate tightly-regulated signaling pathways with complex cross-talk, and elucidation of the mechanisms conferring

CLL B-cell protection in the microenvironments is critical to understanding disease

pathogenesis and developing therapeutics to overcome anti-apoptotic mechanisms and

immunosuppressive hallmarks of CLL.

1.2.4 Innate Immunosuppression by CLL B-cells

CLL patients demonstrate profound immune defects, found throughout both innate and adaptive immune responses resulting in increased susceptibility to recurrent infections and failed anti-tumor response leading to death. The suppression is both intrinsic to disease and can be acquired during therapy.

The innate immune system is the immediate response to attacks on the host without long-lasting protection against pathogens. Phagocytic cells like neutrophils, macrophages, and dendritic cells (DCs), as well as granulocytes, and natural killer (NK) cells comprise

the lymphocyte classes of innate immunity and are referred to as effector cells due to their

ability to target cancer cells for killing.

One of the leading risks associated with disease is recurrent and often severe

infections predominantly due to Staphylococcus aureus, Streptococcus pneumoniae, and

Haemophilus influenza within the respiratory tract, and Escherichia coli implicated within

the urinary tract [85]. Up to 47% of patients suffer from recurrent infections and account

for approximately 30% of deaths in CLL patients [86, 87]. Reduced levels of serum

complement proteins is documented in an estimated 40% of patients and could explain, in

part, the high risk of infection due to the impaired ability of complement C3b protein to

opsonize bacterial pathogens [88] and the deficiency in complement proteins directly limits

efficacy of CD20 monoclonal antibody therapy [89].

In addition to a dysfunctional complement system, cellular immune defects also

provide an opportunistic environment for infections. Neutrophil deficiencies include

impaired phagocytosis of bacteria and reduced chemotaxis towards C5a [90]. Notably,

monocyte count is increased by over 60% in CLL patients; however, these cells express monocyte-nonclassical CD14+ CD16++ surface markers and deregulation of genes involved in phagocytosis and inflammation characteristic of immunosuppression [91].

Stimulation of these monocyte with CLL-derived nicotinamide phosphoribosyltransferase

(NAMPT) promotes M2 macrophage polarization. M2 cells can secrete immunosuppressive cytokine IL10 and upregulate expression of ligands that inhibit T cells such as PDL1 and PDL2 [92, 93].

NK cell counts are predictive of disease progression and increased NK to CLL B-

cell ratio is correlated better prognosis including mutated IGHV status and longer time to

treatment [94, 95]. CLL patient NKs are marked with numerous defects including reduced

killing activity and cytotoxic granule release, impaired CD107 expression upon activation,

and decreased cytokine production levels, all of which are critical for properly eradicating

tumors [96]. NK cell activity from CLL patients is impaired as demonstrated by minimal

response against K562 cells [97]. However, NK activity can be restored when activated by

glyco-engineered variant antibodies, or pretreated with a cocktail of IL2/IL15 [98, 99].

Several mechanisms lead to NK cell impairment in hematological malignancies including

imbalanced expression of activating and inhibitory receptors or deregulated expression of

ligands on target cells. NKG2D and natural cytotoxicity receptors NKp30, NKp44, and

NKp46 are the main receptors that mediate NK-cell activation and killing of targets. These

receptors recognize major histocompatibility (MHC) class I proteins and viral proteins

[100, 101] and patrol through the blood and lymphatics for the pathogen-infected or transformed cells distinguishing “self” from “non-self” [102]. NK cells do not possess one dominating activation receptor but rather rely on a repertoire of activating and inhibitory receptors that are tightly balanced and cooperate to initiate effector functions. This was demonstrated by individual triggering of each activating receptor and monitoring lytic activity and calcium flux, which cytotoxicity was only induced by CD16 activation and not by NKp46, NKG2D, CD244, DNAM-1, or CD2 [103]. Additional activating receptors include NKp30, NKp44, CD16 (FcγRIIIa), and 2B4 that preferentially bind ligands with deregulated expression on “non-self” cells. Inhibitory receptors predominantly bind HLA- class I ligands, which include killer Ig-like receptors (KIRs), CD94/NKG2A heterodimer receptor, and Ig-like transcript 2 (ILT2). In order to kill target cells, NK cells redistribute the activating and inhibitory receptors into clusters through actin rearrangement and lytic granule polarization to form a lytic synapse with a target cell [104, 105]. Reactive oxygen species (ROS), may be produced by tumor related myeloid cells and contributes to cancer immunosuppression by inhibiting NK cell mediated antibody dependent cellular cytotoxicity (ADCC) against CLL B-cells [106] leading to an immunosuppressive innate phenotype in CLL.

1.2.5 Adaptive Immunosuppression by CLL B-cells

Hypogammaglobulinemia, a decrease in IgG, IgM, and/or IgA in patient serum, is observed in the majority of patients and is the main contributor to failed response against

common pathogens and recurrent infections. As discussed above, the clearance of these

pathogens relies on antigen recognition by antibody. Infiltrating malignant cells in lymphoid tissues is one initial proposed mechanism of hypogammaglobulinemia, though this is unlikely since defective humoral immunity can be detected at early stage CLL or small lymphocytic leukemia (SLL) despite low tumor load [107, 108], indicating that more factors than infiltrating leukemic cells contribute to low antibody serum levels.

CLL B-cells share multiple phenotypic markers with B10 cells, which are B regulatory (Breg) cells that secrete immunosuppressive cytokine IL10 [109]. CD40L

stimulation differentiates CD5+ CD24++ CD27+ memory B cells to B10 cells. Serum IL10

levels are elevated in CLL patients and higher IL10-competent cell frequencies were

associated with IgHV mutated patients [110]. IL10 is a key regulatory cytokine of

inflammation, autoimmunity, and mounting an immune response to pathogen [111-113].

CD5 expression on CLL cells activates Erk1/2 signaling and facilitates transient receptor

potential channel 1 (TRPC1) mediated extracellular calcium entry leading to IL10

production independent from BCR signaling [114]. Similarly, in vitro data demonstrate

that CLL cells produce significant amounts of IL10 in response to stimuli like LPS, T-cell

costimulation via CD40L or IL4, and BAFF [110, 115]. Other Breg associated molecules

are found on the surface of CLL cells such as CD200, CTLA4, and PDL1, which are

important for creating an immune synapse with CLL cells [29, 116, 117]. In addition to

IL10, CLL cells secrete TGF-β that suppresses T cell activation [118].

Marked T-cell dysfunction is found in CLL patients and alterations across T-cell

subsets are evidenced with disease progression including production of

immunosuppressive factors, poor tumor cell T cell synapse formation, and expanded T- regulatory (Treg) populations, which correlate with poor prognostic factors but declined in patients in remission [119]. Global gene expression profiling (GEP) of CLL T-cells reveal major alterations compared to healthy donor T-cells predominantly in genes associated with cell differentiation, vesicle trafficking, and cytotoxicity; these changes were dependent on direct contact of the T cells with CLL and not cytokine mediated [120].

Various markers are altered on the surface of T cells in hematologic malignancies attributing the T-cells to an “exhaustive” state both in patients and the TCL1 transgenic mouse model of CLL [121, 122]. Compared to healthy controls, T-cells from CLL patients upregulate exhaustion markers CD244, CD160, and PD1 with an expanded PD1+

BLIMP1++ population [123]. Inhibitory receptors including PD1 as well as Tim3, Lag-3, and CTLA4 come up to the surface after sustained T-cell engagement in order to contest overactive effector function [124, 125]. Therapeutic blockade of these receptors has entered the clinic in effort to reinvigorate T cell response against tumor [126].

1.2.6 Mouse models to study CLL

In addition to in vitro systems, mouse models that develop leukemias resembling

CLL provide tools to investigate the dynamic progression of CLL disease and potential therapeutics. These include the T-cell leukemia/lymphoma-1 (TCL-1) transgenic mouse model and New Zealand Black (NZB) mouse strain.

Overexpression of TCL-1 is observed in many B-cell leukemias and lymphomas and has prompted the development of mouse models with overexpression of human TCL-

1 under control of B-cell specific Ig heavy-chain (IGH) promoter and Eµ (IgH-Eµ)

enhancer [127]. TCL-1 is a 14kDa protein found in immature normal T-cells, T-cell

prolymphocytic leukemia (PLL), and in B cells TCL-1 is expressed in pre-B cells, surface

IgM-expressing naïve B-cells, mantle cells, germinal center B-cells, and is expressed at

high levels in patient tumor-derived B cell lines [128, 129]. TCL-1 overexpressing mice

have an expanded population of B220+ IgM+ CD5+ CD11b+ in the spleen, bone marrow,

and peripheral blood, and elder mice (13-18 months) share numerous features similar to human CLL [127]. The transformed malignant CD5+ B-cells from these mice express relevant therapeutic targets like Bcl-2, Mcl-1, AKT, PDK1, and DNMT1 at levels representative of CLL as well as demonstrate similar clinical disease progression and therapeutic response in vivo to human CLL [130]. TCL-1 mediates its oncogenic functions through phosphorylation of PI3K and phospholipids at the plasma membrane, leading to

Akt activation and regulation of signaling pathways involved in cell proliferation, survival, and death [131, 132].

NZB mice are classical models for autoimmune disease like systemic lupus erythematosus (SLE) but due to their late onset expansion of IgM+ B220+ CD5+ B-cell compartments can be utilized to study B-cell malignancies. NZB mice exhibit splenomegaly and lymphadenopathy [133], have raised serum IgM levels and produce antibodies against red blood cells leading to the development of autoimmune hematolytic anemia (AIHA), a common event in CLL patients [134, 135]. In contrast to other murine models of CLL, the NZB model is a de novo model. Because of its autoimmune biological

18 characteristics, NZB mice provide an important model for studying CLL-like disease within an autoimmune environment, in vivo.

1.3 Antibody Therapeutics

Monoclonal antibodies (mAbs) are an effective non-chemotherapeutic option for patients suffering from immunosuppressive diseases like CLL since most current therapies further exacerbate patients’ frail immunity.

1.3.2 Currently approved mAb for CLL

Combining monoclonal anti-CD20 antibody, rituximab, with chemotherapeutics, fludarabine and cyclophosphamide, significantly improved overall response (OR) and overall survival (OS) for front-line treatment of CLL and founded a prominent role for chemo-immunotherapy in the clinic [17]. Rituximab, ofatumumab, ocaratuzumab, and obinutuzumab target CD20, which is a hydrophobic glycosylated transmembrane protein on the surface of mature B-cells including malignant B-cells but not on stem cells, pro-B cells, or plasma cells [136, 137]. No natural ligand has been found, though CD20 knockout mice show deficient CD19-induced calcium flux and altered BCR signaling [138].

Rituximab is the first therapeutic mAb approved for B-cell malignancies. As an

IgG1 κ chimeric immunoglobulin, which contains murine light and heavy chain variable

regions (Fab) with human constant region (Fc), it exerts cytotoxicity through Fc-mediated mechanisms such as ADCC, antibody dependent cellular phagocytosis (ADCP), and complement dependent cytotoxicity (CDC) [27], summarized in figure 1.5.3. FCR therapeutic regime combines rituximab with full-dose fludarabine and cyclophosphamide and is highly effective as front-line treatment for both treatment-naïve and relapse patients.

It is the “gold standard” for patients deemed “fit” for the high toxicity profile of fludarabine; however, it is not suitable for patients with 17p deletion or TP53 mutation [1].

Ofatumumab binds an epitope of CD20 distinct from rituximab leading to extended target off-rate, and exhibits more potent CDC and ADCC in CLL in vitro models [27, 139] and is also fully humanzied. FDA approval has been granted for ofatumumab for treating

“unfit” patients, who would not be suited for fludarabine-based therapy. Combination with chlorambucil significantly improves OR rate and progression free survival (PFS) compared to chlorambucil monotherapy (82 vs 69%; 22.4 vs 13.1 months) along with improved response rate and unmanageable toxicity was not detected [140, 141]. This combination qualifies as a preferred first-line therapy for elderly, “unfit” patients.

Obinutuzumab is an Fc-engineered humanized IgG1 anti-CD20 mAbs designed for enhanced Fc-mediated effector anti-tumor activity. Compared to rituximab and chlorambucil (R-CLB), obinutuzumab and chlorambucil (G-CLB) outperforms in unfit previously untreated patients as measured by PFS (29.2 vs 15.4 months) paralleled by a significantly higher number of CR (20.7 vs 7.0%) [142, 143]. By virtue of Fc-engineering that reduces the fucose content of the Fc portion of the mAb, Fc-mediated effector activities

of obinutuzumab are enhanced including ADCC, ADP, and cytokine release and this leads

to highly effective target cell eradication by innate effector cells [144, 145].

Other promising immunotherapeutic targets include CD52, CD19, and CD37, which are all being investigated as novel mAb therapy. Alemtuzumab is a fully humanized

IgG1 targeting CD52 that has unrestricted expression outside B-cell lineage and is found on the surface of T cells, NK cells, DCs, granulocytes, monocytes, and macrophages, and clearly leads to profound immune suppression [146]. CDC is the primary mechanism by which alemtuzumab eliminates leukemic cells. Importantly, it exerts its cytotoxicity

independent of TP53 mutational status as demonstrated in vitro and by clinical activity

among the difficult-to-treat 17p deleted patient groups [147, 148]. Currently, alemtuzumab remains a therapeutic option for CLL but was withdrawn from the market by pharmaceutical company, Sanofi, and can only be accessed via a compassionate use program [1]. CD19 is highly expressed on CLL B-cells and various anti-CD19 mAb have come forward with promising pre-clinical efficacy attributed to the CD19’s co-stimulatory role with BCR signaling and ability to modulate calcium flux [149]. Also under investigation for mAb targeting is the mature B-cell specific marker, CD37. A member of the tetraspanin membrane-spanning family of proteins, CD37 expression levels are highest on peripheral blood B-cells and it is essential for B cell survival and driving adaptive immune response [150, 151]. The exciting clinical potential of targeting these novel antigens as well as modifications to antibody structure has refocused pre-clinical drug development to maximizing the mechanisms by which antibodies elicit an immune response.

1.3.3 Anti-tumor mechanisms of mAb

Antibodies are comprised of a variable region that is specific for a target antigen

and a constant region (Fc) that is specific to the immunoglobulin subclass. Most therapeutic

antibodies are IgG to facilitate binding to FcγRs and activate effector cells like NK cells or

monocytes. IgG subclasses have defining characteristics: IgG1, IgG2, IgG4 half-lives in the blood exceed 20 days, while IgG3 is approximately 7 days. Each subclass is capable of eliciting various degrees of complement and show varying degrees of binding affinity for

FcγRs, all of which are critical to consider when designing therapeutic mAb. Most approved mAbs are IgG1 due to its highest affinity to bind FcγRIIIa (CD16) and therefore are exceedingly potent at mediating NK-cell ADCC. Eradication of target by ADCC depends on the antibody’s Fab binding to the target cell that flags the tumor for effector cells to recognize and bind the Fc portion of the antibody. Cross-linking of CD16 and antibody forms a lytic synapse that activates NK cell to release perforin and granzyme containing granules to lyse the target cell in addition to producing IFN-γ [152]. Upon Fab binding and flagging a target cell, phagocytes can bind the Fc portion of the mAb and mediate ADCP of the target cell, or can recruit complement proteins to activate CDC.

Blocking a critical receptor or ligand is another way to design an effective mAb such as antibodies against CD37 or CD19 as discussed above, or by neutralizing a soluble target.

One example of this is belimumab, which targets and quenches soluble BAFF and is used to treat autoimmune B-cell diseases [153]. Antibody-drug conjugates (ADC) link a cytotoxic molecule to the Fc of an antibody to kill target cells as has been demonstrated

with anti-CD37, IMGN529, in which a maytansine-derivative DM1 is attached via a stable

SMCC linker [154].

In addition to selecting an appropriate target, properly engineering the Fc domain for sufficient immune response is critical to mAb effectiveness. Glyco-engineering is the process of altering the Fc domains glycosylation status to enhance FcγR binding on effector cells and raise anti-tumor response. Three structurally homologous Fc receptors have been identified, which are FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), in addition to the neonatal FcR (FcRn) [155]. The antibody Fc region is a homodimer of covalent inter- heavy chains bonded with a disulphide hinge region, non-covalently paired CH3 domains with distinct oligosaccharide moieties covalently attached at asparagine 297 (Asn297) within the non-paired CH2 domains. Specifically, the oligosaccharide attached to IgG1-Fc domain is a biantennary complex comprised of a mannose-fucose core structure, a bisecting

N-acetylglucosamine (GlcNAc), and a terminal galactose and sialic acid [156]. Depleting the fucose from the oligosaccharide at Asn297 enhances binding between IgG1 and

FcγRIII [157] and raises ADCC levels with altering antigen binding or CDC activity [158].

Generation of defucosylated antibodies utilize Chinese hamster ovary cells (CHO) with knockout of FUT8, which encodes a fucosyl transferase that catalyzes the transfer of fucose to GlcNAc of the Fc domain [159]. Anti-CD20 obinutuzumab is defucosylated and demonstrates superior efficacy in vitro at activating NK cells compared to rituximab [27] and was approved in 2013 by the FDA for treatment of CLL for use in combination with chlorambucil [160]. Single-agent activity of obinutuzumab has been demonstrated in one randomized phase 2 study in symptomatic, untreated CLL to assess dose response [161].

Long-term follow up of these defucosylated antibodies is warranted to cement this subtype of antibody therapy in the clinic as a potential chemotherapy-free treatment option.

1.4 Conclusion and Significance

Patients with Chronic Lymphocytic Leukemia (CLL) demonstrate profound immune suppression allowing for tumor evasion from the patient’s immune response and a significant increase in susceptibility to infectious diseases, which can be further exacerbated by current front-line therapies. NK cells are critical to the anti-tumor responses and are majorly impaired in CLL. Our goal is to define the mechanisms employed by antibody therapeutics to overcome NK cell paralysis in CLL and establish a basis of monoclonal antibody drug combinations for treating CLL and lifting NK-cell immune suppression.

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1.6 Figures

Figure 1. 1 Proximal B-cell receptor signaling pathway The B-cell antigen receptor (BCR) is composed of an immunoglobulin (red) and associated

Igα/Igβ (blue) transmembrane heterodimer with ITAM signaling motifs. Engagement with immunoglobulin initiates the signaling cascade leading to phosphorylation (yellow stars) of ITAM and aggregation. This activates the Src family kinases Lyn (blue box), Syk (green box) and formation of the signalosome including BLNK, Grb2, and Btk (green boxes) as well as CD19. Signaling enzymes including PLCγ2 (brown box) leading to activation of

PKC (brown box) and PI3K (oval). Downstream signals include activation of AKT (red box), NF-κB (brown box), and MEK/ERK 1/2 (brown box).

Figure 1. 2 BAFF/BAFF-R ligation activates alternative NF-κB Upon BAFF binding BAFF-R, TRAF3 (blue box) disassociates from NIK (green box) and

NIK phosphorylates IKKα leading to subsequent phosphorylation of p100. P100 is then

ubiquitinated and degraded by proteasomal processing to p52. P52 dimerizes with RelB

and this complex translocates to the nucleus, binds to NF-κB transcriptional elements inducing target gene expression including Bcl2 anti-apoptotic family members.

Figure 1. 3 Mechanisms of antibody mechanisms to target and clear malignant cells Four mechanisms of antibody anti-tumor response are depicted. A malignant cell, here

CLL B-cell (purple), expresses target antigen that the antibody’s variable region is

designed against. Upon binding, effectors cells that have affinity for the antibody Fc

domain are recruited including macrophages (gray bubble) and NK cells (orange oval).

Activation of macrophages induces the effector to phagocytose the CLL B-cell while

activation of NK cells induces ADCC characterized by release of perforin and granzyme

granules (orange circles) onto the CLL B-cell that lyses the target cells membrane. Fc domains can be bound by complement C1q proteins (blue diamond) and opsonizes the target cell for complement-mediated killing.

Chapter 2: Glyco-Engineered anti-BAFF-R, B-1239, Increases NK-Cell Activation and

Combines with Ibrutinib to Block Multiple Pro-Survival Signals and Eradicate CLL B-

cells

2.1 Introduction

Chronic Lymphocytic Leukemia (CLL) is an incurable lymphoid malignancy and is the most common adult leukemia. The disease is characterized by the clonal expansion and accumulation of mature CD5+ B-lymphocytes in the blood, bone marrow (BM), spleen, and lymph nodes (LN). Un-mutated IGHV status, lack of ZAP-70 methylation, and presence of high-risk genomic features including complex karyotype, del(17)(p13.1), and del(11)(q22.3) are used to risk-stratify patients into more aggressive disease prognosis [1-

5] and contribute to a highly variable clinical course [6, 7]. Deep remission is achievable combining first-line chemotherapy with anti-CD20 monoclonal antibodies [8-10]. Anti-

CD20 monoclonal antibody, rituximab, is a vital treatment for CLL patients as first line therapy, maintenance, or salvage therapy and is arguably the most effective and widely used therapeutic antibody to date. Rituximab and other antibody therapeutics engage immune effector cells like Natural Killer (NK) cells, granulocytes, monocytes, and macrophages via the antibody’s Fc domain to deplete target cells by binding FcγRs and

inducing antibody mediated cellular cytotoxicity (ADCC) or antibody dependent cellular

phagocytosis (ADCP), by binding serum complement protein C1q to activate complement

mediated cell lysis (CDC), or direct binding of the antibody’s variable region to antigen on

the target cell to induce apoptosis independent from effector cells [11, 12]. Second-

generation CD20 antibodies contain novel antigen binding sites and/or engineered Fc

domains that enhance effector cell killing of targets: ofatumumab and ocaratuzumab target a distinct epitope on CD20 that binds more tightly compared to rituximab and mediates more cytotoxic tumor killing by CDC and ADCC, while obinutuzumab binds a similar region of CD20 epitope compared to rituximab it is glyco-engineered to be devoid of

fucose resides on its Fc domain leading to increased ability to activate NK cells and mediate

ADCC [11, 13, 14]. Obinutuzumab combined with chlorambucil in one randomized phase

3 trial demonstrated superior efficacy over rituximab and chlorambucil as measured by

both depth of response and progression free survival [10]. A wide body of evidence has

demonstrated that anti-CD20 therapies substantially improved CLL patient care, however

virtually all patients relapse and become resistant to treatment as neoplastic B-cells are not dependent on CD20 for survival and CD20 shedding [15, 16]. Development of novel-

targeting therapeutic antibodies in CLL remains a priority. Designing an antibody against

a tumor surface protein with defined oncogenic properties would not only abate tumor

fitness but could potentially circumvent resistance mechanisms.

One such tumor surface protein may be the interaction of B-cell activating factor

(BAFF)/ BAFF-Receptor (BAFF-R). BAFF is a member of the tumor necrosis factor

(TNF) superfamily that supports normal B-cell development and proliferation [17, 18].

BAFF-R engagement activates pro-survival activity in B cells by exclusively binding

BAFF with high affinity [19-21] driving anti-apoptotic gene transcription of Bcl-2 family members via NF-κB inducible kinase (NIK) mediated alternative NF-κB signaling [22-24].

Importantly, BAFF is critical for migration and homing of CLL cells to microenvironments

within the bone marrow, spleen, and lymph nodes that protect tumor cells from apoptosis

[25]. These niche environments, comprised of bone marrow stromal cells (BMSCs),

support survival of the malignant clone by producing a proliferation-inducing ligand

(APRIL) and BAFF [26] and protect CLL cells from apoptosis [27]. In addition, tissue- resident CLL B-cells display aberrant B-cell receptor (BCR) signaling and thus display constitutively activated kinase activity, including phosphoinositude-3 kinase (PI3K) and

Bruton’s tyrosine kinase (Btk), further promoting tumor cell survival via activation of NF-

κB, and AKT. Small molecules that target these kinases have emerged to impede on the overactive BCR signaling cascades including ibrutinib, a first-in-class irreversible BTK inhibitor, and idelalisib that inhibits p110δ, an isoform of phosphoinositude 3-kinase

(PI3K), both of which are downstream of BCR activation [28, 29]. While both show promising activity in CLL, complete responses are infrequent and patients develop resistance to therapy [30].

A study by Enzler et al. found that crossing the Eμ-TCL-1 mouse model of CLL

[31] with stromal cell expressing -BAFF transgenic mice lead to development of CLL disease at a younger age concomitant with rapid disease progression and shorter survival time compared to Eμ-TCL-1 animals thus indicating that BAFF is a driving factor in disease progression [32]. We hypothesized that BAFF-R would be an ideal

immunotherapeutic target due to its restricted ligand BAFF, ubiquitous expression on B- cells, and critical biologic function that mediates pro-survival signals independent from

BCR stimulation and could be a promising combination therapy with BCR signaling inhibitors such as ibrutinib.

Here, we evaluated the in vitro and in vivo efficacy of fully human anti-BAFF-R

mAb, B-1239. B-1239 was produced by an engineered CHO line to be devoid of fucose

residues in the antibody constant region (Fc) and facilitates enhanced binding to the

FCγRIIIa, (CD16a) on NK cells [33]. Based upon these modifications, we hypothesized

that it would be effective not only with chemotherapy, but also combined with BTK

inhibitor ibrutinib [34], which we and others have shown can antagonize NK cell ADCC

[35-37]. In this report, we demonstrate BAFF-R as a promising antibody target

ubiquitously expressed on malignant B-cells that can be blocked with B-1239 to inhibit

BAFF/BAFF-R mediated survival via NF-κB pathways in conjunction with enhanced

ADCC via Fc- engineering, and effective combination of B-1239 with ibrutinib. Our

findings demonstrate that B-1239 is a potent ADCC and ADCP -inducing antibody that

blocks BAFF-mediated signaling along with highly effective in vivo activity as a

monotherapy and synergistic activity when combined with ibrutinib.

2.2 Materials and Methods

2.2.1 Human sample preparation and cell culture

Blood samples were obtained from normal donors or CLL patients in accordance

with the Declaration of Helsinki. All subjects provided written, informed consent under an

Ohio State University Institutional Review Board- approved protocol. All patients had

immunophenotypically defined CLL [38] and had been without prior therapy for a minimum of 30 days. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation (Ficoll-Paque Plus, GE Healthcare, Uppsala, Sweden).

Enriched CLL and normal donor samples were prepared via negative selection for B cells or NK cells with RosetteSep (STEMCELL Technologies, Vancouver, BC, Canada)

according to the manufacturer’s protocol.

Cell culture conditions for various cell types were 37°C with 5% CO2 atmospheric

conditions cultured in RPMI 1640 (Life Technologies, Grand Island, NY) and

supplemented with 10% heat-inactivated fetal bovine serum (Sigma, St. Louis, MO, USA),

2 mM L-glutamine (Invitrogen, Carlsbad, CA), and 56 U/mL penicillin with 56 μg/mL

streptomycin (Invitrogen) and this is referred to as “complete media” throughout this

report. NK cell and monocyte purity ranged from 75-90%, and purity of B cells were

greater than 90%. The OSU-CLL cell line was immortalized in our laboratory as described

[39] and cultured in complete media.

Monocytes were positively selected with CD14 microbeads from normal donor

PBMCs using MACS system (Cat# 130-050-201,Miltenyi Biotec, Cambridge, MA). To

promote differentiation into monocyte-derived macrophages (MDMs), selected monocytes

were cultured at 1x107 monocytes in 10 mLs in 10 cm2 dishes with complete media and

treated every two days with fresh media and 20 ng/ml monocyte-colony stimulating factor

(M-CSF; R&D Systems, Minneapolis, MN) for a minimum of 7 days.

2.2.2 Reagents

Therapeutic antibodies used in indicated experiments including alemtuzumab

(ALE), rituximab (RTX), ofatumumab (OFA), obinutuzumab (OBN), and trastuzumab

(TRA) were purchased from the OSU pharmacy. Novartis (East Hanover, NJ, USA) provided: B-1239, B-1239 labeled -PE, IgG1 isotype labeled -PE, B-1239 Fc-variant N297,

B-1239 labeled -Dylight633, IgG1 isotype labeled -Dylight633, and recombinant human

BAFF (PeproTech, Rocky Hill, NJ). Ibrutinib for in vivo studies was sourced from Acorn

PharmaTech, LLC (Redwood City, CA), and was confirmed to be 99% pure by Ohio State

University's Medicinal Chemistry Shared Resource (Columbus, OH) prior to use.

2.2.3 Flow cytometry and reagents

Beckman Coulter FC500 and Gallios flow cytometers were used for all flow experiments (Brea, CA, USA) and flow cytometric data were analyzed using Kaluza software (Beckman Coulter). BAFF-R expression and antibody binding assays were conducted with fluorochrome- labeled monoclonal antibodies purchased from BD

Pharmingen (San Diego, CA, USA). Mouse anti-human flow antibodies included CD19-

PE (BD Pharmingen, cat#555413, clone HIB19), CD5-FITC (ebioscience, clone L17F12),

CD56-APC (Miltenyi, order# 130-104-997, clone AF12-7H3), CD45-Pacific Blue

(Beckman Coulter, item#A74765, clone J.33), CD45-APC (BD Pharmingen, clone HI30),

BCMA-PE (Biolegend, clone19F2), TACI-APC (BD Pharmingen, clone 1A-K21-M22),

BAFF-PE (Biolegen, clone T7-241), BAFF-R-PE (BD Pharmingen, clone 11C1), CD3-

FITC (BD Pharmingen, cat#555332, clone UCHT1), and CD14-PE (Beckman Coulter, item # A07764). Antibodies generated in rat against mouse CD19 (BD Pharmingen, cat#

553786, clone 1D3), CD5-FITC (BD Pharmingen, cat# 553021, clone 53-7.3), CD45-APC

(BD Pharmingen, cat# 559864, clone 30-F11). The BD Pharmingen Anti-BAFF-R –PE

(11C1) with wild type Fc domain (anti-BAFF-R-wtFc) binding was compared to B-1239

–PE to observe B-1239 engaging cells via its variable region, BAFF-R, or by its glyco-

engineered defucosylated –Fc domain. Gating was verified with appropriately matched

isotype controls and/or with Fluorescence Minus One (FMO). To compare binding of

antibodies to cells, delta MFI (ΔMFI) was calculated: ΔMFI = (MFI experimental) – (MFI isotype); negative MFI reported as 0.0.

2.2.4 Assessment of apoptosis

Viability assays were conducted by flow cytometry using fluorescein isothiocyanate (FITC) conjugated AnnexinV and propidium iodide (PI) in 1X AnnexinV-

binding buffer (BD Biosciences). Cells were collected at indicated time points and 100 μL

of cells were stained with saturating amounts of AnnexinV (5 μL) and PI (4 μL) with 100

μL of binding buffer, covered and at room temperature for 15 minutes. After staining

incubation, 300 μL of binding buffer was added to bring the total volume to 500 μL, and

run on flow cytometers. Absolute cell counts were determined by quantitative flow

cytometry using CountBright absolute counting beads (Invitrogen). Briefly, 1 x 106 cells

were washed with cold PBS and stained with antibody against surface marker of interest

as indicated with 100 μL of staining buffer (PBS + 0.1% NaN3 sodium azide + 5% FBS).

Staining was incubated for 15 minutes in the dark on ice followed by two washes with cold

PBS. CLL B-cell viability was determined by plating 5 x 106 cells in complete media either treated with BAFF (500 ng/mL), B-1239 (10 μg/mL), or with B-1239 pretreatment for 15 minutes prior to BAFF stimulation. Cells were harvested at 24, 48, and 72 hours post-

BAFF stimulation and immediately run on flow cytometers. Percent of viable cells was reported as cells double negative for AnnexinV and PI (Ann/PI) and normalized to untreated control and > 10,000 cellular events w collected for each condition.

2.2.5 Antibody Dependent Cellular Cytotoxicity assay

Antibody Dependent Cytotoxicity Assays (ADCC) were performed using standard

4-hr 51Cr-release assay (CRA). Target cells (primary CLL B-cells or Raji cell line) were

loaded with 100 µCi of 51Cr per 1 x 106 target cells, incubated for 30 minutes at 37°C and

6 5% CO2, washed twice with warm media, resuspended to 1 x 10 cells/mL in complete

media, treated with therapeutic antibody at 10 µg/mL for 30 minutes at 37°C and 5% CO2,

washed again with media, and re-suspended to 5 x 105 cells/mL. Loaded targets at 5 x 105

were plated into 96 well plates at 100 µL per well. Co-cultured conditions were at indicated effector to target ratios (E: T) in 200 µL total volumes in 96 well plates. After 4 hours of incubation at 37°C, 5% CO2 co-culture supernatants were collected and run on a Perkin

Elmer (Waltham, MA) Wizard γ counter. Maximum release of 51Cr was determined by

lysing cells with 100 µL of 10% SDS. Spontaneous release was determined from

supernatants of cultured loaded targets only in complete media. All conditions were run in

triplicate and mean relative target cell lysis was determined. Percentage of target cell lysis

= 100 X (ER – SR)/ (MR – SR) where ER, SR, and MR are experimental, spontaneous,

and maximal release, respectively.

2.2.6 Complement Dependent Cytotoxicity assay

Primary CLL B-cells were cultured at 1 x 106 cells/mL in complete media with 30% autologous patient plasma or 30% heat-inactivated autologous patient plasma. Plasma was heat inactivated by 57°C water bath incubation for 30 minutes and was used as a negative control where complement protein activity is abolished. Cells were treated with indicated therapeutic monoclonal antibodies at 10 μg/ml and incubated at 37°C, 5% CO2. After 1

hour of incubation, cells were pelleted, washed and stained with near IR Live/Dead Stain

(Sigma) and % viability was measured by cells negative for near IR Live/Dead stain by

flow cytometry.

2.2.7 Antibody Dependent Cellular Phagocytosis assay

For Antibody Dependent Cellular Phagocytosis (ADCP) assays, 5 x 106 CLL B-

cells were used per condition per monocyte derived macrophage (MDM). CLL B-cells

were pelleted and labeled with PKH67 fluorescent dye diluted according to manufacturer’s

protocol (Sigma). Cells were stained at room temperature for 5 minutes and gently vortexed

every minute. Staining was stopped by incubating with 500 µL of FBS for 3 minutes,

washed with complete media, and resuspended to 1 x 107 cells/ml in complete media. CLL

B-cells were treated with indicated therapeutic monoclonal antibodies at 10 μg/ml for 1

hour on ice. After treatment, cells were washed twice with PBS and resuspended to 1 x 107

cells/ml with complete media. Separately, MDM effector cells were spun down to a 100

μL packed cell volume and labeled with diluted Min-Claret dye according to manufacturer’s protocol (Sigma). Staining was incubated at room temperature for 5 minutes with gentle vortex every minute. Staining reaction was stopped by incubating with

500 μL of FBS for 3 minutes at room temperature. Labeled MDMs were washed once with complete media and resuspended to 2 x 106 cells/mL and co-cultured with labeled CLL B-

cells at E: T of 1: 5 (1 x 106 MDMs, 5 x 106 CLL B-cells, total co-culture volume = 1 mL,

per condition). Co-cultures were spun down and incubated for 30 minutes at 37 ̊C. Samples

were fixed with 1% paraformaldehyde after co-culture. Analysis for phagocytosis was performed using flow cytometry. Cells that were double positive for Claret and PKH67 stains indicated phagocytosis of the CLL cells by the MDMs. The results are presented as relative phagocytosis = (% Claret-positive MDMs becoming PKH67-positive in the treatment condition) − (% Claret-positive MDMs becoming PKH67+ in the untreated control).

2.2.8 Cytokine assays

To determine activation of monocytes and macrophages, cells were enriched from fresh CLL patient blood and cultured for 24 hours. Cell-free culture supernatants were collected after 24 hours of stimulation with indicated therapeutic monoclonal plate-bound antibodies (10 μg/mL) and analyzed for TNFα levels using Quantikine Human TNFα

enzyme-linked immune-sorbent assays (ELISA) performed according to the

manufacturer’s protocol (R&D Systems, Minneapolis, MN). Assessment of plasma levels

from patients for TNFα, IFN- γ, and BAFF used ELISA (R&D Systems) per manufacturer's

directions in triplicate.

2.2.9 Single cell fluorescent microscopy

Transient transduction using Lenti-viral construct: To determine whether ibrutinib

or B-1239 altered BAFF receptor-mediated induction of NF-κB in single OSU-CLL cells,

~2.5 x 105 OSU-CLL cells were transduced with either the Lenti-viral Cignal-GFP-NF-κB

reporter construct (transcriptional response element (5’-GGGACTTTCC -3’) or Cignal

negative and positive control (pCMV promoter) constructs (~25 multiplicity of infection

(MOI)) (Qiagen, Valencia, CA) according to manufacturer’s instructions. The tandem

repeat (TRE) element has been previously reported to activate both classical and alternative

NF-κB signaling pathways by its ability to bind both p65 and p52[40] . Transduction

efficiency was determined after 48 hours (~10% by fluorescence microscopy (488 nm

excitation)). Expired cells were excluded using 7-AAD (BD Biosciences, San Jose, CA)

(561 nm excitation). After 48 hours, the cells were washed twice with PBS and re- suspended in complete RPMI medium containing 10% FBS (Atlas Biologicals, Fort

Collins, CO) and 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco) and 2 mM

6 L-glutamine (Gibco) at 1 x 10 cells/ml and incubated at 37°C and 5% CO2 for 30 minutes upon addition of ibrutinib (1 μM) or DMSO. The cells were washed twice with clear RPMI

1640 medium (Gibco) and re-suspended at ~1.0 x 105 cells/ml with clear complete RPMI

1640 in the presence of ~1.5% FBS and pretreated with B-1239 (10 μg/ml) for 15 minutes

prior to the addition of soluble BAFF (500 ng/ml) and incubated for 16 hours. After

incubation, cells were plated onto 8-well imaging plates (Thermo Fisher, Waltham, MA)

that were pretreated with 0.01% poly-L-lysine (Trevigen) followed by 7-AAD (1:100) (BD

Biosciences) addition for viability exclusion. Cells were visualized via epifluorescence

microscopy (Nikon, Eclipse Ti2) where 488 nm light excitation was employed for GFP-

NF-κB activity. Bright field and 561 nm excitation for 7-AAD was used to determine viable

OSU-CLL cells. The level of GFP-NF-κB dependent fluorescence within single OSU-CLL

cells was measured via Nikon Elements software. In order to identify transduced single

OSU-CLL cells, relative fluorescent units (RFUs) were measured among single cells

treated with the negative control GFP reporter construct under each treatment group.

Similar to a flow cytometry gate, the top 5% of RFU values among the single cell

population treated with the negative control GFP reporter construct were considered auto

fluorescence. The top RFU value of the remaining single cell population was used to

subtract auto fluorescence among single cell populations transduced with the Cignal-GFP-

NF-κB reporter construct across each treatment group. RFUs from single OSU-CLL cells

containing at least 3 orders of magnitude were considered transduced. The number of cells

transduced was normalized across treatment groups and expressed on a per cell basis. The

data are expressed as the mean Fold Change + SEM relative to approximately 100s of

untreated cells from single OSU-CLL cells. For cell surface staining experiments, PE-

B1239 and PE-species-/isotype-matched antibodies were cultured with single OSU-CLL

cells at 1.0 x 106 /ml in 1X PBS. The cells were washed once and re-suspended in clear

RPMI 1640 and visualized under 561 nm excitation via fluorescent microscopy as

described above.

2.2.9 Lysates preparation and immunoblot

Isolation of nuclear and cytoplasmic lysates was performed using NE-PER Kit

(Thermo-Scientific, Rockford, IL) to detect cytoplasmic and nuclear localization of

proteins involved in signaling cascades where increased protein levels as well as

localization indicates activation in a sensitive system. This sensitivity is important due to

the hyperactive pro-survival signals in cancer cells. Briefly, 1 x 108 enriched B-cells from

patient CLL blood was treated with or without BAFF (500 ng/mL) and 10 μg/ml of B-1239

for 16 hours. Cells were harvested and separated by cytoplasmic or nuclear lysates

according to manufacturer’s protocol. Immunoblot experiments were performed using

established methodology as previously described [11]. Equal amounts of protein from

cytosolic and nuclear fractions were separated on polyacrylamide gels and transferred onto

nitrocellulose membranes. The following antibodies (Abs) were used for detection:

p100/p52 increased levels as well as nuclear localization indicates activation of alternative

NF-κB (Millipore); actin (I-19; Santa Cruz), and LaminB (Santa-Cruz) were used as loading controls. After Ab incubations, proteins were detected with chemilluminescent substrate (Thermo Fisher Scientific, Rockford, IL) and quantified using ChemiDoc system with AlphaView software (Protein Simple, San Jose, CA).

2.2.10 Animal Studies

All animal procedures were performed in accordance of Federal and Institutional

Animal Care and Use Committee (IACUC) requirements. All mice were housed in micro

isolator cages under controlled temperature and humidity. Eμ-TCL-1 transgenic mice

(C57BL/6 background) have been previously described as a model of CLL [31, 41].

For in vivo efficacy of B-1239, TCL-1 mice were subjected to submandibular weekly bleeds and enrolled into randomized treatment groups of B-1239 (100 mg/kg) or vehicle (PBS) after blood levels reached high leukemic burden (> 60% CD45+ CD5+

CD19+). B-1239 or vehicle, sterile PBS, retro-orbital injections continued once per week

for two weeks and blood was obtained via submandibular vein 24 hours after injection.

Weekly bleeds were performed in order to track disease progression for a maximum of 100

days.

We utilized a leukemia engraftment murine model to determine in vivo survival

advantage of B-1239 and the efficacy of B-1239 combined with ibrutinib. Spleens from a

moribund, leukemic B6/TCL-1 donor with splenomegaly were harvested, pulverized, and

purified for B-lymphocytes by Fiqoll-Paque density gradient and re-suspended in sterile

phosphate-buffered saline for intravenous lateral tail vein injection of 200 μL containing 1

x 107 cells. “C.B17 scid” female mice were purchased from Taconic Farms, Inc., (Hudson,

NY). These mice are homozygous for the Prkdcscid spontaneous mutation (referred to as

SCID). The animals were randomly assigned to the following treatment groups (n = 13-15

per group): PBS injection (vehicle), B-1239 injection, ibrutinib (Acorn PharmaTech LLC)

drinking water (in 10% β-cyclodextrin, at 0.16 mg/mL, via water bottle), or combination

B-1239 injection with ibrutinib drinking water. Leukemia onset was defined when

54 peripheral blood CD45+ cells > 20% CD5+ CD19+ leukemic B-cells or if splenomegaly was determined by palpitation. Disease was monitored weekly by flow cytometric analysis of leukemia populations in the blood, spleen palpitation, white blood cell counts, and weight. Study mice were euthanized by CO2 upon reaching IACUC early removal criteria

(i.e. weight loss > 20%, labored breathing, hunched posture). Blood, spleen, and bone marrow were harvested from euthanized mice and, if possible, from mice that died, to determine if leukemia was the cause of death. Criteria for leukemic death was defined as >

10% CD5+ CD19+ cells of the CD45+ population in one of the three tissues analyzed.

2.2.11 Histopathological Analysis

Two mice from each treatment group (vehicle, B-1239, ibrutinib, B-1239 + ibrutinib) were sacrificed at week 6 post-treatment, and all mice upon death were analyzed by histopathological analysis for leukemic infiltration and gross anatomy of spleens.

Spleens and bone marrow were harvested from mice, fixed in 10% neutral buffered formalin, paraffin embedded, section at 4 µM onto glass slides, stained with hematoxylin and eosin, and reviewed by a board certified pathologist for leukemic infiltration, mitotic figures, and to confirm death was due to leukemia.

2.2.12 Statistical Considerations

As many of the measurements use samples from the same patients, mixed-effect models were used for analysis to take into consideration the dependency of these observations. Survival studies using the Eμ-TCL-1 SCID engraftment mouse model are

illustrated by Kaplan-Meier plots and analyzed using log-rank tests to compare treatment

groups. Trend analysis was used to compare the rates of leukemic cell depletion among

groups [42]. Bonferroni procedure [43] was used to control the family-wise error rate at

0.05. Statistical significance was determined using Graphpad Prism software (La Jolla,

CA). In this report, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

2.3 Results

2.3.1 Anti-BAFF-R enhanced NK-cell ADCC and cytokine production

Glyco-engineered monoclonal antibodies were previously shown to outperform non-engineered antibodies at activating FcγR immune effector functions demonstrated by in vitro head to head comparisons of anti-CD20 rituximab (RTX) versus glyco-engineered obinutuzumab (OBN) whereby OBN significantly enhances cell death, ADCC, and improves overall survival in vivo compared to RTX [44]. Similar to OBN, B-1239 is glyco- engineered and its Fc-domain is defucosylated leading us to first investigate the ability of

B-1239 to mediate ADCC. 51Chromium release assay (CRA) were performed with

antibody treated primary CLL target B-cells co-cultured with healthy donor allogeneic or

patient-matched autologous effector NK cells at different effector to target ratios (E: T). At

concentrations of 10 μg/mL, B-1239 mediated ADCC at E: T ratios of 6:1, 12:1, and 25:1, and significantly elevated cytotoxicity over RTX with both allogeneic (Fig 2.1A, p <

0.0001; n = 4 normal donor (ND), 2 CLL; 2 independent experiments) and autologous (Fig

2.1B, p < 0.05; n = 4 CLL) NK-ADCC assays. Trastuzumab (TRA), an antibody against

HER2, was used an irrelevant antibody control against the HER2-negative target cells and no ADCC was detected (Fig 2.1A,B). Likewise, no significant killing was detected in conditions with antibody alone (data not shown). Next, we performed CRA comparing

ADCC mediated by B-1239 to other clinically relevant antibody therapeutics as well as antibody efficacy at sub-therapeutic concentrations. B-1239 mediated strong ADCC using

healthy donor NK-cells against CLL B-cell targets (mean relative cytotoxicity = 85.6%),

and this was significant over ofatumumab (OFA) (40.2%), RTX (39.3%), and TRA

(21.5%) (Fig 2.1C, p < 0.001, n = 8). B-1239 was then diluted to sub-therapeutic concentrations where B-1239 mediated superior NK-ADCC over glyco-engineered CD20 antibody, OBN, at 1 μg/mL (83.5% vs. 61.3%, p < 0.05), down to concentrations of 0.0001

(Fig 2.1C, 55.4% vs. 3.8%, p < 0.001). Compared to RTX, OFA, and OBN that showed virtually no killing at concentrations of 0.0001 B-1239 mediated killing activity of 26.2%

(Fig 2.1C, p < 0.01, n = 8). Even at concentrations of 0.0001 μg/mL B-1239 significantly outperformed rituximab with respect to relative cytotoxicity at 10 μg/ml (Fig 2.1C, 41.5% versus 32.6%, p < 0.001) strongly suggesting B-1239’s highly effective ADCC at concentrations much lower than is typically used in clinical setting for mAb therapy.

Likewise, ADCC efficacy by B-1239 remained unchanged when concentrations were titrated from 10 μg/mL to 0.01 μg/mL in allogeneic (Fig 2.1C, 93.8% vs. 91.2%, n = 6) or autologous (Fig 2.1D, 54.8% vs. 54.9%, n = 4) assays. These findings strongly suggest that

B-1239 mediated potent and superior NK-ADCC activity against CLL B-cells compared to CD20 antibodies RTX, OBN, and OFA. Importantly, the enhanced ADCC activity by

B-1239 was observed at sub-therapeutic concentrations of rituximab and OBN attained in clinical trials.

The superior ADCC activity observed with B-1239 to OBN was unexpected due to

their similarly defucosylated Fc domains. Quickly after activation cytolytic CD56dim

CD16+ NK cells secrete IFN-γ, which can elevate surface expression of ICAM-1 on target

cells and promotes NK: target cell conjugation leading to increased target cell lysis [45,

46]. Therefore, we hypothesized that one mechanism of B-1239 enhanced NK cell mediated killing of targets was through increased IFN-γ release. ELISA for IFN-γ was performed on supernatants from NK cells activated by plate-bound B-1239 or OBN. IFN-

γ production was significantly detected in NK cells cultured with B-1239 compared to

OBN at 10 μg/mL (1468.0 vs. 490.3 pg/mL) where statistical significance was observed at

1.0 and 0.1 μg/mL (Fig 2.1E, p < 0.05, n = 3, 2 independent experiments) suggesting that the ability of B-1239 to induce NK IFN-γ release could partially explain for the enhanced killing of targets. Clearly, defucosylated B-1239 is a potent activator of cytolytic CLL NK- cells even compared to defucosylated OBN and we are further investigating the mechanism of enhanced NK cell activity.

2.3.2 Anti-BAFF-R activates innate immunity

Tumor cell lysis by myeloid-derived innate immune cells including monocytes and macrophages is governed by interactions between FcγRI, FcγRIIa, FcγRIIb, or FcγRIIIa with the antibody Fc domain [47]. To test the ability of B-1239 to activate cytokine production of innate myeloid cells, we evaluated whether CLL patient monocytes or

monocyte-derived-macrophages (MDMs) could release TNFα after culture with plate-

bound B-1239. As shown in figure 2.2A and B, monocytes and MDMs displayed increased

levels of TNFα cytokine release after plate-bound antibody stimulation (B-1239 vs. no antibody, monocytes: p < 0.01; MDMs: p < 0.01, n = 3) with no statistical significance between cytokine production with B-1239 and rituximab. Incubation of CLL cells with B-

1239 and autologous patient serum failed to trigger complement-mediated-lysis of CLL B- cells, using anti-CD52 alemtuzumab as a positive control [11] (Fig 2.2C, alemtuzumab

(ALE) vs. B-1239 or TRA, p < 0.01, n = 6), however B-1239 triggered macrophage antibody-dependent-cellular-phagocytosis (ADCP) of CLL cells comparable to antibody therapeutics RTX, OBN, and OFA that was significant over untreated conditions (Fig 2.2D,

p < 0.05, n = 3) and this is in agreement with previous reports demonstrating similar ADCP

levels between RTX and defucosylated CD20 antibody OBN stimulated macrophages [11].

Collectively, this provides evidence that B-1239 activated an innate immune response using CLL patient cells by stimulating cytokine production and ADCP via engagement of

FcγRs expressed on monocytes and macrophages while activation was comparable to RTX and OBN innate immune activation.

2.3.3 Specificity of anti-BAFF-R on B cells and FcγR on NK cells

Since previous work demonstrated uniform BAFF-R expression on normal B-cells and CLL cells [21, 48], we evaluated BAFF-R expression via flow cytometry of commercial anti-BAFF-R antibody with wild type Fc domain (anti-BAFF-R-wtFc) and glyco-engineered BAFF-R antibody therapeutic, B-1239. B-1239 bound to the CLL B-cell

surface in a robust manner relative to species, isotype-matched control Ab (Fig 2.3A, representative analysis from CLL patient) and mean ΔMFI demonstrating B-1239 binding over isotype was quantified (Fig 2.3B, Mean ΔMFI: B-1239 = 6.3; Isotype = 0.6, n = 22; p < 0.0001). Extending this finding, we stratified patient cytogenetic analysis with B-1239 surface expression. The data revealed increased B-1239 binding and a more variable binding pattern on CLL B-cells with unmutated IGHV than the mutated IGHV group (Fig.

3C; mean ΔMFI: unmutated = 10.2, n = 10; mutated = 3.0, n = 6; p < 0.05). However, the levels of B-1239 binding were not correlated with increased white blood cell (WBC) counts in patient peripheral blood suggesting that B-1239 binding is independent from disease aggressiveness (Fig 2.3D, ρ = 0.0096). These findings suggest that B-1239 is an appropriate treatment for all patients’ disease subtype or mutational status.

Next we assessed the binding profile of B-1239 to effector immune cells in order to determine what cells B-1239 may additionally activate with in vivo administration. B-

1239 failed to bind T-cell populations from healthy samples and this corresponded to the lack of BAFF-R surface expression as confirmed using anti-BAFF-R-wtFc (Fig 2.3E, representative shown, n = 4). We unexpectedly identified B-1239 binding to primary CLL

NK-cells, a cell type not known to express BAFF-R (Fig 2.3F) and hypothesized that this binding was by virtue of its glyco-engineered Fc domain specifically modified to exhibit higher affinity for FcγRIIIa, (CD16a). Indeed, the B-1239 variant control (N297) with mutated Fc domain that abolishes FcγR binding failed to bind to NK cells (Fig 2.3F; representative shown, B-1239 ΔMFI 2.67, N297 ΔMFI 0.00, n = 8). Concurrently, B-1239 and anti-BAFF-R-wtFc antibody failed to bind NK-92 cell line that characteristically does

not express FcγRIIIa [49] (Fig 2.3G), both antibodies failed to bind FcγRIIIa (CD16a) negative primary CLL NK-cells (Fig 2.3H) while only B-1239 bound to the CD56dim

CD16+ NK cells (Fig 2.3I), and no detectable BAFF-R mRNA was found in NK cells (data not shown) indicating that B-1239 is binding with strong affinity to CLL NK-cells via

FcγRIIIa.

2.3.4 B-1239 obstructed alternative NF-κB and decreased cell viability

Since engagement of the BAFF-R by BAFF is known to counter apoptosis via triggering pro-survival signaling in CLL B-cells [25, 50, 51], we postulated that targeting the BAFF/ BAFF-R survival axis on CLL cells with B-1239 could interfere with BAFF-R- mediated survival. In order to test this B cells enriched from CLL patient whole blood cultured with or without BAFF stimulation were harvested at indicated time points and viability was determined by Annexin V/ PI staining and flow cytometry analysis, CLL B- cell viability progressively decreased in a time dependent manner due to spontaneous apoptosis (Fig 2.4A, mean percent viable cells, normalized to untreated: 100% at 0 hours,

83.7% at 24 hours, and 71% at 48 hours, untreated vs. 48 hours p < 0.001, n = 21). A representative example of the flow data analyzed for Annexin V/ PI staining is shown in figure 2.4B. BAFF-R engagement via BAFF treatment was sufficient to protect CLL cells from apoptosis as seen by the increase in viability at 48 hours from 71% in the untreated conditions to 87.2% (Fig 2.4A, p < 0.001, n = 21). While B-1239 failed to directly kill

CLL cells, pretreatment withB-1239 blocked BAFF-dependent protection of CLL cells.

Viability of cells in B-1239 + BAFF conditions were decreased by 10% compared to BAFF

61 stimulated at 48 hours (p < 0.01). These experiments highlight the impact of BAFF-R on promoting tumor cell survival, an effect that can be blocked by anti-BAFF-R glyco- engineered mAb, B-1239, to ultimately reduce BAFF-mediated survival of CLL B-cells.

Additionally, this provides strong rationale to block the BAFF/BAFF-R molecular axis for the treatment of CLL.

Disruption of BAFF-mediated survival by B-1239 was due to blocking its interaction with BAFF-R, which next lead us to explore whether or not BAFF/BAFF-R signaling was affected. Previous evidence has demonstrated that BAFF/BAFF-R ligation results in NF-κB-dependent pro-survival signaling [23] and primarily characterized by activation of the alternative NF-κB inducible kinase (NIK) mediated NF- κB pathway, which is deemed active by p100 degrading to its subunit p52, nuclear translocation of p52/RelB, and binding of p52/RelB to NF-κB transcription factors [22]. To determine whether B-1239 blocked BAFF-R mediated alternative NK- κB signaling, we stimulated primary CLL B-cells with BAFF and observed degradation of p100 to p52 and nuclear translocation of p52 via immunoblot analysis (Fig 2.4H). Densitometric analysis of protein levels in the presence of BAFF relative to untreated conditions revealed a decrease in cytoplasmic p100 levels (0.5 fold, p < 0.01) with corresponding increased p52 levels in the cytoplasm (1.8 fold, p < 0.05) and nucleus (3.3 fold, p < 0.05). While B-1239 treatment alone did not alter p100 or p52 levels, pretreatment of CLL cells with B-1239 prior to

BAFF stimulation prevented the BAFF-R dependent p100 cytoplasmic degradation (1.0 fold compared to untreated, p < 0.01), and inhibited the BAFF-R induced increase of p52 levels in the cytoplasmic (1.0 fold compared to untreated, p < 0.05) and nuclear (0.7 fold

62 compared to untreated, p < 0.05) fractions (Fig 2.4I, J, n = 3 CLL patients, cytoplasmic; n

= 4, nuclear; 3 independent experiments). Additionally, similar trends of B-1239 blocking

BAFF-R mediated tumor cell survival were observed in viability assays from these patient samples (Fig 2.4K). Evidenced here, B-1239 directly prevents BAFF binding and anti- apoptotic signaling through the BAFF-R, and B-1239 is established as a BAFF-R antagonizing antibody.

Although our findings have revealed B-1239’s ability to antagonize the BAFF-R to ultimately disrupt pro-survival in CLL cells via inhibition of the alternative NF-κB signaling pathway at the bulk protein level, the level of alternative NF-κB signaling variability at the single cell level remained unclear. Recent findings demonstrated that evaluating an intracellular signaling response at the single cell level not only allows for increased measurement sensitivity, but also a more representative precision evaluation of the cell population since single cell populations are inherently heterogeneic [52]. In addition, single cell analysis allows for a direct comparison of equal cell numbers across treatment groups allowing for data analysis on a per cell basis. To confirm BAFF activated

NF-κB at the single cell level and to determine the effect of B-1239’s ability to block BAFF modulation of NF-κB activation, we transduced the OSU-CLL cell line, characterized in

[39], with a NF-κB driven GFP- reporter Lenti-Viral construct that contains tandem repeat elements for NF-κB transcriptional response elements allowing us to measure relative fluorescence units (RFU) of GFP expression. We confirmed B-1239 bound to BAFF-R on the OSU-CLL transduced cell line via flow cytometry (Fig 2.4C, ΔMFI = 11.7) and by single cell epifluorescence microscopy (Fig 2.4D). Single cell fluorescent microscopy

analysis of OSU-CLL cells transfected with NF-κB driven GFP- reporter construct

confirmed BAFF- induction of NF-κB signaling (mean reporter activity = 719.3 RFU) over

untreated conditions (mean reporter activity = 499.1 RFU) (Fig 2.4E, p < 0.001). Likewise,

pretreatment of OSU-CLL cells with B-1239 inhibited BAFF induced NF-κB driven GFP

expression as seen by the decreased reporter activity in B-1239 + BAFF (mean reporter

activity = 580.8 RFU) compared to BAFF alone (719.3 RFU), (Fig. 4E p < 0.001, n = 465

single cells per condition). In figure 2.4F, anti-CD20 RTX was used to prove that this effect

was mediated by specifically blocking BAFF-R and not by virtue of antibody binding to

cells as is indicated by BAFF inducing NF-κB reporter activity with RTX pretreatment.

Together, these findings confirm that BAFF engagement of the BAFF-R triggers NF-κB

activation pro-survival signaling at the single cell level in OSU-CLL cells, an effect that is

blocked by B-1239. These findings support and extend the bulk immunoblot NF-κB protein

findings from CLL patient samples and lend further rationale to B-1239 as a therapeutic

antagonizing antibody to block BAFF-R induction of NF-κB activation and, therefore, pro-

survival signaling in CLL cells.

2.3.5 Murine activity of anti-BAFF-R and survival advantage, in vivo

Given the enhanced potency of ADCC activity combined with the ability of B-1239

to block BAFF-mediated cell protection, we next tested the efficacy of glyco-engineered anti-BAFF-R in vivo using the Eμ-TCL-1 transgenic mouse model of CLL, previously described [31, 41]. Binding of B-1239 to the BAFF-R on the surface of TCL-1 splenocytes was confirmed (Fig 2.5A). We recruited Eμ-TCL-1 mice with high disease burden in the

peripheral whole blood (> 60,000 CD5+ CD19+ cells/ μL). Mice were treated once per

week for two weeks with B-1239 at the highest repeatable dose, 100 mg/kg, given to mice

with “no observable adverse effects level” (NOAEL). Absolute counts and percent of

peripheral CD5+ CD19+ leukemic B-cells was evaluated pre and post-treatment, and

monitored weekly until the animals met disease burden criteria and were removed from the

study. A representative animal’s flow analysis displays the rapid clearance of leukemic cell

burden in the peripheral blood 24 hours post treatment (Fig 2.5B). Peripheral blood B-cells

in pretreatment compared to post treatment revealed a dramatic reduction in cell counts of

circulating leukemic cells from 3.6 x 107 cells/ml at pretreatment to 2.7 x 106 cells/ml 24

hours post treatment (Fig 2.5C, B-1239, n = 3; Vehicle, n = 2), while the mean percent of

circulating leukemic lymphocytes dropped from 78.1% to 5.2% (Fig 2.5D) and low

leukemic counts were maintained even up to 80 days post treatment. After verifying that

B-1239 was not cytotoxic to TCL-1 splenocytes at 24 or 48 hours (Fig 2.5E, n = 6), we

tested if B-1239 could provide a survival advantage, and at a lower concentration than the

NOAEL. Utilizing injection concentrations of 10 mg/kg and SCID mice engrafted with

splenocytes from a disease burdened TCL-1 mouse as a model, we were able to circumvent the development of anti-human-antibody response to B-1239, which is a fully human antibody. Mice received 6 weekly injections of B-1239 at 10 mg/kg and provided a survival advantage over vehicle control with a median survival of 42 vs. 30 days (Fig 2.5F, p <

0.05; B-1239, n = 3; Vehicle, n = 4). The potent eradication of leukemic burden in vivo with B-1239 strongly supports its move into clinical trials to assess safety and preliminary

efficacy as a monotherapy for CLL as well as combination of B-1239 with small molecule

inhibitors to simultaneously target BAFF-R and overactive oncogenic pathways.

2.3.6 Conserved BAFF-mediated NF-κB activity in ibrutinib treated CLL patient cells

Our findings thus far suggested that B-1239 is effective in blocking BAFF-R

intracellular pro-survival signaling in vitro and is potent in its ability to clear circulating

leukemia cells in vivo. Therefore, we investigated potential therapeutic combinations that

would further antagonize leukemic cell survival. Ibrutinib is an irreversible BTK inhibitor

that inhibits downstream B cell receptor (BCR) pro-survival signaling, prevents

lymphocyte adhesion and homing, and impedes on microenvironment stimuli that protect

CLL cells from apoptosis [53-55]. Despite promising clinical results complete responses

are infrequent, patients inevitably relapse and develop drug resistance [30]. Our group and

others have discovered mechanisms to ibrutinib resistance that involves mutations that

allow for hyperactive levels of NF-κB. Specific mutations identified include the C481 site

on BTK, mutations in the immediate BTK downstream signaling molecule PLCγ2, or

mutations in the scaffold protein CARD11 [56-58]. Various reports postulate cross talk

signaling between BCR and BAFF-R, both of which are essential to B-cell survival,

proliferation, and activation of NF-κB. A report by Shinners et al. demonstrates that BTK regulates B-cell survival and NF-κB cascade in response to BAFF and that NF-κB and

BTK couple BAFF-R and BCR to NF-κB in mice with xid phenotype and Btk-/- knockouts.

Interestingly, BTK-/-, plcγ2-/-, and p50-/- mice had reduced levels of p100, the precursor of the alternative NF-κB transcription factor, validating the necessity of classical NF-κB

signaling to produce the critical alternative pathways signaling components. In BTK-/- mice

BAFF activated alternative NF-κB signaling was shown to be negligible due to reduced

p100 levels, though anti-CD40 stimulation rescued p100 levels and stimulated p100

processing to p52 [59].

First, B-1239 –mediated ADCC was increased relative to controls using NK cells from CLL patients on ibrutinib therapy (Fig 2.6A, p < 0.01 vs. TRA control, n = 8).

Importantly, we saw no change in BAFF serum levels from patients on ibrutinib comparing pretreatment to cycle 2 day 1 (C2D1) and cycle 3 day 1 (C3D1) (Fig 2.6B), implicating

BAFF/BAFF-R’s relevance to disease and that persistence of BAFF could in part diminish long-term effectiveness of ibrutinib providing a strong rationale for combining B-1239

with ibrutinib. In order to test whether BAFF-R induction of alternative NF-κB signaling

was BTK-dependent in primary CLL B-cells, patient samples were treated with or without

ibrutinib, followed by pre-treatment with B-1239 and BAFF stimulation. The level of

alternative NF-κB signaling was determined via immunoblot analysis. As seen in figure

2.6E, BAFF induced p100 processing and p52 nuclear translocation in ibrutinib in vitro

treated patient cells. This activation was inhibited with B-1239 pretreatment demonstrating that BAFF alternative NF-κB activity in ibrutinib treated cells is restricted through BAFF-

R. In order to extend this finding, we utilized single cell fluorescence microscopy analysis

at the single cell level in OSU-CLL cells. BAFF –mediated NF-κB signaling was not compromised in ibrutinib treated OSU-CLL cells. Mean GFP -reporter activity of BAFF

stimulated cells compared to untreated conditions was 719.2 RFU vs. 540.8 RFU in vehicle

treated conditions (p < 0.05) and 883.6 RFU vs. 388.4 RFU in ibrutinib treated conditions

(p < 0.0001) clearly demonstrating that BAFF did activate NF-κB in OSU-CLL cells.

Similarly, B-1239 blocked NF-κB reporter activity in BAFF stimulated cells as seen by the decrease from 883.6 RFU to 518.6 RFU (p < 0.001), which further supports BAFF-R as the primary mode of BAFF activation of NF-κB in ibrutinib treated or naïve cells (Fig

2.6C, n = 106 single cells / condition). In this same model ibrutinib decreased NF-κB reporter activity in anti-IgM stimulated control conditions compared to vehicle conditions

(Fig 2.6D, p < 0.05, n > 15 single cells / condition), thus validating BAFF activation of alternative NF-κB signaling in ibrutinib treated cells through the BAFF-R.

2.3.7 Anti-BAFF-R and ibrutinib combination enhances efficacy and survival advantage, in vivo

Previous work from our laboratory has shown that ibrutinib targets IL-2–inducible kinase (ITK) [60], and that ibrutinib impairs antibody -mediated NK cell anti-tumor activity [36, 37]. Since our findings thus far suggested potent ADCC activity with B-1239, we hypothesized that the increased affinity for FcγRIIIa on NK cells could overcome ibrutinib-mediated NK cell impairment, and effectively combine in CLL murine models beyond single therapy alone. To address this, SCID mice were engrafted with splenocytes from a disease burdened TCL-1 mouse and randomly assigned to vehicle, B-1239, ibrutinib, or combination B-1239 + ibrutinib treatment groups. Mice were enrolled once leukemic CD5+ CD19+ peripheral blood populations reached > 20%. Remarkably, median survival of B-1239 + ibrutinib combination treatment group was 170 days, (n = 9) and increased significantly compared to monotherapy alone (Fig 2.7A, vs. B-1239 = 115 days,

p < 0.05, n = 13; vs. ibrutinib = 79 days, p < 0.001, n = 13). Animal tissues were harvested

at six weeks post enrollment and analyzed by immunohistochemistry and histopathology

to observe leukemia infiltration in lymphoid organs (Fig 2.7B). Spleens from vehicle

treated or ibrutinib treated mice are markedly enlarged compared to B-1239 or B-1239 + ibrutinib treatment conditions. Additionally, we detected expanded periarteriolar lymphoid sheaths (PALS) by leukemia cells (architecture indicated by arrows), which were visible as white foci in enlarged spleens. Notable infiltration by leukemic cells was observed in all treatment groups, but is most extensive in vehicle and ibrutinib treatment conditions where nearly all cells are leukemic and mitotic figures are visible (indicated by *). Likewise, white blood cell (WBC) counts and percent of CD5+ CD19+ leukemic lymphocytes were

monitored weekly in the peripheral blood of all mice and further demonstrate the potency

of B-1239 to eradicate circulating leukemia as well as the efficacy when combined with

ibrutinib. In the presence of vehicle and ibrutinib treatment conditions all mice were

removed due to high leukemic burden or leukemic death by week 10. Interestingly,

ibrutinib slowed the rate of leukemia while B-1239 monotherapy completely eliminated

peripheral leukemia through week 10 (Fig 2.7C, D). Mouse spleen and bone marrow were analyzed post-mortem for percent of CD5+ CD19+ lymphocytes. Analogous to the data from weekly blood monitoring, mice receiving B-1239 + ibrutinib treatment showed less disease burden then monotherapy alone. In the bone marrow, percent of leukemic cells for combination therapy was 5.2% compared to 15.4% in B-1239 and 23.3% in ibrutinib treated mice (Fig 2.7E) while the percent of leukemic cells in the spleen for combination was 15.1% compared to 63.8% in B-1239 and 58.0% in ibrutinib treated mice (Fig 2.7F).

This study underlines B-1239’s potent leukemia depletion in vivo that promotes overall

survival in a murine CLL model, even in the absence of continuous therapy, and B-1239

remains highly effective when combined with ibrutinib.

2.4 Conclusions and Discussion

The goal of the current study was to evaluate the efficacy of a BAFF-R-specific glyco-engineered antibody, B-1239, both in vitro and in vivo alone and in the presence of

a potent tyrosine kinase inhibitor, ibrutinib. Findings from our study revealed the novelty

of targeting BAFF-R in CLL with antibody therapeutic, B-1239, and assessed the efficacy of its multiple modes of tumor cell killing. Additionally, we provided evidence that BAFF-

R pro-survival signaling is active in protecting CLL cells from apoptosis in ibrutinib treated patient samples. The defucosylated Fc-domain of B-1239 engaged NK cells to deliver potent ADCC to CLL cells, even at therapeutic concentrations exceedingly lower than what is achieved with mAbs in the clinic, and the killing activity mediated by B-1239 was

superior to that of RTX and OBN. Similarly, B-1239 enhanced IFN-γ production by CLL

NK-cells over OBN, while innate immune cell activation was demonstrated by pro-

inflammatory cytokine release and ADCP, without affecting complement activation. In

BAFF stimulated cells, B-1239 blocked NF-κB activation and reduced leukemic cell

viability. In vivo, we observed dramatic reduction of peripheral blood TCL-1 leukemia with

B-1239 monotherapy single injection that was complemented by improved survival with

2-weeks of B-1239 treatment. In vitro, ibrutinib failed to compromise BAFF-R mediated

NF-κB activity, though, blocking BAFF-R with B-1239 impaired BAFF-R mediated viability and NF-κB signaling. Combining B-1239 and ibrutinib in vivo synergistically improved survival and disease burden of leukemia engrafted SCID mice. Collectively, findings from this study provide strong justification for further clinical development of B-

1239 as a therapeutic for relapsed/refractory CLL, and provide pre-clinical evidence for the combination of B-1239 with ibrutinib.

A previous report by Parameswaran et al. showed that B-1239 is highly effective to deplete human Acute Lymphoblastic Leukemia (ALL) cells in vitro and decrease tumor burden in vivo, and demonstrated that B-1239 binds to BAFF-R epitopes that are part of the BAFF ligand-binding site [61]. The blocking ability of B-1239 on CLL cells is a novel concept for immunotherapy. Though most antibody therapeutics are designed to bind to a cell specific target, here we highlight the advantage to designing the variable region to block the target’s ability to propagate survival signals concomitant with cell specific targeting. The full extent to which blocking BAFF or BAFF-R will contribute to CLL cell eradication remains unclear. In vitro, the presence of BCMA-Fc decoy receptors reduces

CLL B-cell viability by soaking up soluble BAFF and APRIL; however BAFF-R-Fc decoy receptor was unable to reduce viability [50]. Demonstrated herein, BAFF protected CLL cells from spontaneous apoptosis, and anti-BAFF-R treatment with B-1239 was able to inhibit the BAFF-enhanced viability. This suggests that blocking interaction of BAFF with

BAFF-R, not just quenching BAFF in the serum, is essential to impede on leukemia evasion of apoptosis. Furthermore, BAFF/BAFF-R warrants more consideration as a

critical mediator of neoplastic B-cell survival. B-1239 interferes with the BAFF/BAFF-R

survival axis, thus facilitating highly effective and rapid antibody-mediated clearance of

target cells. Here, utilizing B-1239, we demonstrate the innovative approach to depleting

BAFF-R expressing cells by obstructing BAFF ligation to its pro-survival receptor in

addition to simultaneously engaging NK-effector cells to potently target and kill leukemic cells.

Beyond survival in CLL cells, BAFF has been recently reported to impair NK cell tumor killing by protecting leukemic cells [62], reinforcing the requirement to develop

BAFF/BAFF-R antagonist therapeutics for B-cell malignancies. Wild et al. found that NK

cells produce BAFF that can enhance metabolic activity of CLL B-cells and reduce their

sensitivity to antibody- mediated NK-directed lysis. Ibrutinib impairs NK cell lytic activity,

which can be explained, in part, by ibrutinib binding critical kinases involved in NK cell

activation, like ITK [36, 37, 60], though the comprehensive mechanism of ibrutinib –

impaired NK cell activity is unknown. It is tempting to propose a mechanism where

ibrutinib limits tumor killing by altering both effector cells and tumor cells. First, that

ibrutinib targets kinases critical to NK cell activation and secondly, that inhibiting BTK in

CLL B-cells shifts the tumor cells to depend on pro-survival signals that are independent from BCR activation, like BAFF-R. Supporting this hypothesis, we show that BAFF is plentiful in serum from patients on ibrutinib therapy (Figure. 6B). Multiple mutations have been associated with ibrutinib –resistance in CLL such as acquired cysteine 481 missense mutations (C481) that disrupts ibrutinib binding to this site [56, 57]. In mantle cell lymphoma (MCL), primary resistance to ibrutinib is associated with mutations that over-

activate alternative NF-κB driving tumor growth independent of BTK inactivity. Based

upon this, novel small molecules inhibitors against NIK are being studied to specifically

target this pathway in the context of ibrutinib –resistance and -sensitivity [63]. The dependence between BTK, BTK mutations, and alternative NF-κB signaling cross talk is not fully understood and warrants further testing to manage appropriate treatment options for patients. Nonetheless, we demonstrate the prevalence of BAFF in ibrutinib patients and that via BAFF-R engagement BAFF can activate pro-survival signaling, and consequently

BAFF/BAFF-R can enhance tumor cell fitness in ibrutinib patients.

In conclusion, our evidence provides insight into the potent NK- ADCC function by B-1239 and its impact on blocking BAFF/BAFF-R interaction mediated CLL survival advantage in vitro and in vivo and in combination with ibrutinib. This multi-modal anti- tumor activity of B-1239 against the target cell viability and enhanced engagement of innate immune effector cells including NK cells, monocytes, and macrophages warrant further clinical development of this reagent and similar multi-functional BAFF-R targeted therapies. This study provides strong support for future clinical development of B-1239 in

CLL and other related lymphoid malignancies that express BAFF-R and in combination with ibrutinib.

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2.6 Figures

Figure 2. 1 B-1239 enhances ADCC and NK cell activation 51Cr release assays comparing ADCC mediated by B-1239. ADCC of B-1239 vs. rituximab

(RTX) at 25, 12, 6: 1 E: T ratios against CLL B-cell targets (continued on the next page) 80

Figure 2. 1 continued with (A) allogeneic normal donor (ND) (p < 0.0001, n = 4 ND; n = 2 CLL) or (B) autologous NK cells (p < 0.05, n = 4). Trastuzumab (TRA) was used as a negative control.

E: T ratios titrated from no effectors (white bars), 6: 1 (light gray bars), 12: 1 (dark gray bars), and 25: 1 (black bars). (C, D) ADCC of B-1239 (black line, solid squares) is compared to RTX (black line, solid circles), obinutuzumab (OBN, black line, solid diamonds), ofatumumab (OFA, black line, open triangles), alemtuzumab (ALE, dotted gray line, solid triangles), and trastuzumab (TRA, dotted gray line, open circles). (C)

Allogeneic ADCC: B-1239 compared to RTX, OFA, and HER at concentrations 10, 1, 0.1,

0.01, 0.001, 0.0001 -μg/mL, p < 0.001; B-1239 compared to OBN at 1 μg/mL p < 0.001 and at 0.1, 0.01, 0.001, 0.0001 -μg/mL p < 0.0001; n = 9. (D) Autologous ADCC: B-1239 compared to TRA at concentrations 10, 1, 0.1, 0.01, 0.001 -μg/mL, p < 0.001; n = 3 separate experiments. Mixed effect model was used for data analysis. (E) ELISA data of IFN-γ

(pg/mL) release by NK cells incubated with plate bound B-1239 vs. OBN at 10, 1.0, 0.1 -

μg/mL; p < 0.05, n = 3 NKs, 2 separate experiments.

Figure 2. 2 B-1239 activates innate immunity of monocytes and macrophages ELISA data comparing TNFα (pg/mL) release by (A) monocytes or (B) monocyte-derived macrophages (MDMs) stimulated with plate-bound B-1239, OBN, OFA, RTX, ALE, or

IgG (n.s., n = 3). (C) CDC was analyzed using flow cytometry for viable CLL B-cells incubated with autologous serum and B-1239, TRA, or ALE (p < 0.01: ALE vs. B-1239, vs. TRA, n = 6). (D) ADCP was observed using flow cytometry and measuring the percent of cells double positive for Claret and PKH67 (p < 0.05: Untreated vs. RTX, vs. OBN, vs.

OFA, vs. B-1239, n = 3). Alemtuzumab = ALE; rituximab = RTX; ofatumumab = OFA; obinutuzumab = OBN.

Figure 2. 3 B-1239 engages surface molecules on CLL B and NK -cells Enriched cells from primary CLL or healthy donor blood was stained with anti -CD45

APC, -CD19 PE, -CD56 APC, -CD5 FITC or -CD3 FITC, and fluorochrome conjugated

B-1239ll Dylight633, anti-BAFF-R-wtFc, or isotype control. (A) Leukemic B-cells

(CD45+, CD19+, CD5+) were analyzed by flow cytometry to show B-1239 and anti-

BAFF-R-wtFc binding to CLL B-cells (continued on the next page)

Figure 2. 3 continued

(B-1239 average ΔMFI = 6.3, p < 0.001, n = 22; anti-BAFF-R-wtFC ΔMFI = 12.58, n =

10). Paired t-tests were used to compare the difference in binding affinity of CLL cells to

B-1239 and Isotype. (B, C, D) MFI of B-1239 vs. Isotype of CLL patient B-cells, and mean

ΔMFI was calculated. (B). Summary of all patients MFI with B-1239, isotype, and ΔMFI.

(C) Patients ΔMFI was stratified by IGHV mutational status (p < 0.05 mutated vs. unmutated; mutated, n = 6; unmutated, n = 10; unknown status, n = 6). Unpaired t-test was used to compare mean ΔMFI. (D) ΔMFI was correlated with patient white blood cell

(WBC) counts (n.s., ρ = 0.0096, n = 22). (E) Flow cytometry analysis of B-1239 or anti-

BAFF-R-wtFc binding to healthy T-cells (CD45+, CD3+, CD19-, n = 10) or (F) binding to CLL NK-cells (CD45+, CD56+, CD3-, n = 8) or (G) binding to NK-92 cell line or (H)

CLL NK-cells negative for CD16 or (I) mature CLL NK-cells positive for CD16+

CD56dim.

Figure 2. 4 BAFF-mediated survival is blocked by B-1239 pretreatment of CLL B- cells (continued on the next page)

Figure 2. 4 continued

(continued on the next page)

Figure 2. 4 continued

(A) Time course comparison of primary CLL cells’ viability at 0, 24, and 48 hours treated

with BAFF (500 ng/mL), B-1239, or cells pre-treated with B-1239 subsequently stimulated with BAFF (p < 0.001: 48 hours, BAFF vs. Untreated; p < 0.01: 24 hours, B-1239 + BAFF vs. BAFF). Inhibitory effect of B-1239 at each time point was tested by interaction contrast

(BAFF + B-1239 subtracting BAFF vs. B-1239 subtracting Untreated; 48 hours, p < 0.01).

Data were analyzed by mixed effect model and and Holm’s method was used to adjust multiplicity (n = 21). (B) Representative Ann/PI FACS analysis of a CLL patient’s B- cell

viability at 72 hours. (C, D, E) Western blot analysis of p100 and p52 protein levels in primary CLL patient B-cells. Treatments were soluble BAFF (500 ng/mL) for 16 hours

with or without B-1239 (10 μg/mL) pre-treatment. Activation of alternative NF-κB was

determined by separate cytoplasmic and nuclear protein fractions, loading controls actin

(cytoplasmic) and laminB (nuclear). Quantification of p100 or p52 protein by densitometry

analysis was normalized to loading control and Untreated conditions. (p < 0.01: nuclear

p52, B-1239 + BAFF vs. BAFF; p < 0.05: p100 and p52 cytoplasmic levels, BAFF vs.

Untreated, B-1239 + BAFF vs. BAFF; n = 5 CLL patients, 3 independent experiments).

(F) Viability of CLL patient B-cells treated with BAFF, B-1239, combination, or untreated

from matched patients used for immunoblot assays. (G) Flow cytometry histogram

demonstrating binding of B-1239 –PE vs. isotype –PE control on OSU-CLL cell line. (H)

Images from single cell epifluorescence microscopy analysis of B-1239 –PE binding to

OSU-CLL cells vs. isotype –PE control, (continued on the next page)

Figure 2. 4 continued

DIC = differential interference contrast, (n = 3 independent experiments). (I, J, K) OSU-

CLL cells were transduced with Cignal-GFP-NF-κB Lenti-viral reporter construct. GFP-

NF-κB activity (488 nm) was measured via fluorescent microscopy under 40x magnification. Analysis of GFP-NF-κB activity was performed using Nikon Elements software. Data are expressed as relative florescence unit (RFU) mean fold change + SEM relative to untreated control. Mean background fluorescence was subtracted from single

OSU-CLL cells that were transfected with the negative control construct among the same conditions. The data represent five independent experiments. A one-way ANOVA followed by Bonferroni post-hoc analysis was performed to determine statistical significance between groups. (I) Representative stitched 7 x 7 image from five independent experiments is shown. (J) Cells were treated with B-1239 with or without 16 hour BAFF stimulation (500 ng/mL) (n = 435 single cells). (K) To show this effect was specific to blocking BAFF-R, RTX was used as a non-BAFF-R antibody in BAFF stimulated cells (n

> 100 single cells).

Figure 2. 5 In vivo efficacy of B-1239 in the TCL-1 mouse model of CLL (A) Histogram FACS of B-1239 binding over isotype to CD5+ CD19+ double positive

TCL-1 mouse splenocytes. (B) Representative FACS of CD5+ CD19+ % double positive leukemia lymphocyte population in peripheral blood samples from a leukemia burdened

Eμ-TCL1 transgenic mouse. Mice were injected weekly for two weeks total with 100 mg/kg of B-1239. Blood was collected 1 day after treatment (continued on the next page)

Figure 2. 5 continued

and weekly. (C, D) Peripheral blood from Eμ-TCL1 mice were collected 24 hours before (Pre

tx) and 24 hours after (Post tx) injection of B-1239 at 100 mg/kg or PBS vehicle control and

CD5+ CD19+ double positive leukemic cells were (C) counted and (D) percent was analyzed

by flow cytometry. (E) 24 and 48 hour direct cytotoxicity FACS assay using Ann/PI viability

staining of Eμ-TCL1 mouse splenocytes treated with B-1239 (10 μg/mL). OSU2S (5 μM) was

used as a positive control, trastuzumab (TRA) was used as a negative control, and results were

normalized to untreated conditions at each time point. (n = 6 mice, 2 independent experiments).

(F) Survival analysis of SCID mice engrafted with leukemia burdened TCL-1 splenocytes from

a single donor. SCID mice were enrolled in the study when CD5+ CD19+ % lymphocytes from

peripheral blood reached > 20% within 9 weeks of engraftment. Mice received weekly

injections of B-1239 (10 mg/kg) or PBS vehicle control for 6 weeks (p = 0.0169: B-1239, n =

3, vs. Vehicle, n = 4).

Figure 2. 6 BAFF induced NF-κB signaling in CLL treated with ibrutinib (A) Allogeneic ADCC using NK cells enriched from patients on ibrutinib therapy against

Raji targets at 12: 1 E: T ratio (B-1239 vs. trastuzumab (TRA) p < 0.05; B-1239 vs. No

Antibody p < 0.01, n = 8). (B) Serum from CLL patients on ibrutinib therapy was collected at the indicated time points (Pre = Cycle 1 Day 1, prior to (continued on the next page)

Figure 2. 6 continued

ibrutinib therapy; C2D1 = Cycle 2 Day 1; C3D1 = Cycle 3 Day 1) and ELISA was used to

determine BAFF levels. (C, D) Western blot analysis of p100 and p52 protein levels in

primary CLL patient B-cell cytoplasmic-enriched protein lysates. Treatments were

ibrutinib (1 μM) or DMSO vehicle control for one hour and washed out, and followed by

with or without 16 hours of BAFF (500 ng/mL), B-1239 (10 μg/mL), or B-1239 + BAFF.

Phosphorylated BTK (pBTK) and phosphorylated PLCγ2 (pPLCγ2) were probed as positive controls for ibrutinib inhibition of BTK and downstream signals. (E, F) OSU-CLL

cells transduced with Lenti-viral Cignal- GFP-NF- κB reporter were treated with ibrutinib

(1 μM) or DMSO vehicle control followed by treatments of BAFF (500 ng/mL), B-1239

(10 μg/mL), or B-1239 + BAFF. GFP-NF-κB activity was measured via fluorescent microscopy under 40x magnification 15 hours post-treatment. A representative stitched 7 x 7 image is shown (n = 106 single cells, 3 independent experiments). Data are expressed as the mean RFU. A one-way ANOVA followed by Bonferroni post-hoc analysis was performed to determine whether statistical significance existed between groups.

Figure 2. 7 B-1239 combines effectively with ibrutinib in vivo Tumor-derived splenocytes from disease burdened TCL-1 mice were engrafted into SCID mice. (A) Kaplan-Meier survival plot receiving weekly (continued on the next page) 93

Figure 2. 7 continued

B-1239 (10 mg/kg) retro-orbital injections, vehicle control injections, ibrutinib drinking water, or combination B-1239 + ibrutinib, for up to six injections of B-1239 or vehicle, and

continuous ibrutinib drinking water provided through study. Leukemic death was confirmed

upon death by FACS analysis of blood, spleen, and bone marrow CD5+ CD19+ % of

lymphocytes (B -1239 + ibrutinib vs. ibrutinib, p < 0.0001; B -1239 + ibrutinib vs. B -1239, p

= 0.06, n = 14 / group). Adjustments for multiple comparisons for the log rank test were used

for data analysis. (B) After 6 weeks 2 animals per group were sacrificed and sent for

immunohistochemistry and histopathology analysis. Periarteriolar lymphoid sheaths (PALS)

expanded by leukemia cells are visible as white foci in the enlarged spleens. PALS areas

(arrows) and red pulp (arrow heads) are infiltrated and expanded by leukemia cells in all

specimens. Left column: photomicrographs at low power magnification to demonstrate level

of infiltration. Right column: high power magnification to demonstrate cellular detail. Mitotic

figures are indicated with an asterisk (*). Bottom row: gross anatomy images of spleens. (C)

WBC counts from blood smears and (D) CD5+ CD19+ % of circulating lymphocytes were

followed weekly for leukemia progression. (E, F) At death, spleen and bone marrow were

analyzed by FACS for % lymphocytes CD5+ CD19+.

Chapter 3: Therapeutic CD94/NKG2A Blockade Improves Natural Killer Cell

Dysfunction in Chronic Lymphocytic Leukemia

3.1 Introduction

CLL is a malignant proliferation of morphologically mature CD5+, CD19+,

CD20+, CD23+ B lymphocytes and is the most frequent leukemia diagnosed in adults, with

18,960 new cases estimated in the US in 2016 [1]. Despite significant advances in therapy,

CLL remains incurable with current standard therapies. The B cell receptor (BCR)

signaling is essential for proliferation and survival of malignant CLL B-cells [2].

Conversely, cytotoxic T-cells as well as NK-cells may play an important role in the control

of the disease. NK-cell count is predictive of disease progression in newly diagnosed CLL

[3]. NK-cells, although functionally competent, appear to exert weak cytotoxicity against

CLL cells, potentially resulting from both up-regulation of HLA-E and low expression of

NK-cell activating ligands [4].

Monalizumab (IPH2201) is a humanized monoclonal antibody (mAb) of the immunoglobulin-4 (IgG4) subtype produced by recombinant technology in Chinese

Hamster Ovary (CHO) cells. It has a non-depleting and purely blocking activity directed with high affinity and specificity against the NKG2A subunit of the inhibitory,

heterodimeric CD94/NKG2A receptor with its ligand HLA-E. CD94/NKG2A is expressed by large subsets of NK-cells as well as cytotoxic T-lymphocytes (CTLs) like activated αβ

CD8+ T-cells, γδ T-cells and NKT-cells [5]. The role of NKG2A on these cells appears to be a negative feedback loop to limit T-cell Receptor (TCR) activation. TCR engagement leads to increased expression of NKG2A on CTLs, subsequently, ligand engagement with

NKG2A limits TCR activation, and this regulatory loop is specific to the NKG2 family of inhibitory receptors [6]. Likewise, NKG2A is found highly expressed in the intestinal micro-environment indicating that the NKG2A/HLA-E negative feedback loop is utilized to regulate T-cells exposed to high antigenic load [7].

The natural ligand of CD94/NKG2A is HLA-E, a non-classical major histocompatibility complex (MHC) class I molecule, which is over-expressed by malignant cells in a variety of cancers, including in chronic lymphoid leukemia (CLL) [4]. Higher levels of HLA-E on primary B-CLL cells, as compared to normal B-cells, were also suggested. Binding of HLA-E to CD94/NKG2A induces inhibitory signals that suppress the cytokine secretion and direct cytotoxicity of effector cells against malignant cells and this mechanism plays a significant role in the immune escape of certain tumor cells [8-10].

Conversely, by suppressing the inhibitory signal transduced by NKG2A, monalizumab enhances the anti-tumor functions, including lytic activity of these immune effector cells, as shown ex vivo and in vivo in several experimental models [8-10]. Herein, we report the increase of HLA-E on CLL tumor cells and demonstrate promising pre-clinical activity of monalizumab to enhance NK-cell activity by specifically blocking the NKG2A/HLA-E

interaction in CLL patients.

3.1 Materials and Methods

3.1.1 Cells and Culture

Blood samples were obtained from normal donors (ND) or CLL patients in accordance with the Declaration of Helsinki. All subjects provided written, informed consent under an Ohio State University Institutional Review Board- approved protocol. All patients had immunophenotypically defined CLL [11] and had been without prior therapy for a minimum of 30 days. CLL samples were collected after obtaining written informed consent as part of an institutional review board (IRB) approved clinical trial whereas normal leukopaks were obtained as part of an exempt IRB approved protocol. Ficoll separation protocol was used to collect peripheral blood mononuclear cells (PBMCs) or enriched B- or NK- cells, and is as follows: whole blood is diluted with PBS (15 mL blood added to 30 mL PBS), layered over 10 mL of Ficoll-Paque PLUS (GE Healthcare Life

Sciences, Uppsala, Sweden) & centrifuged for 30 minutes at 450 xG. The leukocyte layer is then pulled, washed with RPMI media, re-pelleted & re-suspended in media for counting.

Cells are pelleted at 650 xG for 10 minutes and washed with PBS. Enriched CLL and normal donor fractions were prepared via negative selection for B cells or NK cells with

RosetteSep (STEMCELL Technologies, Vancouver, BC, Canada) according to the manufacturer’s protocol prior to ficoll separation. Cells were cultured in RPMI 1640 (Life

Technologies, Grand Island, NY) media supplemented with 10% heat-inactivated fetal

bovine serum (Sigma, St. Louis, MO, USA), 2mM L-glutamine (Invitrogen, Carlsbad,

CA), and 56 U/mL penicillin with 56 μg/mL streptomycin (Invitrogen), and cells were

maintained at 37° Celsius with 5% CO2 atmospheric conditions.

Derived from a bone marrow sample of a patient with Chronic Myelogenous

Leukemia, K562 cells are lymphoblast line (CCL-243, ATCC, Manassas VA) widely used

as a highly sensitive in vitro target for Natural Killer (NK) cell assays [12]. In order to

investigate the role of HLA-E expression on target cells in NK cell assays, K562 cells,

which are negative for HLA-E, were cloned to overexpress HLA-E and selected for low

levels of surface CD32 and called K562-E6 (cloned and provided by Innate Pharma S.A.).

3.1.2 Flow Cytometry, HLA-E & NKG2a surface expression

The following types of cells were used per reaction tube, 1x106 cells of: K562 cell

line (CCL-243, ATCC, Manassas VA), K562-E6 cell line (provided by Innate Pharma,

S.A.), tumor B-cells from CLL patients treated at The Ohio State University Medical

Center James Cancer Hospital, or whole blood from leukopacks (American Red Cross,

SER-BC, Zen-Bio). Cells for HLA-E staining were stained for 30 minutes at 4ºC with the following: Live Dead Near IR (L010119, Life Technologies), CD45 Pacific Blue (A74765,

Beckman Coulter), CD3 PC7 (6607100, Beckman Coulter), CD19 FITC (555412, BD

Bioscience), HLA-E PE (12-9953-42, eBiosciences). Cells for NKG2A staining were

stained for 30 minutes at 4ºC with the following: Live Dead Near IR (L010119, Life

Technologies), CD45 Pacific Blue (A74765, Beckman Coulter), CD3 PC7 (6607100,

Beckman Coulter), CD16 FITC (IM0814U, Beckman Coulter), CD56 APC (555518, BD

Bioscience) and CD159a PE (IM3291U, Beckman Coulter). Cells were pelleted again, washed with PBS and fixed with 2% paraformaldehyde. Fixed cells were run on Gallios flow cytometer (Beckman Coulter) and Kaluza software (Beckman Coulter) was used for analysis. To compare binding of antibodies to cells, adjusted MFI (ΔMFI) was calculated:

ΔMFI = (MFI experimental) – (MFI isotype); negative MFI reported as 0.0. Percent HLA-

E or NKG2A positive cells were determined using fluorescence minus one (FMO) controls and gating on cells shifted right of the FMO peak.

3.3.3 Real-time PCR

RNA was isolated from the respective selected NK or B cells using a RNA Easy mini kit (74106, Qiagen) and following the manufacturer’s instructions a total of 2 µg of

RNA per reaction was used to make cDNA by adding random hexamer to the RNA, incubating for 2 minutes at 70ºC and then adding the cDNA master mix containing 6 µl of

5X buffer, 3 µl of 0.1M DTT, 1.5 µl of 10 mM dNTP, 1.5 µl of M-MLV and 0.9 ul of

RNAse-Out per reaction from the TaqMan Reverse Transcription Reagent kit (N8080234,

Life Technologies). Tubes were placed on the thermocycler for 42ºC for 1-hour and 5 minutes at 95ºC before going to 4ºC. The cDNA (1 µl) was added to a qPCR master mix of 5 µl 2X TaqMan Fast Advanced universal PCR Master Mix (4444557, Life

Technologies) with 3.5 µl of nuclease-free water and 0.5 µl of TaqMan Gene Expression

Primers HLA-E (Hs03045171_m1, Invitrogen), NKG2A (Hs00970273_g1, Invitrogen),

Beta-Actin or GAPDH (4331182, Life Technologies) per reaction. The plate was then run

on for 40 cycles of 95ºC for 15 seconds & 60ºC for 1 minute after a 10-minute 95ºC pre- amplification step.

3.3.4 NK cell killing assays (Direct Cytotoxicity and ADCC)

Assessment of NK-cell killing activity was performed using standard 4-hour 51Cr

release (CR) assay. Briefly, target K562 parental or K562-E6 cells were labeled with

radioactive chromium -51 for 1 hour at 37°C, washed, and plated on 96 –well flat bottom

plates. Antibodies monalizumab, isotype, or PBS were added to NKs and then co-

cultured with chromium-51 -labeled target cells at a 12: 1 and/or 6: 1 effector to target (E:

T) ratio as indicated in each experiment. Supernatants were collected after 4 hours of co-

culture and counted on a Perkin Elmer (Waltham, MA) Wizard γ counter. Specific lysis

was determined by % lysis = 100 x (ER – SR)/(MR –SR) where ER, SR, and MR are

experimental, spontaneous, and maximum release, respectively.

3.3.5 Statistical Considerations

The difference in the percent of positive HLAE and NKG2A between groups were

compared by using Mann-Whitney tests [13]. The difference in mRNA expression of HLA-

E and NKG2A was compared by t-tests. The associations of the percent of HLAE or

NKG2A and its ∆MFI and the percentage of HLAE and NKG2A were evaluated by using

Spearman’s rank correlation coefficient (ρ). Additionally, for the experiments that treated

sample from the same subject with various conditions, mixed-effect models were used for

100 analysis to take into consideration the dependency of these observations [14]. Holm’s procedure [15] was used to control the family-wise error rate at 0.05.

3.3 Results

3.3.1 HLA-E and NKG2A landscape in CLL

Previous studies have demonstrated ubiquitous HLA-E expression across a number of primary cells and cell lines and up regulation in various cancers (reviewed in [16]). First, we compared surface HLA-E expression on freshly isolated CLL patient to normal donor

B-cells. CLL B-cells appeared to have variable expression of HLA-E, possibly as a reflection of their immune deregulation, as compared to normal donor (ND) B-cells as shown by percentage of cells positive for HLA-E (Figure 3.1A, CLL B-cells median=97.3%, n=36; ND B-cells median= 50.4%, n=12; p=0.005), or intensity of HLA-

E as determined by adjusted mean fluorescence intensity (MFI) (Figure 3.1B, CLL B-cells median=4.87, n=36; ND B-cells median=2.86 n=12; p=0.017). Similarly, CLL B-cells had similar levels of HLA-E mRNA compared to ND B-cells (Figure 3.1C, CLL HLA-E median=2.3, n=10; ND HLA-E median=3.2, n=5; p=0.24) suggesting that the regulation of this gene expression between normal and CLL patients was predominately with post- transcriptional mechanism. The increased surface expression hints at a putative role of

HLA-E in allowing CLL B-cells to evade NK-cell mediated lysis. Induction of HLA-E mRNA was also confirmed by interferon (IFN)-γ treatment of CLL B-cells (Figure 3.1D,

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no treatment adjusted mean fold change=1.4, treated mean fold change=4.13; n= 6,

p=0.032), which has been shown to occur through distinct STAT1α and GATA3 response

elements [17, 18]. HLA-E expression was also found to be unrelated to patient treatment status (Figure 3. 1E, untreated median=99.5%, n=9; treated median=97.4%, n=27; p=0.05)

or IGHV mutation status (Figure 3.1F, unmutated median=99.3%, n=14; mutated

median=97.5%, n=16; p=0.4).

Surface NKG2A expression was confirmed on CLL patient NK-cells and compared

to normal donors. No significant differences were observed between CLL and ND NK- cells with wide variability in percent positive cells (Figure 3.1G, CLL NK-cell median=53.4%, n=37; ND NK-cells median=39.3%, n=13; p=0.59) and MFI (Figure 3.1H,

CLL NK-cells median=1.12, n=36; ND NK-cells median=1.75, n=13; p=0.33). CLL patient NK-cells tended to have a similar expression of NKG2A mRNA compared to normal donor NK-cells (Figure 3.1I, ND NKG2A median=0.2, n=5; CLL NKG2A median=0.1, n=7; p=0.19). NKG2A expression was not related to patient treatment status

(Figure 3.1J, untreated median=54.3%, n=32; mutated median=42.5%, n=5; p=0.60) or

IGHV mutation status (Figure 3.1K, unmutated median=53.4%, n=23; mutated median=65.7%, n=6; p=0.10).

Moreover, no significant correlation was observed between HLA-E and NKG2A expression within matched CLL patient samples by percent cells positive for HLA-E vs.

NKG2A (Figure 3.1L, n=11, rho-ρ=0.23, p=0.48) or MFI (Figure 3.1M, n=11, rho-ρ=0.15, p=0.65). As anticipated, patients who had higher percentage of positive cells had higher

MFI for HLA-E (Figure 3.1N, n=36, ρ=0.74, p<0.0001) on CLL HLA-E positive B-cells

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and NKG2A (Figure 3.1O, n=37, ρ=0.5, p<0.002) on CLL NKG2A positive NK-cells.

Collectively, these data suggest that CLL B-cells express more HLA-E, which could

potentially contribute to and enhance immune evasion by their NK-cells.

3.3.2 Monalizumab, blocks NKG2A and enhances CLL NK-cell activity

Monalizumab is a first-in-class humanized IgG4 antibody targeting NKG2A and blocking its interaction with HLA-E thus preventing the propagation of inhibitory signals that is utilized by certain cancer cells as immune escape mechanism. In order to determine the role of tumor cell’s expressing HLA-E on NK-cell function, a HLA-E overexpressing

K562 cell line (K562-E6) was utilized (Figure 3.2A, Adjusted MFI K562-E6 vs.

K562=32.81) to allow serial comparison of CLL patient NK-cell function with blockade of NKG2A. Additionally, CLL tumor cells have variable numerous other immune checkpoint inhibitors in addition to HLA-E expression in varied amounts that would prevent dissecting out the influence of this ligand receptor interaction. CLL patient NK- cells showed diminished direct cytotoxicity against K562-E6 cell line as compared to the parental K562 cell line that lacks HLA-E expression (Figure 3.2B, mean % relative cytotoxicity: K562 vs. K562-E6 at 25:1=63% vs. 43.3%, 12:1=54.9% vs. 32.9%;

6:1=43.5% vs. 21.7%; n=15, p=0.03, <0.01, <0.01 respectively). Similar effect was also observed when utilizing normal donor (ND) NK-cells confirming the impact of overexpression of HLA-E on diminishing NK-cell mediated direct cytotoxicity (Figure

3.2C, mean % relative cytotoxicity: K562 vs. K562-E6 25:1=60.6% vs. 32.5%,

12:1=48.1% vs. 26.6%; 6:1=36.7% vs. 18.8%. n=8, p=<0.01, <0.01, 0.03 respectively).

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The addition of monalizumab significantly increased CLL NK-cell mediated direct cytotoxicity against HLA-E expressing targets (K562-E6) (Figure 3.2D, monalizumab vs. isotype at 25:1=54.0% vs. 46.3%, n=12, p=0.04; at 12:1=43.3% vs. 35.1%, n=14, p=0.02; at 6:1=31.2% vs. 23.2%. n=12, p=0.05). This effect was specific to the blockade of

NKG2A/HLA-E interaction since monalizumab was unable to enhance direct cytotoxicity in non-HLA-E expressing target cells (Figure 3.2E, mean % relative cytotoxicity of monalizumab vs. isotype at 25:1= 62.5% vs. 69.0%, n=12, p=0.26; 12:1=48.1% vs. 58.4%, n=13, p=0.1; at 6:1=37.2% vs. 46.8%. n = 12, p=0.2). To further establish the specificity of monalizumab, Fc-gamma receptor blocking antibodies were utilized in direct cytotoxicity experiments with Fc-blocking-treated K562-E6 targets cultured with CLL

NK-cells (Figure 3.2F, CLL NK-cells, mean % relative cytotoxicity of monalizumab - Fc- block vs. + Fc-block at E:T 25:1=54% vs. 55.1%, n=12, p=0.6, at 12:1=43.3% vs. 46.1%, n=14, p=0.8, at 6:1=31.2% vs. 33.2%, n=12, p=0.87). Similarly, mixed effect interaction tests proved no interaction of adding Fc-blocker on monalizumab or isotype on CLL NK- cells relative cytotoxicity when cultured with K562 targets (Figure 3.2G, CLL NK-cells,

mean % relative cytotoxicity of monalizumab - Fc-block vs. + Fc-block at 25:1=62.5% vs.

69.8%, n=12, p=0.09, at 12:1=48.1% vs. 56.3%, n=13, p=0.19, at 6:1=37.2% vs. 45.2%, n=12, p=0.17). We further demonstrate that monalizumab does not impair NK-cell mediated antibody dependent cellular cytotoxicity (ADCC). CLL B-cells treated with obinutuzumab or trastuzumab control and ND NK-cells treated with monalizumab or isotype at E: T ratio of 25:1 showed similar mean % relative cytotoxicity as compared to isotype (25% vs. 24.1%, respectively, n=15, p=0.83, Figure 3.2H). Similar effect was also

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observed when autologous conditions were utilized where B-cells and NK-cells were derived from the same CLL patient (mean % relative cytotoxicity of monalizumab vs. isotype with obinutuzumab=47.2% vs. 47%, n=3, p=0.99, Figure 3.2I). These experiments confirm our hypothesis that the impact of monalizumab in enhancing CLL NK-cell mediated direct cytotoxicity is not mediated through engagement with Fc-gamma receptors on B-cells. Monalizumab is shown here to be specific in blocking NKG2A and enhancing

CLL NK-cell lytic activity against HLA-E expressing targets without impacting ADCC.

3.4 Conclusion and Discussion

CLL NK-cells exhibit profound immune deregulation and mediate weak killing of tumor targets [19-21]. In an effort to elucidate the role of NKG2A/HLA-E in CLL immune evasion from NK-cell mediated killing, we performed surface expression and transcript analysis on CLL patient samples. Various in vitro functional assays using CLL patient NK- cells were carried out to demonstrate the deregulated immune effector activity mediated by

NKG2A/HLA-E.

We describe the variable expression of both HLA-E on CLL B-cells and NKG2A on NK-cells, and demonstrate no correlation of their surface expression to either treatment status or IGHV mutational status. In agreement with previous reports by Veiullen et al.

[22] HLA-E was higher on CLL compared to normal B-cells, but again CLL patients showed variable expression. Additionally, no correlation was found between both HLA-E

105

and NKG2A expression with treatment or IGHV mutational status implying that immune

evasion via NKG2A/HLA-E deactivating NK-cells does not discriminate subsets of CLL

patients. Furthermore, we show for the first time that blocking NKG2A/HLA-E interaction was sufficient to restore CLL NK-cells’ ability to lyse HLA-E expressing target cells.

The primary role of HLA-E, a non-classical HLA-class I molecule, is to identify

“self” cells and tissue to surveying NK-cells, protecting the cell expressing HLA-E from

NK-cell killing [23]. As compared to classical HLA molecules like HLA –A, -B, and -C that have restricted expression to specific cells and tissues, HLA-E is expressed on all human cells and tissues with significant expression on endothelial and immune cells and the highest mRNA expression levels reported in resting T-cells [24]. HLA-E is overexpressed in various cancers and overexpressed in CLL as others have demonstrated and we have shown within this report [22]. We found no correlation between prior treatment status or IGHV mutation change versus germ-line with expression of HLA-E or

NKG2A implying that regardless of a patient’s treatment history or disease stage, NKG2A is actively involved in interacting with HLA-E and deactivating NK-cell lytic activity.

Additionally, we showed that IFN-γ induced expression of HLA-E transcript in 6 of 7 patients tested. IFN-γ is reported to be abundant in CLL patient serum [25] and may potentially limit CLL B-cell apoptosis in vitro through both autocrine and paracrine pathways. Indeed, IFN-γ expression is increased in CLL patients with advanced disease, according to Rai staging, and overexpression has been linked with higher lymphocyte count as well as total tumor mass score [26, 27]. Here we add that IFN-γ can up regulate HLA-E

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transcript in CLL and demonstrates that IFN-γ is more tumorigenic than previously reported and acts through multiple mechanisms to protect CLL B-cells.

Human NKG2A expresses two ITIMs, which become phosphorylated upon

NKG2A binding with HLA-E. The phosphorylated ITIMs recruit and activate phosphatase

SHP-1, which suppress the signals generated from ITAM containing NK-cell activating

receptors [28]. Functional studies have showed that HLA-E binding NKG2A interferes

with CD16 association with spleen tyrosine kinase (SYK) and inhibits activation of SYK

as well as extracellular regulated kinases (ERK) therefore decreasing NK-cell activation,

direct killing, and ADCC [29]. Maintenance of inhibitory receptor expression is critical to

prevent self-destruction and by overexpressing NKG2A, the cancer cell can protect itself

from NK-cell lytic activity. Using NKL and NK92 cells lines, it has been shown that IFN-

α can decrease NKG2A surface expression leading to increased lysis of MICA+ targets

and by this same measure, IFN-γ increased surface expression of NKG2A and thus reduced

MICA+ target killing [30]. Here we demonstrate that this mechanism of evading NK-cell

detection and killing is active in CLL patients and that the NKG2A/HLA-E interaction can be blocked leading to increased lytic activity of CLL NK-cells without compromising NK-

cell mediated ADCC. Furthermore, high IFN-γ production by CLL cells may explain our

observed increased surface expression though transcripts between normal and CLL are

comparable.

In acute myeloid leukemia (AML) where HLA-E is expressed by all blasts,

blocking NKG2A restores lysis against AML blasts [31]. In contrast to this study, which

utilized an IgG2b anti-NKG2A mAb, monalizumab is of IgG4 subtype and thus would not

107 activate complement or innate immune effector functions; it would act purely as a blocking antibody [32] and we postulate would mediate the same effects on restoring NK-cell mediated lysis against AML. CLL NK-cells, which exert weak lytic activity against HLA-

E targets, have improved function by blocking NKG2A with monalizumab. The studies described above have specifically addressed the efficacy of blocking NKG2A on CLL NK- cells, however, activated αβ CD8+ T cells, γδ-T cells and NKT cells also express NKG2A

[5, 33] and the efficacy of blocking NKG2A on these cells remains to be studied. Based upon the data with monalizumab restoration of NK-cell direct cytotoxic function, clinical trials with this agent as monotherapy or combination with other immune agents seems warranted.

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3.5 References

1. Siegel, R.L., K.D. Miller, and A. Jemal, Cancer statistics, 2016. CA Cancer J Clin, 2016.

2. Awan, F.T. and J.C. Byrd, New strategies in chronic lymphocytic leukemia: Shifting treatment paradigms. Clin Cancer Res, 2014.

3. Palmer, S., et al., Prognostic importance of t and nk-cells in a consecutive series of newly diagnosed patients with chronic lymphocytic leukaemia. Br J Haematol, 2008.

4. Veuillen, C., et al., Primary b-cll resistance to nk cell cytotoxicity can be overcome in vitro and in vivo by priming nk cells and monoclonal antibody therapy. J Clin Immunol, 2012.

5. Arlettaz, L., et al., Activating cd94:Nkg2c and inhibitory cd94:Nkg2a receptors are expressed by distinct subsets of committed cd8+ tcr αβ lymphocytes. European Journal of Immunology, 2004.

6. Jabri, B., et al., Tcr specificity dictates cd94/nkg2a expression by human ctl. Immunity, 2002.

7. Jabri*, et al., Selective expansion of intraepithelial lymphocytes expressing the hla-e–specific natural killer receptor cd94 in celiac disease. Gastroenterology.

8. Braud, V.M., et al., Hla-e binds to natural killer cell receptors cd94/nkg2a, b and c. Nature, 1998.

9. Borrego, F., et al., Recognition of human histocompatibility leukocyte antigen (hla)-e complexed with hla class i signal sequence-derived peptides by cd94/nkg2 confers protection from natural killer cell-mediated lysis. J Exp Med, 1998.

10. Lee, N., et al., Hla-e is a major ligand for the natural killer inhibitory receptor cd94/nkg2a. Proc Natl Acad Sci U S A, 1998.

11. Hallek, M., et al., Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: A report from the international workshop on chronic

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lymphocytic leukemia updating the national cancer institute–working group 1996 guidelines. Blood, 2008.

12. Pross, H.F. and M.G. Baines, Spontaneous human lymphocyte-mediated cytotoxicity againts tumour target cells. I. The effect of malignant disease. Int J Cancer, 1976.

13. Mann, H.B. and D.R. Whitney, On a test of whether one of two random variables is stochastically larger than the other. 1947.

14. Verbeke, G. and G. Molenberghs, Linear mixed models for longitudinal data. 1 ed. 2000: Springer-Verlag New York.

15. Hsu, J., Multiple comparisons: Theory and methods. 1996: CRC Press.

16. Wieten, L., et al., Clinical and immunological significance of hla-e in stem cell transplantation and cancer. Tissue Antigens, 2014.

17. Gustafson, K.S. and G.D. Ginder, Interferon-γ induction of the human leukocyte antigen-e gene is mediated through binding of a complex containing stat1α to a distinct interferon-γ-responsive element. Journal of Biological Chemistry, 1996.

18. Marusina, A.I., et al., Gata-3 is an important transcription factor for regulating human nkg2a gene expression. The Journal of Immunology, 2005.

19. Ziegler, H.-W., N.E. Kay, and J.M. Zarling, Deficiency of natural killer cell activity in patients with chronic lymphocytic leukemia. International Journal of Cancer, 1981.

20. Kay, N.E. and J.M. Zarling, Impaired natural killer activity in patients with chronic lymphocytic leukemia is associated with a deficiency of azurophilic cytoplasmic granules in putative nk cells. Blood, 1984.

21. Jaglowski, S.M., et al., The clinical application of monoclonal antibodies in chronic lymphocytic leukemia. Blood, 2010.

22. Veuillen, C., et al., Primary b-cll resistance to nk cell cytotoxicity can be overcome in vitro and in vivo by priming nk cells and monoclonal antibody therapy. Journal of Clinical Immunology, 2012.

23. Rodgers, J.R. and R.G. Cook, Mhc class ib molecules bridge innate and acquired immunity. Nat Rev Immunol, 2005.

24. Koller, B.H., et al., Hla-e. A novel hla class i gene expressed in resting t lymphocytes. The Journal of Immunology, 1988.

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25. Buschle, M., et al., Interferon gamma inhibits apoptotic cell death in b cell chronic lymphocytic leukemia. The Journal of Experimental Medicine, 1993.

26. Podhorecka, M., A. Dmoszynska, and J. Rolinski, Intracellular ifn-γ expression by cd3+/cd8+ cell subset in b-cll patients correlates with stage of the disease. European Journal of Haematology, 2004.

27. Gallego, A., et al., Production of intracellular il-2, tnf-α, and ifn-γ by t cells in b- cll. Cytometry Part B: Clinical Cytometry, 2003.

28. Carretero, M., et al., Specific engagement of the cd94/nkg2-a killer inhibitory receptor by the hla-e class ib molecule induces shp-1 phosphatase recruitment to tyrosine-phosphorylated nkg2-a: Evidence for receptor function in heterologous transfectants. European Journal of Immunology, 1998.

29. Palmieri, G., et al., Cd94/nkg2-a inhibitory complex blocks cd16-triggered syk and extracellular regulated kinase activation, leading to cytotoxic function of human nk cells. The Journal of Immunology, 1999.

30. Zhang, C., et al., Opposing effect of ifnγ and ifnα on expression of nkg2 receptors: Negative regulation of ifnγ on nk cells. International Immunopharmacology, 2005.

31. Nguyen, S., et al., Nk-cell reconstitution after haploidentical hematopoietic stemcell transplantations: Immaturity of nk cells and inhibitory effect of nkg2a override gvl effect. Blood, 2005.

32. Irani, V., et al., Molecular properties of human igg subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases. Molecular Immunology, 2015.

33. Gunturi, A., et al., The role of tcr stimulation and tgf-β in controlling the expression of cd94/nkg2a receptors on cd8 t cells. European Journal of Immunology, 2005.

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3.6 Figures

Figure 3. 1 HLA-E and NKG2A landscape in CLL (continued on the next page)

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Figure 3. 1 continued

(continued on the next page)

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Figure 3. 1 continued

B-cells or normal donor (ND) B-cells were stained with anti-HLA-E or matched isotype

control antibodies. Samples were stained with and gated on live cells, CD45+, CD3-,

CD19+, and CD5+ cells for CLL or CD5- for ND B-cells and analyzed by FACS. Fig 3.1A

shows the increased surface expression of HLA-E on CLL B-cells as compared to ND B-

cells (median 97.7% vs 50.4%, n = 36 and 12 respectively, *p = 0.005, adjusted to

fluorescence minus one (FMO)). (B) Demonstrates increased surface expression intensity of HLA-E as shown by change in the mean fluorescence intensity when compared to FMO

(adjusted MFI) in CLL B-cells and ND B-cells (continued on the next page)

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Figure 3. 1 continued

(median 4.8 vs 2.8, n = 36 and 12 respectively, *p = 0.01). RT-PCR was used to compare transcript levels of HLA-E in B-cells and NKG2A in NK-cells. Fig 3.1C shows CLL B- cells HLA-E expression (median = 2.29, n = 10) as compared to ND B-cell HLA-E expression (median = 3.15, n = 5; p = 0.24), both normalized as fold change to GAPDH.

Fig 3.1D shows the increased surface expression of HLA-E mRNA in CLL B-cells after

24-hr interferon-γ treatment (untreated mean = 1.44, IFN-γ treated mean = 4.13; n = 6, *p

= 0.03, both normalized as fold change to GAPDH. Fig 3.1E depicts HLA-E expression on

CLL B-cells with respect to treatment status (median 99.5% vs 97.4%, p = 0.05, n = 9 and n = 27, for previously clinically treated and untreated patients, respectively) and Fig 3.1F depicts HLAE expression on CLL B-cells with respect to IGHV mutational status (median

97.5% vs 99.3%, p = 0.4, n = 16 and n = 14, for IGHV mutated and unmutated patients, respectively). CLL NK-cells or ND NK-cells were stained with antiNKG2A or matched isotype control antibodies. Samples were stained with and gated on live cells, CD45+,

CD3-, CD56+/CD16+. Fig 3.1G shows the surface expression of NKG2A on CLL NK- cells as compared to ND NK-cells (median 53.4% vs 39.2%, n = 37 and n = 13 respectively, p = 0.5). Fig 3.1H denotes surface expression intensity of NKG2A as demonstrated by change in the MFI in CLL NK-cells and ND NK-cells (median 1.1 vs 1.7, n = 37 and 13 respectively, p = 0.3). Fig 3.1I depicts CLL NK-cell NKG2A mRNA expression (median

= 0.1, n = 7; p = 0.19) as compared to ND NK-cell NKG2A expression (median = 0.2, n =

5); both normalized as fold change to GAPDH). Fig 3.1J depicts NKG2A expression on

CLL NK-cells with respect to treatment status (continued on the next page)

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Figure 3. 1 continued

(median 42.4% vs 54.2%, p = 0.6, n = 5 and n = 32, for previously treated and untreated

patients, respectively) and Fig 3.1K depicts NKG2A expression on CLL NK-cells with respect to IGHV mutational status (median 65.7% vs 53.4%, p = 0.1, n = 6 and n = 23, for

IGHV mutated and unmutated patients, respectively). B-cells and NK-cells from the same patient were analyzed for HLA-E and NKG2A expression respectively. Fig 3.1L compares the surface expression and Fig 3.1M compares the MFI. No correlation was found between expression of HLA-E and NKG2A (ρ = 0.23, p = 0.4, n = 11) and MFI (ρ = 0.15, p = 0.6, n = 11), respectively. Significant correlation was observed between HLA-E expression and

MFI (ρ = 0.74, p<0.01, n = 36) on matched CLL B-cells (Fig 3.1N) and NKG2A expression and MFI (ρ = 0.5, p = 0.002, n = 37) on matched CLL NK-cells (Fig 3.1O).

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Figure 3. 2 Monalizumab blocks NKG2A and enhances CLL NK-cell mediated cytotoxicity (continued on the next page)

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Figure 3. 2 continued

(A) Overexpression of surface HLA-E on K562-E6 compared to K562 parental line. FACs analysis of anti-HLA-E vs isotype. Adjusted MFI = 32.81. (continued on the next page)

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Figure 3. 2 continued

(B) Direct cytotoxicity CR assay with CLL NK-cells and K562 or K562-E targets. Mean

% relative cytotoxicity: K562 vs. K562-E6 25:1 = 63% vs. 43.3%, 12:1 = 54.9% vs. 32.9%;

6:1 = 43.5% vs. 21.7%. n = 15, *p = 0.03, <0.01, <0.01 respectively. (C) Direct cytotoxicity

CR assay with ND NK-cells and K562 or K562-E targets. Mean % relative cytotoxicity:

K562 vs. K562-E6 25:1 = 60.6% vs. 32.5%, 12:1 = 48.1% vs. 26.6%; 6:1 = 36.7% vs.

18.8%. n = 8, *p = <0.01, <0.01, 0.03 respectively. (D) Direct cytotoxicity CR assay with

CLL NK-cells against K562-E6 targets. Mean % relative cytotoxicity: monalizumab vs. isotype at 25:1 = 54.0% vs. 46.3%, n = 12, *p = 0.04; at 12:1 = 43.3% vs. 35.1%, n = 14,

*p = 0.02; at 6:1 = 31.2% vs. 23.2%. n = 12, *p = 0.05. (E) Direct cytotoxicity CR assay with CLL NK cells against K562 targets. Mean % relative cytotoxicity monalizumab vs. isotype at 25:1 = 62.5% vs. 69.0%, n = 12, p = 0.26; 12:1 = 48.1% vs. 58.4%, n = 13, p =

0.1; at 6:1 = 37.2% vs. 46.8%. n = 12, p = 0.2. (F) Direct cytotoxicity CR assay with CLL

NK-cells treated with monalizumab or isotype and co-cultured with K562-E6 targets treated with or without Fc-block. Mean % relative cytotoxicity of monalizumab - Fc-block vs. + Fc-block at 25:1 = 54% vs. 55.1%, n = 12, p = 0.6, at 12:1 = 43.3% vs. 46.1%, n =

14, p = 0.8, at 6:1 = 31.2% vs. 33.2%, n = 12, p = 0.87. (G) Direct cytotoxicity CRA with

CLL NK-cells treated with monalizumab or isotype and co-cultured with K562 targets treated with or without Fc-block at E:T ratio of 25:1, 12:1 or 6:1. Mean % relative cytotoxicity of monalizumab - Fcblock vs. + Fc-block at 25:1 = 62.5% vs. 69.8%, n = 12, p = 0.09, at 12:1 = 48.1% vs. 56.3%, n = 13, p = 0.19, at 6:1 = 37.2% vs. 45.2%, n = 12, p

= 0.17. (H) Antibody dependent cell meditated cytotoxicity (continued on the next page)

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Figure 3. 2 continued

(ADCC) CR assay with CLL B cells treated with obinutuzumab or trastuzumab control and

ND NK-cells treated with monalizumab or isotype at E:T ratio of 25:1. Mean % relative cytotoxicity of monalizumab vs. isotype with obinutuzumab = 25% vs. 24.1%, n = 15, p =

0.83. (I) Antibody dependent cell meditated cytotoxicity (ADCC) CR assay with CLL B-

cells treated with obinutuzumab or transtuzumab control and autologous NK-cells treated with monalizumab or isotype at E:T ratio of 25:1. Mean % relative cytotoxicity of monalizumab vs. isotype with obinutuzumab = 47.2% vs. 47%, n = 3, p = 0.99. (J) Effect of monalizumab was not mediated through an induction of interferon-γ production by NK-

cells after 24-hrs of treatment; optical density (OD) by ELISA of PBS = 260.6 vs. isotype

= 261 vs. monalizumab = 255.4, n = 5, p = 0.9.

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Chapter 4: Conclusions and Future Perspectives

4.1 Conclusions

Collectively, we have evaluated mechanisms to overcome NK cell

immunosuppression in CLL patients using monoclonal antibody (mAb) therapeutics in

order to restore and enhance NK killing activity against tumor B-cells. We described

BAFF-R as a novel target antigen of mAb therapy that is ubiquitously expressed on CLL

B-cells, and eliminates malignant B-cells through a variable region against BAFF-R that blocks critical pro-survival signals concomitant with a glyco-engineered Fc domain that mediates potent NK-cell ADCC. Furthermore, we demonstrated that BAFF/BAFF-R activates pro-survival NF-κB in CLL B-cells even when treated with BTK inhibitor, ibrutinib, indicating that the BAFF/BAFF-R pathway is independent from BTK signaling.

Additionally, we confirmed HLA-E overexpression on CLL B-cells and deregulated

NKG2A inhibitory signals as a mechanism of CLL tumor evasion from NK cell killing that can be overcome by blocking NKG2A with mAb. These findings deepen our understanding of CLL NK-cell biology allowing for improved mAb and small molecule inhibitor combination therapies as well as attests to the significance of designing antibody therapeutics to overcome tumor evasion and achieve maximal NK cell anti-tumor response.

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First, we validated BAFF-R as a promising target antigen for mAb and detected

BAFF-R ubiquitously expressed on CLL B-cells. Anti-BAFF-R, B-1239, mediated ADCC by healthy donor and CLL NK-effector cells indicating that glyco-engineering Fc-domain modifications potently activated NK-ADCC to kill tumor cells and activate innate immune mechanisms. This demonstrates that glyco-engineered antibodies can overcome CLL NK- cell paralysis and restore the effector cell’s ability release cytokines and kill targets through

ADCC. B-1239 also induced high levels of IFN-γ production in response to engaging the glyco-engineered antibody. We observed increased activity over anti-CD20 obinutuzumab, which was unexpected since they are similarly glyco-engineered to be defucosylated. In order to assess the degree of NK cell activation that is Fc-mediated by B-1239, TCL1 mouse splenocytes could be engrafted into NOTAM mice. NOTAM mice express normal surface levels of FcγR but are incapable of signaling due to mutations in the ITAM motif within the FcγRs [1]. Utilizing NOTAM and FcγR-/- mice, Boross et al. observed that anti-

CD20 rituximab or ofatumumab were effective at reducing tumor load when mice were engrafted with low tumor burden, however antibody efficacy was fully abrogated when mice were engrafted with high tumor burden as demonstrated with engraftment of high or

low cell counts of lymphoma cell lines [2]. This suggests that functional FcγR signaling

mechanisms are essential for clearing high disease burden tumors by mAbs. Testing the

efficacy of glyco-engineered anti-BAFF-R, B-1239, in the NOTAM mouse model

engrafted with TCL1 leukemic splenocytes would demonstrate the role of functional FcγRs

in the ability of B-1239 to clear leukemia or reduce tumor burden. Furthermore, blocking

BAFF-R may exploit CLL B-cells’ vulnerability to NK-ADCC, thus providing deeper

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rational of anti-BAFF-R therapy. Wild et al. documented CLL NK-cells produce BAFF in

response to FcR triggering by rituximab, which subsequently enhanced metabolic activity

of primary CLL B-cells and impaired rituximab mediated NK-ADCC. Addition of neutralizing anti-BAFF, belimumab, prevented increased metabolic activity in CLL cells and restored susceptibility to NK-cell direct killing and rituximab-mediated ADCC, even in autologous assays [3] and suggests a role for BAFF in CLL tumor evasion from NK cells. Possibly, blocking BAFF-R increases CLL B-cell susceptibility to NK-ADCC by preventing activated NK-derived BAFF to bind BAFF-R and this warrants further investigation into the production of BAFF from NK cells in response to glyco-engineered and non glyco-engineered antibodies engaging FcR. Additionally, B-1239 would confirm if BAFF-R was the sole BAFF receptor mediating protection of CLL cells from NK cell killing.

BAFF-R expression is ubiquitously expressed on CLL B-cells and ligation with

BAFF, its exclusive ligand, activates critical pro-survival signals [4, 5]. Microenvironment niches produce large amounts of BAFF that CLL cells extensively rely upon to activate

NF-κB, drive pro-survival signals, and overcome apoptosis [6, 7]. Together, these findings implicate BAFF-R as a promising candidate to target and eliminate CLL B-cells. Targeting the BAFF-R pro-survival axis with anti-BAFF-R, B-1239, demonstrates promising efficacy in models of Acute Lymphocytic Leukemia (ALL) by enhancing NK cell ADCC as well as by binding an epitope on the receptor that abrogates BAFF ligation [8]. In CLL, some patient groups fail to qualify for therapeutics including some mAbs due to the immunosuppressive mechanisms associated with disease. Ibrutinib, a small molecule

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irreversible inhibitor of BTK approved for front-line therapy in CLL, antagonizes ADCC rendering combination studies with most antibodies ineffective [9, 10]. Additionally, B cells from a murine model with inactivated Btk failed to activate alternative NF-κB signaling in response to BAFF, suggesting that BAFF signaling depends on functional BTK

[11]. Due to these reports, it was unknown whether anti-BAFF-R would be an effective

CLL monotherapy or in combination with BTK inhibitor ibrutinib. Herein, we reported that BAFF/BAFF-R activated alternative NF-κB signaling and protected CLL B-cells from apoptosis in vitro. Interestingly, BAFF stimulation activated alternative NF-κB signaling through BAFF-R in primary CLL cells that was independent of BTK inhibition by ibrutinib. Blocking BAFF ligation to BAFF-R with B-1239 obstructed alternative NF-κB signaling. In vivo, B-1239 rapidly cleared leukemic B-cells from peripheral blood and effectively combined with ibrutinib for an additive survival advantage compared to either monotherapy in CLL murine models. These studies suggest that B-1239 and ibrutinib could block both BAFF-R and BTK downstream pro-survival signals if used in combination leading to effective eradication of CLL tumor.

Recently, alternative NF-κB has been implicated in driving resistance mechanisms in ibrutinib therapy and chemotherapy. In a phase 1/2 clinical trial, ibrutinib produced complete or partial response in 37% (14/38 patients) of patients with activated B cell-like

(ABC) subtype of diffuse large B cell lymphoma (DLBCL). ABC DLBCL fosters chronically active BCR signaling, and these patients can have mutations in BCR signaling molecules such as myeloid differentiation primary response 88 (MYD88), CD79a and

CD79b leading to more activated NF-κB. Also in this clinical trial, the authors found that

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ibrutinib was ineffective in patients with mutations in the BCR pathway caspase

recruitment domain family member 11 (CARD11) scaffold molecule [12] suggesting a role for CARD11 in ibrutinib resistance and that chronic BCR pathway activation is independent of BTK activity. CARD11, mutated or wild type, is required for constitutive

NF-κB activity and survival in ABC DLBCL [13, 14]. Phosphorylation of CARD11 activates the recruitment of adapter protein B-cell CLL/lymphoma 10 (BCL10) and mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) forming the

CARD11/BCL10/MALT1 (CBM) complex. The CBM complex further recruits activating factors and kinases leading to NF-κB and c-Jun N-terminal kinase (JNK) activation [15].

Aberrant CBM signaling is sufficient to drive lymphoproliferation in vivo and can mediate proliferation and NF-κB signaling in malignant B-cells treated with inhibitors of SYK,

BTK, PI3K, or ERK [16]. BAFF-R surface expression is decreased on CARD11-deficient murine B-cells [17]. Together, this suggests that overactive CARD11 signaling could increase surface BAFF-R expression and increase B cells dependence on BAFF-R pro- survival signaling. Indeed, because CARD11 mutations are associated with ibrutinib resistance, ibrutinib resistant cells may be dependent on both CARD11 and BAFF-R for survival. In addition to CARD11 mediating overactive NF-κB signaling, the recurrent chromosomal translocation 14;19 (t14;19), which is found in CLL patients and involves the BCL3 locus [18], also promotes B cell survival and proliferation via NF-κB [19] and is implied in ibrutinib resistance mechanisms. Interestingly, BAFF-induced activation of alternative NF-κB is not altered in B cells from Bcl3-/- mice [19] and further supports BAFF

125 as a driver of NF-κB over activation in B-malignancies that could mediate ibrutinib resistance.

We verified through multiple CLL models and tools that BAFF signals in the presence of ibrutinib, and further investigation of this mechanism and impact on CLL survival is warranted since our findings counter indications from other groups, and the cross-talk between BAFF-R and BCR is far from elucidated [20, 21]. BCR and CD40L stimulation drives transcription of proteins, specifically p100, that are critical building blocks of alternative NF-κB signaling cascades [11]. Quantitation of p100 and other critical

BAFF-R mediated alternative NF-κB building blocks like NIK and TRAF3 could be measured at the protein and RNA level in patients on ibrutinib therapy. If ibrutinib treated

CLL B-cells have sufficient levels of p100, NIK, and TRAF3 for alternative NF-κB signaling, then BAFF could protect these cells from apoptosis through BAFF-R. Further analysis of CLL B-cell protection could be analyzed by viability assays of cells treated with ibrutinib and stimulated with BAFF. Additionally, many studies are lacking regarding the downstream gene transcription of BAFF stimulation in CLL patients. Microarray analysis comparing primary CLL B-cell RNA that has been stimulated with BAFF, and with or without ibrutinib treatment would add to our understanding of BAFF-driven CLL disease and the alterations occurring in patients on ibrutinib.

Lastly, these studies would greatly benefit from understanding the mechanism of ibrutinib and B-1239 additive survival advantage, in vivo. Evaluating the potential of BAFF to protect cells and counter ibrutinib could be examined utilizing the BAFF-transgenic

(BAFF-Tg) mouse. When crossed with the TCL1 mouse, the BAFF-Tg mouse developed

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CLL-like disease at a significantly faster rate and shorter survival than TCL1-Tg mice,

indicating a role for BAFF driving disease progression [22]. In order to examine this, TCL1

splenocytes could be engrafted into BAFF-Tg mice compared to non-BAFF-Tg litter-mate

control (C57Bl/6 background) and treated with or without ibrutinib, with additional

experimental arms for B-1239 treatment. If ibrutinib provided a survival benefit and

reduced tumor load in control non-BAFF-Tg mice but not in BAFF-Tg mice, then this

would support our hypothesis that BAFF can signal and drive leukemia in ibrutinib

treatment settings, in vivo. Additionally, if blocking BAFF-R with B-1239 re-sensitized the leukemic cells to ibrutinib then this would confirm that BAFF-R mediates critical survival signals that can lead to ibrutinib resistance. NK cells can be isolated from the spleen once mice die, and be immunophenotyped by flow cytometry for activating and inhibitory receptors to discover if treatments lead to expression alterations providing further insight into NK cell immunosuppression, in vivo, with these treatments.

Our reports on anti-BAFF-R in CLL emphasize the improved efficacy of antibody therapeutics designed with multi-modal anti-tumor activity. This includes Fc modifications to enhance NK cell activation concomitant with targeting antigens that are essential for tumor survival, especially targeting pathways that are independent of BTK signaling and could lead to additive combination therapy. Ibrutinib provides an advantage over standard

CLL therapies as measures by progression free survival (PFS) and overall survival (OS), and is approved for both relapsed and treatment-naïve patients; but, is limited by the development of clinical resistance in some patients and there is a strong need for indefinite treatment since patients relapse with aggressive disease once they discontinue ibrutinib

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[23-26]. Because BAFF-R signals pro-survival NF-κB in ibrutinib treated CLL B-cells, we hypothesize that the addition of B-1239 to ibrutinib would overcome the limitations of ibrutinib monotherapy. Indeed, the glyco-engineered Fc domain of B-1239 would enhance the patient’s compromised NK cells to mediate ADCC while also blocking BAFF-R-

induced NF-κB and ibrutinib would inhibit BTK-mediated survival pathways. Clinical trials are underway to evaluate this combination to improve response of patients who have not achieved complete response with minimal residual disease negative status (MRD -), or patients with developed mutations associated with ibrutinib resistance. Additionally, patients would be evaluated for depth of response with combination with the goal to discontinue therapy. Planned correlative studies include observing the impact of anti-

BAFF-R on blocking BAFF-R mediated NF-κB signaling in vivo, analyzing BAFF-R

surface expression of patient B-cells, and analyzing NK and T cells activation status for

overcoming immunosuppression of these effector cells that is intrinsic to disease and

associated with ibrutinib therapy. Blocking BAFF-R with glyco-engineered mAb

demonstrates great clinical potential with multi-modal tumor eradication mechanisms and

further investigation of its efficacy as monotherapy and in combination is warranted.

Additionally, we demonstrated that directly targeting NK cell immunosuppression

through blocking inhibitory NKG2A is sufficient to restore NK immune response to kill

malignant cells in CLL. Profound immune deregulation is observed in CLL such as

expression of immune escape “self” markers that leads to weakened NK activation and

failed tumor-killing [27, 28]. The surface receptor and ligand expression profile on NK

cells is tightly regulated; each activating signal is combated with an inhibitory signal in

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order to properly kill pathogen or malignant invaders and evade killing of “self”. The

primary inhibitory receptor of NK cells, NKG2A and its ligand, HLA-E, are manipulated in many cancers and mediate tumor immune escape [29, 30]. In CLL, blocking NKG2A effectively combines with rituximab to reduce tumor load [31]. Corroborating this report, we provide an in-depth analysis of NKG2A and HLA-E surface expression and RNA levels on B- and NK- cell subsets as well as expression profiles of patient risk groups compared to normal donors. Specifically, CLL B-cells showed higher expression of HLA-E compared to normal with no correlation of expression to treatment status, IGHV mutational status, or cell subsets. Furthermore, our studies demonstrate that CLL NK-cell weakened killing activity can be restored by blocking NKG2A with monalizumab and disrupting

HLA-E mediated suppression. This finding is significant due to the profound NK cell suppression in CLL, and that blocking NKG2A overcomes this suppression allowing NK cells to lyse tumor targets.

Analysis of blocking NKG2A with Tec-kinase inhibitors, including ibrutinib, would examine the impact of this receptor in controlling activation. The extent of ibrutinib inhibiting NK cell activity is unknown, though it has been suggested to abrogate ADCC

[9], which more selective BTK inhibitors do not affect [32]. The impact of these inhibitors on direct killing of tumors by NK cells is unknown. By treating NK cells with various

BTK, ITK, or RLK inhibitors NK cell activity can be measured to find the inhibitor with the lowest impact on NK cell direct killing. As mentioned previously, NK cells express a tightly regulated network of inhibitory and activating receptors and deciphering if this network is altered with Tec-kinase inhibitors should be studied. If ibrutinib is polarizing

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NK cells by binding ITK or RLK, like it polarizes CLL T-cells to Th1 subsets [33], and upregulates NKG2A expression, then this warrants combination with anti-NKG2A, monalizumab. These findings would provide insight to appropriate combination treatments to enhance NK-cell anti-tumor response, elevate patient’s immunity, and ensure treatment efficacy.

4.2 Future Perspectives

These studies demonstrate the significance of enhancing NK cell tumor killing with antibody therapeutics and the innovation of small molecule inhibitors combination therapy to develop novel and effective therapies for CLL patients. NK cells are critical to host- rejection of tumor or virally infected cells. These cells express many activating and inhibitory receptors that are deregulated in cancers leading to tumor evasion. Monoclonal antibodies interact with and activate NK cells that can alleviate the intrinsic immunosuppression of CLL disease. Using monoclonal antibodies to target specific antigens in CLL patients, the findings presented here deepen our mechanistic understanding of CLL tumor evasion; however our comprehension of NK cell biology in malignancy and how these cells are transformed to a suppressed state is minimal.

Realistically, enhancing one NK cell effector mechanism with antibody therapy is insufficient to properly eradicate malignant B-cells. Ideally, the next generation of antibody therapeutics will be designed with superior NK cell activation concomitant with selecting

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target antigens critical for tumor vitality. Potential target antigens include pro-survival

molecules like BAFF-R, or immunosuppressive signaling molecules like NKG2A.

Trastuzumab, an anti-HER2 humanized antibody is one clinically successful example of

this. Its target, HER2/neu is a tyrosine kinase receptor that mediates proliferation and

survival. By blocking HER2, survival signaling is prevented [34]. Moreover, isotype and

subclass of antibody are important to consider in antibody design to activate certain

mechanisms by specific effector cell types. For example, choosing an antibody structure

that activates NK-ADCC may be more desirable if tumor burden is high in peripheral

blood, while macrophage activation would be important for tumors that infiltrated tissues.

Chimeric-antigen-receptor (CAR) engineered T-cells is another promising and highly

effective clinical tool. CARs are derived from monoclonal antibody single chain Fv

fragments that are expressed on the surface of T cells [35]. Currently, CAR-T cells in the clinic for B-cell malignancies target CD19. Due to the success of designing antibody therapeutics against pro-survival target antigens, CAR-T cells could be engineered to target critical survival molecules like BAFF-R. Indeed, antibodies and immune-based targeted therapies with multi-modal mechanisms of action may be the most efficacious for eradicating tumor, and requires specific antibody engineering to strategically target tumor.

Although decades of in vitro and in vivo work has been done on NF-κB, the overlap of BCR signaling with other pathways driving NF-κB in B-cell malignancies remains unresolved. Small molecule inhibitors against BTK, PI3K, and SYK provide insight to what molecules are critical for effective downstream signaling and which are overactive or even mutated and providing a hyperactive pro-survival signal. Moreover, blocking

131 antibodies against BAFF-R demonstrate that BAFF is a critical pro-survival signaling molecule in CLL. By targeting both BCR and BAFF-R we hypothesize that CLL B-cells will undergo apoptosis due to lack of pro-survival signaling; however other NF-κB signaling molecules may continue to keep the malignant cell alive, like other BAFF receptors TACI or BCMA, raising the question of which NF-κB pathways are the most critical to target and will this lead to tumor cell death. It would therefore be of interest to combine inhibitors of BCR signaling molecules with BAFF receptor blocking antibodies to determine which are most vital for tumor survival and the most effective combination.

CLL cells secret many soluble factors that can lead to tumor evasion from NK cell killing, including through exosomes. CLL cells release NK activating ligands, like BAG6, in exosomes or in soluble form mediating dysregulated NK cell activation and tumor evasion [36]. In addition to BAG6, there are many known factors that are raised in CLL patient serum and compromise NK cell function. Also, overexpression of inhibitory ligands on CLL cell surface like HLA-E was verified and that blocking its receptor, NKG2A, could alleviate NK cell immunosuppression and activate NK killing of targets. No doubt, CLL immune evasion mechanisms target various aspects of the host’s immune system and these mechanisms could be different depending on the tissue compartment. It is unknown whether NKG2A/HLA-E drives inhibitory signals to NK cells throughout the host, or if

CLL expression of HLA-E changes based on tissue localization. The relative importance of each of these immunosuppressive factors that limit therapeutics is unknown. Further analysis of patients’ soluble serum factors, exosomes, and surface receptors and ligands

132

while on therapy or during disease progression will allow for further understanding of CLL

disease mediated immunosuppression.

The work presented in this thesis provides proof of concept that NK cell activity is

suppressed via multiple mechanisms in CLL, though this can be overcome to potently

target and kill leukemic cells. The ultimate significance of this work is to emphasize

antibody drug design with multiple modes to target pro-survival pathways in malignant cells, and to highlight the power of harnessing NK cell killing properties to treat cancers.

Overall, alleviating NK cell immunosuppression using antibody therapeutics, with the potential to further enhance patient’s innate anti-tumor response shows great promise for treating patients with CLL and other B-cell malignancies. Combination therapies that overcome NK cell immunosuppression and mediate killing of tumors while also inhibiting multiple critical survival signals opens exciting new opportunities for treating CLL and beyond.

133

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4.4 Figures

Figure 4. 1 A model of overcoming CLL NK-cell immunosuppressive mechanisms NK cells from CLL patients are limited in their killing capacity due to suppressive soluble factors and dysregulated activating and inhibitory surface molecules. CLL cells receive multiple pro-survival signals through BCR (red surface molecule) and BAFF-R (orange surface molecule). NK cells mediate tumor killing through antibody mediated cellular cytotoxicy (ADCC) which depends on release of perforin and granzyme containing granules (orange circles) from the NK cell that attacks the CLL B-cell membrane. Anti-

BAFF-R, B-1239 (blue antibody structure) is glyco-engineered for enhanced binding to

FcγRIIIa on NK cells and demonstrates potent activation of (continued on the next page)

137

Figure 4. 1 continued

NK-ADCC and killing of target CLL B-cells. By blocking BAFF-R, BAFF/BAFF-R

mediated pro-survival signaling is blocked. Ibrutinib blocks downstream BCR signaling

molecule BTK (green box) and inhibits pro-survival signaling. When cells were treated

with ibrutinib BAFF/BAFF-R signaling was active. NK cells mediate direct killing by

detecting dysregulated surface “self” markers like MHC class I molecule HLA-E (blue surface molecule). HLA-E binds to NKG2A (black surface molecule) that sends inhibitory signaling to the NK cell and leads to deactivation of activating receptors NKG2C, E, H

(black surface molecules). Anti-NKG2A (red antibody structure) blocks NKG2A/HLA-E ligation and allows the NK cell activating receptors to mediate NK cell direct killing of target.

138

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