Novel Immunotherapeutic Strategies For

NOVEL IMMUNOTHERAPEUTIC STRATEGIES FOR

CHRONIC LYMPHOCYTIC LEUKEMIA

DISSERTATION

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

Kyle Beckwith, B.S.

Graduate Program in Biomedical Sciences

The Ohio State University

2016

Dissertation Committee:

John Byrd MD (Co-Advisor)

Natarajan Muthusamy DVM, PhD (Co-Advisor)

Michael Caligiuri MD

Abhay Satoskar MD, PhD

Kyle A. Beckwith

2016

ABSTRACT

Chronic lymphocytic leukemia (CLL) is malignancy characterized by the progressive accumulation of neoplastic B-cells that are phenotypically mature, but functionally incompetent. It is the most prevalent adult leukemia in the United States, and has an incidence of approximately 18,000 new cases per year. The introduction of

CD20-targeted monoclonal antibody rituximab dramatically altered how this disease is managed, and immunotherapeutic strategies have become a mainstay of CLL therapy ever since. Newer immunotherapies approved by the FDA include anti-CD52 alemtuzumab and two anti-CD20 antibodies with improved activity. Due to expression of

CD52 on T-cells, alemtuzumab therapy puts an already immunocompromised population at greater risk for infection (which is already the most common cause of CLL patient death). Unfortunately current therapeutics are not curative, and resistance/relapse is inevitable. Given the limitations of these therapies, particularly when confronted with the challenge of treating relapsed/refractory disease, it is important to expand our immunotherapeutic arsenal beyond targeting CD20. Therefore, the overall aim of this thesis is to develop novel approaches to CLL immunotherapy that can be successfully translated to the clinic. One of the most promising alternative targets for immunotherapy is CD37. This tetraspanin protein is largely B-cell restricted and highly expressed in CLL and Non-Hodgkins Lymphoma. Furthermore, the single agent clinical activity of anti-

CD37 otlertuzumab in CLL validates this therapeutic target. A number of CD37-targeted therapies have been developed recently, but our ability to study them preclinically is significantly hampered by the lack of suitable animal models that can accurately

ii recapitulate CLL. Ideally we would use the TCL1 mouse model of CLL, which has been extensively characterized as a drug development tool, but the antibodies targeting human CD37 (hCD37) do not cross react with murine CD37. This problem was addressed by developing a model of murine leukemia expressing hCD37, crossing hCD37 transgenic mice developed by our laboratory with the TCL1 mouse. These hCD37xTCL1 mice facilitated the in vivo study of IMGN529, a CD37-targeting antibody- drug conjugate. Using our new model, I demonstrate that IMGN529 can not only improve overall survival, but is also capable of eliminating the proliferative subset of CLL B-cells

within lymphoid tissues. I go on to investigate combination strategies that can improve

the efficacy of anti-CD37 therapeutics, including addition of a phosphoinositide 3-kinase

(PI3K) inhibitor, which improves cytotoxicity against primary CLL B-cells (and is equally

effective against cells from high risk patients). I also investigate whether a CD37-

targeted bispecific antibody can effectively recruit T-cells to kill malignant B-cells, despite

the profound T-cell defects observed in CLL. Unfortunately, it appears to be inferior to

bispecific antibody formats and is only effective at high effector-to-target ratios. Finally, I

use a model of CD37-deficient CLL to explore the function of this tetraspanin in B-cell

malignancy. These mice exhibit decreased survival that suggests a tumor suppressor

role in this context. Overall, this thesis represents a comprehensive inquiry into both the

function of CD37, and a wide variety of approaches to targeting CD37 which provide

valuable information relevant to future clinical trials.

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DEDICATION

This dissertation is dedicated to my family and to my current and former mentors.

ACKNOWLEDGMENTS

First, I would like to acknowledge Dr. Michael Uhler for his mentorship while I was at the University of Michigan. His trust and encouragement to seek out answers to my own questions fostered a love of research I did not discover until very late in college.

Without his mentorship and the (relative) freedom he afforded me, it is unlikely I would have continued to pursue laboratory research. This experience encouraged me to continue to seek out research opportunities in medical school, which led me to Dr. Phillip

Popovich’s laboratory. The short time I spent in this lab working closely with John

Gensel ultimately prompted me to delay entrance to Med 3 to pursue additional research opportunities.

I would like express my deepest thanks to my mentors Dr. John Byrd and Dr. Raj

Muthusamy for their guidance and support. Without the opportunity to pursue a

Pelotonia fellowship in the lab, I would not be seeking a PhD. Your neverending enthusiasm for science and mentorship inspires me. If I train future students half as well as you have, it would be considered a major success. To all members of the Byrd lab

(past and present) who have helped or provided insight along the way – thank you.

I am grateful to many faculty members at OSU for providing their expertise and support, but I would like to specifically extend thanks to Dr. Michael Caligiuri and Dr.

Abhay Satoskar for taking the time to be part of my committee. Thank you for your support and feedback throughout this entire process.

I would also like to thank my parents for their amazing support over the many years of my education. Finally, I am especially thankful to my brilliant wife Puneet for her endless support, insight, patience, and encouragement. You make me strive to be a better person and scientist.

VITA

2009 ...... B.S. Biochemistry, with distinction

University of Michigan

2009 to present ...... Graduate/Medical Student

Ohio State College of Medicine

Biomedical Sciences Graduate Program

PUBLICATIONS

1. *Fraietta JA, *Beckwith KA, Patel PR, Ruella M, Zheng Z, Barrett DM, Lacey SF, Melenhorst JJ, McGettigan SE Cook DR, Zhang C, Xu J, Do P, Hulitt J, Kudchodkar SB, Cogdill AP, Gill S, Porter DL, Woyach JA, Long M, Johnson AJ, Maddocks K, Muthusamy N, Levine BL, June CH, *Byrd JC and *Maus MV. Ibrutinib Enhances Chimeric Antigen Receptor T-cell Engraftment and Efficacy in Leukemia. Blood. Pre-published online January 26, 2016. doi: http://dx.doi.org/10.1182/blood-2015-11-679134. Asterisk (*) indicates equal contribution by these authors. 2. Gensel JC, Wang Y, Guan Z, Beckwith KA, Braun KJ, Wei P, McTigue DM, Popovich PG. Toll-Like Receptors and Dectin-1, a C-Type Lectin Receptor, Trigger Divergent Functions in CNS Macrophages. J Neurosci. 35(27):9966-76, 2015. doi: 10.1523/JNEUROSCI.0337-15.2015 3. Beckwith KA, Byrd JC, and Muthusamy N. Tetraspanins as therapeutic targets in hematological malignancy: a concise review. Front. Physiol. 6:91, 2015. doi: 10.3389/fphys.2015.00091 [Review article] 4. Hing ZA, Mantel R, Beckwith KA, Guinn D, Williams E, Smith L, Williams KE, Johnson AJ, Lehman A, Byrd JC, Woyach JA, Lapalombella R. Selinexor is effective in acquired resistance to ibrutinib and synergizes with ibrutinib in chronic lymphocytic leukemia. Blood. 125(20):3128-32, 2015. doi: 10.1182/blood- 2015-01-621391

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5. Liu T, Ling Y, Woyach JA, Beckwith K, Yeh Y, Hertlein E, Zhang X, Lehman A, Awan F, Jones JA, Andritsos LA, Maddocks K, MacMurray J, Salunke SB, Chen C, Phelps MA, Byrd JC, Johnson AJ. OSU-T315: a novel targeted therapeutic that antagonizes AKT membrane localization and activation of chronic lymphocytic leukemia cells. Blood. 125(2):284-95, 2015. doi: 10.1182/blood- 2014-06-583518 6. Beckwith KA, Frissora FW, Stefanovski MR, Towns WH, Cheney C, Mo X, Deckert J, Croce CM, Flynn JM, Andritsos LA, Jones JA, Maddocks KJ, Lozanski G, Byrd JC, and Muthusamy N. The CD37-targeting antibody-drug conjugate IMGN529 is highly active against human CLL and in a novel CD37 transgenic murine leukemia model. Leukemia. 28(7): 1501-10, 2014. 7. Zhong Y, El-Gamal D, Dubovsky JA, Beckwith KA, Harrington BK, Williams KE, Goettl VM, Jha S, Mo X, Jones JA, Flynn JM, Maddocks KJ, Andritsos LA, McCauley D, Shacham S, Kauffman M, Byrd JC, Lapalombella R. Selinexor suppresses downstream effectors of B-cell activation, proliferation and migration in chronic lymphocytic leukemia cells. Leukemia. 28(5): 1158-63, 2014. 8. Hertlein E, Beckwith KA, Lozanski G, Chen TL, Towns, WH, Johnson AJ, Lehman A, Ruppert AS, Bolon, B, Lozanski A, Rassenti L, Zhao W, Jarvinen T, Senter L, Croce CM, Symer DE, de la Chapelle A, Heerema N, Byrd JC. Characterization of a new chronic lymphocytic leukemia cell line for mechanistic in vitro and in vivo studies relevant to disease. PLoS One. 8(10), 2013. 9. Dubovsky JA, Beckwith KA, Natarajan G, Woyach JA, Jaglowski S, Zhong Y, Hessler JD, Liu T, Chang BY, Larkin KM, Stefanovski MR, Frissora FW, Smith LL, Smucker KA, Flynn JM, Jones JA, Andritsos LA, Maddocks KJ, Lehman AM, Furman R, Sharman J, Mishra A, Caligiuri MA, Satoskar AR, Buggy JJ, Muthusamy N, Johnson AJ, and Byrd JC. Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1 selective pressure in T-lymphocytes. Blood. 122(15): 2539-49, 2013.

FIELDS OF STUDY

Major Field: Biomedical Sciences

Specialization: Immunology

viii

TABLE OF CONTENTS

Abstract ...... ii

Dedication ...... iv

Acknowledgments ...... v

Vita ...... vii

List of Figures ...... x

Chapters:

1. Introduction ...... 1

2. A Mouse Model of Human CD37-Expressing Leukemia ...... 19

3. A CD37-Specific Antibody-Drug Conjugate for B-cell Malignancy ...... 30

4. Developing Strategies to Enhance Anti-CD37 Therapy ...... 60

5. Bispecific Antibodies: Bringing T-cells into the Fold ...... 73

6. Exploring the Function of CD37 in Leukemia ...... 89

7. Conclusions ...... 96

List of References ...... 107

LIST OF FIGURES

Figure Page

1.1 Structural features of tetraspanins ...... 15

1.2 Signaling pathway associated with CD37 ligation by SMIP-016 ...... 16

1.3 CD37-targeted antibody therapeutics ...... 18

2.1 Generation and characterization of human CD37 transgenic mouse ...... 25

2.2 Human CD37 transgenic B-cells are sensitive to anti-CD37 therapy ...... 27

2.3 hCD37xTCL1 mice retain expression of both transgenes and develop CLL ...... 28

3.1 IMGN529 is directly cytotoxic to primary human CLL B-cells ...... 44

3.2 IMGN529 retains the Fc-mediated activities of its unconjugated precursor ...... 46

3.3 Gating strategy for measuring phagocytosis of CLL B-cells...... 48

3.4 Gating strategy for whole blood cytotoxicity assays ...... 50

3.5 IMGN529 demonstrates exceptional cytotoxicity in CLL whole blood ...... 52

3.6 DM1-conjugation improves killing and arrests proliferating B-cells ...... 54

3.7 IMGN529 demonstrates in vivo efficacy against hCD37+ murine leukemia ...... 55

3.8 IMGN529 eliminates splenomegaly and demonstrates CD37-specificity ...... 57

3.9 IMGN529 targets proliferating mouse leukemia cells within lymphoid tissues ..... 58

4.1 Combining TLR 7/8 agonist CL-075 with TRU-016 is not beneficial ...... 68

4.2 BI 836826 effector mediated killing ...... 70

4.3 Combining BI 836826 with idelalisib improves cytotoxicity ...... 71

4.4 Preliminary evaluation of BI 836826 in hCD37 leukemia model ...... 72

5.1 IgG-derived therapeutics constructed from scFV ...... 79

5.2 TSC-TRU recruits and activates T-cells ...... 81

5.3 Cytotoxicity of TSC-TRU with allogeneic or autologous T-cells ...... 82

5.4 T-cell activation by TSC-TRU in CLL PBMC cultures ...... 83

5.5 CLL B-cells evade killing by TSC-TRU in 48 hour PBMC cultures ...... 84

5.6 Longer incubation has minimal impact on TSC-TRU efficacy in PBMCs ...... 86

5.7 Gating strategy for PBMC co-culture experiments ...... 88

6.1 Confirmation of CD37 knockout ...... 93

6.2 CD37 deficiency reduces TCL1 mouse survival ...... 94

6.3 Measurement of leukemic burden in peripheral blood ...... 95

CHAPTER 1

INTRODUCTION

Chronic Lymphocytic Leukemia (CLL)

Chronic lymphocytic leukemia (CLL) is a malignancy characterized by the progressive accumulation of neoplastic B-cells that are phenotypically mature, but functionally incompetent. It is the most prevalent adult leukemia in the United States, and has an incidence of approximately 18,000 new cases per year. Typically, CLL is diagnosed between the ages of 65-74 and is more common in men than women (white men being the most commonly afflicted group). (Siegel et al., 2013)

The highly heterogeneous nature of CLL has substantial impact on how this disease is managed clinically. While patients who are asymptomatic at diagnosis have a median survival of greater than 10 years (often permitting a “watch and wait” approach), the prognosis of those with symptomatic or advanced stage CLL is between 1.5 and 3 years without treatment (Rai et al., 1975). In the years that followed FDA approval of the anti-CD20 monoclonal antibody (mAb) rituximab, chemoimmunotherapy combining mAbs with traditional chemotherapeutics became the favored approach for initial treatment of patients with active disease (Woyach et al., 2012; Gribben, 2010). A regimen that adds rituximab to fludarabine and cyclophosphamide (FCR) improves both progression-free survival (PFS) and overall survival (OS) in previously untreated CLL patients (Hallek et al., 2008), although FCR still benefits patients with relapsed disease

(Wierda et al., 2005).

Despite the impact made by rituximab-based immunotherapy, it is not curative and resistance/relapse is inevitable (Woyach et al., 2011; Rezvani and Maloney, 2011).

In the years following rituximab’s success, several other mAb therapies have received

FDA approval for the treatment of CLL. These include the anti-CD52 mAb alemtuzumab and two CD20-targeting mAbs with improved activity (ofatumumab and obinatuzumab).

Alemtuzumab is a very potent mAb therapeutic, but unfortunately causes profound immunosuppression due to the elimination of CD52 expressing T-cells. While still used in certain cases, a delicate balance must be struck as infection is already the most common cause of death among CLL patients. Due to the limitations of these therapies, particularly when confronted with the challenge of treating relapsed/refractory disease, it is important to expand our immunotherapeutic arsenal beyond targeting CD20. This demand has been the motivation for the work presented herein, which focuses on the tetraspanin CD37 as an alternative target for immunotherapy.

Tetraspanin proteins

The tetraspanin superfamily is comprised of at least 33 members in humans, and these transmembrane proteins are ubiquitous among metazoans (Maecker et al., 1997.

Many of the tetraspanins present on immune cells are also found in a variety of other tissues, but some display hematopoietic-restricted expression, including CD37, CD53,

Tssc6 (TSPAN32), and TSPAN33 (Tarrant et al., 2003; Heikens et al., 2007). The predominant view has been that tetraspanins are facilitators of signal transduction, providing organization to plasma membrane domains through lateral interaction with their numerous partners (Maecker et al., 1997; Hemler, 2005; Charrin et al., 2009).

However, there is recent evidence that certain tetraspanins also recruit signaling

proteins directly (Lapalombella et al., 2012). Tetraspanins have been reported to regulate diverse processes, including cellular migration, adhesion, activation, and apoptosis (Hemler, 2005). Furthermore, several tetraspanins influence cancer metastasis/progression and their functional roles in immune cells can impact anti-tumor immunity (Zoller, 2009; Veenbergen and van Spriel, 2011; Hemler, 2014).

Tetraspanins contain short N-terminal and C-terminal cytoplasmic tails, a small extracellular loop (EC1 domain), a large extracellular loop (EC2 domain) and four transmembrane domains (Figure 1.1). The EC2 domain contains a region conserved among tetraspanins, but also a highly variable region that is frequently involved in the specific interactions between tetraspanins and various non-tetraspanin partners (Yauch et al., 2000; Charrin et al., 2001; Shoham et al., 2006; Zevian et al., 2011). It is typical for tetraspanins to undergo extensive post-translational modification. Covalent attachment of palmitate to intracellular cysteine residues is implicated in mediating tetraspanin-tetraspanin interactions and assembly of tetraspanin-enriched domains that can support signaling (Berditchevski et al., 2002; Charrin et al., 2002; Yang et al., 2002;

Yang et al., 2004). Furthermore, nearly all tetraspanins display extensive N-linked glycosylation at extracellular sites (Maecker et al., 1997). This glycosylation is likely to have functional relevance, as shown with CD82 and CD9, which could only influence motility or apoptosis when glycosylated (Ono et al., 1999; 2000). A variety of glycosylation patterns are observed across cell lines, including those of the same lineage, but it remains unknown whether these differences have any impact on tetraspanin function (Schwartz-Albiez et al., 1988; White et al., 1998).

Numerous cis-interactions occur between tetraspanins and neighboring plasma membrane proteins within what are known as tetraspanin-enriched microdomains

(Hemler, 2005). These microdomains may function as signaling platforms, similar to lipid rafts but generally comprised of distinct components (Claas et al., 2001; Le Naour et al.,

2006a; Mattila et al., 2013; Zuidscherwoude et al., 2014). Many tetraspanin interactions depend on binding to the extracellular EC2 domain (Yauch et al., 2000; Charrin et al.,

2001; Shoham et al., 2006; Zevian et al., 2011), although transmembrane domains are also frequently involved (Charrin et al., 2001; Charrin et al., 2003; Shoham et al., 2006).

While numerous associations have been documented with other transmembrane proteins, there are fewer examples of tetraspanins interacting with cytoplasmic proteins.

This is not surprising, given their short cytoplasmic tails are generally less than 20 amino acids in length (Maecker et al., 1997). This has contributed to the thought that tetraspanins do not directly participate in signal transduction. However, several tetraspanins have been reported to associate with intracellular signaling proteins.

Common cytoplasmic partners include PI4K and PKC (Berditchevski et al., 1997; Zhang et al., 2001; Andre et al., 2006), but tetraspanins have also been shown to interact with several other signaling proteins (Clark et al., 2004; Little et al., 2004; Andre et al., 2006;

Le Naour et al., 2006b; Lapalombella et al., 2012). While tetraspanins clearly influence signaling, it remains possible that some of these interactions could be indirect as a result of association with adapter proteins.

Tetraspanins have been largely discounted as potential cell-surface receptors on the basis of their structure, which protrudes at most 5 nm into extracellular space

(Kitadokoro et al., 2001; Min et al., 2006). While their interactions do primarily occur in cis, recent publications have challenged the notion that tetraspanins cannot also function as receptors. CD9 is reported to have multiple soluble ligands, both acting as an alternative IL-16 receptor (Qi et al., 2006) and binding a placental protein released

during pregnancy (Waterhouse et al., 2002). CD81 has been identified as an essential receptor for Hepatitis C virus (Pileri et al., 1998). CD82 has been described as a receptor for an endothelial cell-surface protein, and this interaction was important for the suppression of tumor metastasis by this tetraspanin (Bandyopadhyay et al., 2006).

However, the capability of tetraspanins to bind endogenous ligands in a trans-fashion remains somewhat controversial in the field.

CD37

This protein is most highly expressed by mature B-cells, although other immune cells express CD37 to a lesser degree (Link et al., 1986; van Spriel et al., 2009; Deckert et al.,

2013). It is absent in the earliest stages of B-cell development and is lost again following differentiation into plasma cells; a pattern mirrored by B-cells malignancies originating from various developmental stages (Barrena et al., 2005). CD37 is highly expressed in mature B-cell malignancies, such as non-Hodgkin lymphoma and CLL, but is low or absent in acute lymphoblastic leukemia and multiple myeloma. The expression pattern of CD37 has led to considerable interest in targeting this tetraspanin therapeutically

(Zhao et al., 2007; Heider et al., 2011; Krause et al., 2012; Dahle et al., 2013; Deckert et al., 2013; Beckwith et al., 2014).

CD37-deficient mice exhibit defective IgG1 production in response to T-cell dependent antigens (Knobeloch et al., 2000), which is a consequence of decreased survival among IgG1-secreting B-cells in the days following antigen exposure (van Spriel et al., 2012). It was demonstrated that CD37 has an important role in clustering α4β1 integrin (also known as VLA-4) on the plasma membrane. Absence of CD37 impaired integrin-dependent Akt signaling that is typically activated through interaction with

follicular dendritic cells expressing a ligand of α4β1 integrin (van Spriel et al., 2012).

Similarly, ligation of CD37 by the antibody-derived peptide SMIP-016 also modulates the

Akt pathway in B-cells (Lapalombella et al., 2012). However, SMIP-016 induces both pro-apoptotic Akt inactivation and opposing pro-survival phosphoinositide 3-kinase δ

(PI3Kδ) activation (Figure 1.2). Analysis of its sequence suggested that the cytoplasmic tails of CD37 contained weak ITIM and ITAM-like motifs. Mutational studies support this function, providing evidence that the N-terminal ITIM can recruit SHP1 (which is capable of dephosphorylating/inactivating Akt) and the C-terminal ITAM can recruit PI3Kδ. While pro-survival and pro-apoptotic pathways are simultaneously induced by CD37 ligation, cellular death is favored. This may explain why several anti-CD37 therapeutics promote high degrees of apoptosis in leukemia cells (Zhao et al., 2007; Heider et al., 2011;

Krause et al., 2012; Lapalombella et al., 2012; Deckert et al., 2013; Beckwith et al.,

2014).

While the expression of CD37 is low in non-B cells (van Spriel et al., 2009;

Deckert et al., 2013), it still has important functions in T-cells, dendritic cells, and macrophages (van Spriel et al., 2004; Meyer-Wentrup et al., 2007; Sheng et al., 2009;

Gartlan et al., 2010; Gartlan et al., 2013). CD37 associates with Dectin-1 and appears to negatively regulate its activity in anti-fungal response, as IL-6 production is dramatically increased in CD37−/− macrophages following Dectin-1 stimulation (Meyer-Wentrup et al., 2007). It is possible that this regulation is accomplished through recruitment of phosphatases by CD37, which can associate with its N-terminal domain (Lapalombella et al., 2012). IL-6 production by CD37−/− cells likely supports the generation of IgA- secreting plasma cells, leading to excessive IgA secretion that ultimately provides these mice with resistance to fungal infections (van Spriel et al., 2009). CD37 plays a complex

role in T-cell responses, as made evident by the seemingly contradictory results of in vitro and in vivo studies. In vitro, CD37−/− dendritic cells are hyperstimulatory toward T- cells, and the data imply that CD37 negatively regulates peptide-MHC presentation

(Sheng et al., 2009; Gartlan et al., 2010). Furthermore, CD37 in T-cells may play a negative regulatory role in T-cell receptor (TCR) signaling. CD37-deficient T-cells proliferate more rapidly in response to TCR stimulation, which could be a result of decreased Lck phosphorylation (van Spriel et al., 2004). While the in vitro data imply a negative regulatory role for CD37 in T-cell responses, the opposite is observed using in vivo models. Mice deficient for CD37 are more susceptible to infection with murine malaria (Gartlan et al., 2010) and fail to reject syngeneic tumor cells transfected to express a foreign antigen (Gartlan et al., 2013). These discrepancies are explained by the observation that dendritic cells from CD37−/− mice have impaired migratory and adhesion capabilities, which clearly overshadows other potential contributions of CD37

(Gartlan et al., 2013). It remains unclear whether the hyperproliferative phenotype of

CD37-deficient T-cells is relevant beyond in vitro studies, but providing CD37−/− mice with wildtype dendritic cells did not appear to significantly increase the number of IFNγ producing T-cells relative to wildtype mice.

Anti-CD37 Therapy in Hematological Maligancy

Antibody-based strategies for treating cancer have rapidly increased in prevalence since anti-CD20 rituximab was introduced to the clinic. More than a dozen antibodies have been approved by the U.S. Food and Drug Administration (FDA) for cancer therapy and hundreds of ongoing human trials are registered at clinicaltrials.gov

(Scott et al., 2012). The first attempts to develop a CD37-targeted therapy predate the

approval of rituximab by nearly a decade, when 131I –labeled murine anti-CD37 antibody was tested in a small cohort of non-Hodgkin lymphoma (NHL) patients (Press et al.,

1989). While the early results were promising, targeting CD20 was quickly becoming the favored approach and CD37 was subsequently neglected for many years. Anti-CD37 therapy has experienced a recent resurgence, with five different targeting approaches being explored in B-cell malignancies. While several tetraspanins may be promising targets for cancer therapy, CD37 is by far the furthest in terms of clinical development.

CD37 is highly expressed on the surface of human B-cells (where its antigen density is at least 15 times greater than on non-B leukocytes) and it is present in the vast majority of CLL and NHL cases (Deckert et al., 2013), making it an attractive target for immunotherapy. Several CD37-targeting antibody-based therapeutics have been developed which are currently being evaluated in the clinic (Zhao et al., 2007; Heider et al., 2011; Deckert et al., 2013). Otlertuzumab was the first of these to begin clinical trials.

This therapeutic is a humanized, antibody-derived CD37-targeting peptide developed using the ADAPTIRTM platform. Mono-specific ADAPTIR molecules are built from a

single-chain variable fragment (scFv; a binding domain formed by linking the heavy and

light chain variable regions of an immunoglobulin), fused to the hinge region and Fc

domain of human IgG1 (Figure 1.3). These molecules form antibody-like dimers that are

smaller than IgG1 (intended to increase tissue penetration), but otherwise retain similar

pharmacokinetics and activity as traditional IgG1. Preclinical studies using SMIP-016, a

tool molecule not fully humanized (but containing human IgG1 Fc), demonstrated

superior NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) compared to

anti-CD20 rituximab. This therapy also directly killed CLL tumor cells through induction

of caspase-independent apoptosis when in the presence of anti-Fc crosslinker (Zhao et

al., 2007). As discussed earlier, SMIP-016 was shown to induce both pro-apoptotic Akt inactivation and (to a lesser extent) pro-survival PI3Kδ activation (Lapalombella et al.,

2012). The simultaneous activation of these opposing signaling pathways provides an opportunity to utilize unique combination strategies for anti-CD37 therapies. Indeed,

Lapalombella et al. showed that SMIP-016 cytotoxicity against CLL B-cells was enhanced by the addition of either a pan-PI3K inhibitor (LY294002) or the PI3Kδ- selective CAL-101 (idelalisib; now FDA approved for CLL therapy). Further investigation of this potential combination is warranted, and should also be explored with newer anti-

CD37 therapies that can more efficiently induce apoptosis without dependence on additional crosslinking (Heider et al., 2011; Deckert et al., 2013).

Otlertuzumab (TRU-016) has been tested in CLL and NHL patients during recent clinical trials (Byrd et al., 2014; Gopal et al., 2014; Pagel et al., 2014). A phase I study in

CLL observed modest single-agent activity and found it to be well tolerated (Byrd et al.,

2014). Peripheral lymphocyte reduction was observed in 75.5% of patients with elevated initial lymphocyte counts, but overall response rate (ORR) was only 23% (19/83 patients) by NCI-96 criteria. Only partial responses (PR) were observed, which were more common among treatment-naïve CLL patients (6/7) or those who received 1 or 2 previous therapies (12/28). These results were encouraging, given that responses to single-agent rituximab are also limited (Byrd et al., 2001; Huhn et al., 2001), but can be dramatically improved by combination with chemotherapy (Keating et al., 2005).

Similarly, an early report from a randomized Phase II trial in relapsed CLL demonstrates

the improved efficacy of otlertuzumab when combined with bendamustine

(NCT01188681). Patients receiving otlertuzumab plus bendamustine had an ORR of

80% (16/20 patients) with 20% achieving a complete remission (CR), while those treated

with bendamustine alone had an ORR of only 42% (10/24) with a CR rate of 4% (Robak et al., 2013). A single-agent trial of otlertuzumab was also performed in NHL patients with follicular lymphoma, Waldenström's macroglobulinemia, or mantle cell lymphoma

(Pagel et al., 2014). Responses were limited to 2 of 16 patients (12%). However, another phase I study evaluated the combination of otlertuzumab, rituximab, and bendamustine in twelve patients with indolent NHL (follicular, mantle cell, and small lymphocytic) who had relapsed after receiving treatment regimens which included rituximab (Gopal et al.,

2014). Two doses of otlertuzumab were tested (10 or 20 mg/kg) with 6 patients per dose. This regimen was well tolerated and achieved an ORR of 83% (10/12), with four

CRs. All of the patients receiving the higher dose (6/6) responded, with 2 CRs and 4

PRs. Overall, these clinical studies highlight the promise of anti-CD37 therapies, particularly in combination with other agents.

Several newer CD37-targeted therapeutics have the potential to surpass the clinical benefits observed with otlertuzumab. To mediate ADCC, IgG1 requires covalent attachment of oligosaccharides at Asn297 within its Fc region, but eliminating fucose from

this carbohydrate structure is known to improve ADCC (Shinkawa et al., 2003). A non-

fucosylated variant of otlertuzumab has been generated which has enhanced binding to

FcγRIIIa, resulting in improved NK cell mediated ADCC and phagocytosis by

macrophages (Rafiq et al., 2013). Alternatively, ADCC can be enhanced by mutating

certain amino acid residues within the Fc region of IgG1 (Lazar et al., 2006). This

approach was taken in the generation of BI 836826 (Figure 1.3), an IgG1 with specific

mutations in the CH2 domain that augment ADCC (Heider et al., 2011; Krause et al.,

2012). This antibody has already entered clinical trials in Europe, with studies also

expected to begin in the United States.

This final broad category of therapies utilizes anti-CD37 antibodies to guide cytotoxic agents to tumor cells. CD37 internalizes moderately faster than CD20 when bound by antibody (Press et al., 1994), yet not so quickly that ADCC is prevented (Zhao et al., 2007; Heider et al., 2011; Krause et al., 2012; Deckert et al., 2013; Beckwith et al.,

2014). IMGN529 is a humanized anti-CD37 IgG1 conjugated to DM1 (Figure 1.3), a drug which inhibits microtubule assembly during mitosis (Deckert et al., 2013). Currently,

IMGN529 is being evaluated in NHL as part of an ongoing clinical trial (NCT01534715).

This trial has encountered some early difficulties, with several patients experiencing grade III/IV neutropenia that can be largely avoided with the addition of corticosteroids and G-CSF (Stathis et al., 2014). While possible that the low level of CD37 expression on neutrophils results in their direct elimination, evidence obtained from mouse models is suggestive of cell redistribution (Deckert et al., 2014). Only relatively low doses of

IMGN529 have been tested thus far, but 4 of 10 relapsed/refractory diffuse large B-cell lymphoma patients have responded to therapy (1 CR, 3 PR). This is expected to improve at higher doses, now that dose escalation is continuing with prophylaxis that addresses the neutropenia.

Similar to the above approach, anti-CD37 antibody can be used to deliver radioactive isotopes to tumor cells. This was first explored over 20 years ago (Press et al., 1989), but the renewed interest in targeting CD37 has led to the reemergence of

CD37-directed radioimmunotherapy (Dahle et al., 2013; Repetto-Llamazares et al.,

2014). There are currently two FDA-approved radiolabeled antibodies that target CD20

(Scott et al., 2012). However, the propensity of CD37 to internalize may make it a superior target, given that this occurs 10 times faster with 177Lu conjugated to anti-

CD37 tetulomab compared to anti-CD20 rituximab (Dahle et al., 2013). A phase I/II trial

recently initiated in Europe is exploring this therapeutic strategy in NHL using Betalutin, a 177Lu-conjugated anti-CD37 antibody. Thus far, 7 of 11 patients on this trial have

responded (ORR of 64%) with 4 CRs and 3 PRs (Kolstad et al., 2014).

A third approach to CD37-targeted drug delivery is the use of immunoliposomes

coated in antibody (Yu et al., 2013). CD37-coated immunoliposomes effectively

delivered cytotoxic drug to cell lines and CLL B-cells, and specificity could be altered by

using a dual targeting approach with an additional CD19 or CD20 antibody. Furthermore,

CD37 immunoliposomes that were not loaded with drug were capable of inducing

apoptosis in CD37+ cells, presumably due to a crosslinking effect. While this approach is

interesting experimentally, transitioning to full clinical development is quite complex due

to formulation related issues.

Eµ-TCL1 transgenic mouse model of CLL

A number of the studies described herein utilize the TCL1 mouse as a model of

CLL. These mice develops a leukemia closely resembling the more aggressive and

treatment-resistant subtype of CLL (Bichi et al., 2002; Yan et al., 2006). This leukemia

has the same CD5+CD19+ phenotype as human CLL in addition to displaying

immunodeficiencies and epigenetic changes that are observed in the human counterpart

(Ramsay et al., 2008; Chen et al., 2009). Overall, it is the best characterized drug

development tool for this malignancy (Johnson et al., 2006). Human CLL is a disease of

the elderly and TCL1 leukemia mimics this and requires a lengthy development period

that can exceed 1 year. However, the leukemia readily engrafts into normal mice on a

C57BL/6 background, which is advantageous from an experimental standpoint. Due to the lack of cross-reactivity of anti-human antibodies with equivalent mouse proteins, it is

challenging to study mAbs using this model (Macor et al., 2008). To address this for

CD37-targeted therapies we have developed a transgenic mouse expressing human

CD37 on its B-cells and crossed it with the TCL1 mouse to provide a model of spontaneous, human CD37-positive leukemia (hCD37xTCL1 mouse). This characterization of this model is the subject of Chapter 2 where it will be discussed in greater detail.

CLL microenvironment

Historically, CLL was viewed as a disease characterized by the slow accumulation of malignant B-cells with defects in apoptosis, but this has changed as the importance of a proliferative component has become increasingly apparent (Chiorazzi et al., 2005;

Messmer et al., 2005; Soma et al., 2006). Current evidence suggests that a subset of transformed B-cells form proliferative centers in lymphoid tissues, which are the source of the malignant cells that accumulate in the blood (Soma et al., 2006; Caligaris-Cappio et al., 2008; Vandewoestyne et al., 2011). In these tissues CLL cell survival, proliferation, and retention can be supported by accessory cells within the tumor microenvironment (Burger et al., 2000; Ghia et al., 2002; Burkle et al., 2007; Burger et al., 2013).

Kinase inhibitors

The tumor microenvironment in CLL is complex, and involves contribution from numerous cells types and signaling pathways. One of the most significant contributions of the lymphoid microenvironment is activation of B-cell receptor (BCR) signaling, which drives expansion of the malignant clone (Woyach et al., 2012). The importance of the

BCR pathway in CLL is highlighted by the clinical activity of drugs that inhibit various components, such as PI3K, Bruton’s tyrosine kinase (Btk), and spleen tyrosine kinase

(Woyach et al., 2012; Burger et al., 2013; Byrd et al., 2013). In particular, the Btk inhibitor ibrutinib has had a major impact on the management of CLL patients and even produces similar responses in high risk patients with deletion 17p (Byrd et al., 2013).

Idelalisib is another recently approved kinase inhibitor which has specificity for PI3Kδ.

These kinase inhibitors (and others still under clinical investigation) are rapidly altering how CLL is treated.

Thesis Objectives

The overall objective of this thesis is to develop novel approaches to CLL

immunotherapy that can be successfully translated to the clinic. In Chapter 2, I describe

a mouse model of human CD37-positive leukemia, providing a valuable preclinical tool

for evaluating anti-CD37 therapeutics. This mouse model enables the thorough study of

a CD37-specific antibody-drug conjugate in Chapter 3. The focus of Chapter 4 is the

development of combination strategies that enhance CD37-targeted therapy, which

includes immunomodulation and use of a kinase inhibitor. Chapter 5 investigates use of

a therapeutic that redirects T-cells to eliminate CD37+ tumor. Finally, Chapter 6

describes ongoing efforts to elucidate the function of CD37 in leukemia.

Figure 1.1. Structural features of tetraspanins.

Several common features of tetraspanins are depicted here. They possess 4 transmembrane domains (which are highly conserved), two short cytoplasmic tails, and two extracellular portions known as the EC1 domain (small extracellular loop) and EC2 domain (large extracellular loop).

Portions of the EC2 domain are conserved between various tetraspanins, but it also contains a highly variable region (shown in red). One of the features of this segment is the presence of 2-4 disulfide bonds (yellow lines) formed between cysteine residues (yellow circles), the number of which depend on the particular tetraspanin. The variable region of the EC2 domain contains binding sites for interactions with partner proteins and is frequently where epitopes for anti- tetraspanin antibodies are found. Many tetraspanins undergo palmitoylation at cysteine residues located near the intracellular border of the four transmembrane proteins. Additionally, most tetraspanins also experience N-linked glycosylation at extracellular asparagine residues (not depicted).

Figure 1.2. Signaling pathway associated with CD37 ligation by SMIP-016.

Lapalombella et al. described several cytoplasmic proteins which can associate with CD37, as depicted. Ligation by SMIP-016 leads to phosphorylation of Tyr13 within an ITIM-like motif in the

continued

Figure 1.2 continued

N-terminal cytoplasmic tail, which associates with a complex of proteins that includes Syk, Lyn,

and SHP1 (which likewise become phosphorylated). In addition, SMIP-016 induces

phosphorylation of an ITAM-like motif (containing Tyr274 and Tyr280) located in the C-terminal cytoplasmic tail that recruits PI3Kδ. Mutational studies suggest that the events requiring the N-

terminal ITIM drive apoptosis, while the C-terminal tail has a role in promoting cell survival. The

proposed mechanism of anti-CD37 induced cellular death involves a balance between these

signals, with preferential SHP1 activation driving apoptosis. SHP1 is capable of inactivating both

PI3K and Akt. SMIP-016 decreases the nuclear localization of Akt, preventing phosphorylation of

Fox O3a (and promoting retention in the nucleus) to allow transcription of pro-apoptotic BIM. An

opposing signal is transduced through PI3Kδ recruited to the C-terminal ITAM, activating Akt and

resulting in the downstream phosphorylation of GSK3β (which permits nuclear translocation of

pro-survival β-catenin). However, the contribution of PI3Kδ to survival can be eliminated by either

combination with a PI3K inhibitor or deletion of the ITAM-containing C-terminal domain of CD37.

While both pro-survival and pro-apoptotic signaling pathways are activated upon ligation by anti-

CD37 SMIP-016, those that promote cellular death predominate. Several other CD37-targeted

antibodies directly induce leukemia cell death, presumably in a similar fashion as SMIP-016/TRU-

016. However, they do not require additional receptor crosslinking (by use of anti-Fc antibody to

amplify the signal) as was observed with SMIP-016.

Figure 1.3. CD37 targeted antibody therapeutics.

Left: Otlertuzumab is an ADAPTIR™ molecule, constructed from an anti-CD37 single-chain variable fragment (scFv; a binding domain formed by linking the heavy and light chain variable regions of an immunoglobulin) which has been fused to the hinge region and Fc domain of human IgG1. Middle: mAb 37.1 is an Fc-engineered IgG1 with specific amino acid substitutions within the Fc region to increase ADCC mediated by effectors such as NK cells and macrophages.

Right: IMGN529 is a humanized anti-CD37 IgG1 (K7153A) conjugated to 3–4 molecules of cytotoxic drug (DM1) by stable thioether bonds. mAb, monoclonal antibody; CHO, Chinese hamster ovary; VH, heavy chain variable region; VL, light chain variable region; CH, heavy chain constant region (1, 2, or 3); CL, light chain constant region; D (orange circles), DM1; SMCC, N- succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate.

CHAPTER 2

A MOUSE MODEL OF HUMAN CD37-EXPRESSING LEUKEMIA

As discussed in Chapter 1, the anti-CD20 mAb rituximab has made a

tremendous impact on the treatment of CLL. Despite the benefits it provides as part of

chemoimmunotherapy regimens, resistance is common and relapse essentially

inevitable (Rezvani and Maloney, 2011; Woyach et al., 2011). Expanding our arsenal

with novel immunotherapies that target alternative antigens could help circumvent

resistance to anti-CD20 therapy.

One of the most promising alternative targets is CD37, a tetraspanin protein

highly expressed on malignant B-cells in CLL and NHL (Link et al., 1986; Schwartz-

Albiez et al., 1988). While a natural ligand for CD37 has not been identified, signaling

induced by the anti-CD37 peptide SMIP-016 has implicated this tetraspanin as a

mediator of the PI3K/Akt survival pathway (Figure 1.2). The interest in this target has led

to the generation of several novel platforms of anti-CD37 therapeutics, including the

small modular immunopharmaceutical peptide otlertuzumab (TRU-016, the fully

humanized version of SMIP-016), an Fc-engineered IgG1 mAb, and an antibody-drug

conjugate (Zhao et al., 2007; Heider et al., 2011; Deckert et al., 2013). These therapies

are being explored for use in a variety of hematological malignancies, but my primary

focus has been their potential use in CLL. An ideal set of preclinical studies to evaluate

such therapies would include in vivo experiments in rodents, but suitable CLL mouse models have been lacking. The study of anti-human CD37 mAbs in vivo is impeded by

the lack of cross-reactivity with the murine protein. Xenograft of human cell lines into immunodeficient mice represents a common strategy in preclinical drug testing, although it is suboptimal as a tool for studying an immunotherapy and few CLL cell lines exist

(and they are rather poor representations of the disease). Therefore, a need exists for an improved mouse model that facilitates the preclinical evaluation of CD37-targeted therapies. To address this, our lab generated a human CD37 transgenic mouse. I have extensively characterized this mouse and used it to generate a spontaneous model of murine CLL expressing human CD37.

Materials and Methods

Mice

The human CD37 transgenic mouse (hCD37-Tg) was generated on a C57BL/6

background at the OSUCCC Transgenic Mouse Facility by conventional methodology as

previously described (Chen et al., 2006). B-cell restricted hCD37 expression is driven by

immunoglobulin (Ig) VH promoter and IgH-μ enhancer elements in the pBH expression

vector (Chen et al., 2006). The transgenic construct was generated by ligating the cDNA

sequence of human CD37 (with an additional FLAG sequence) into EcoRI and NotI sites

within the pBH vector. To generate our leukemia model, I crossed hemizygous hCD37-

Tg mice with homozygous Eμ-TCL1 mice (C57BL/6 background) that have been

previously described (Bichi et al., 2002; Johnson et al., 2006). Mice were housed in

microisolator cages under controlled temperature and humidity. All animal procedures

were performed in accordance with Federal and Institutional Animal Care and Use

Committee (IACUC) requirements.

Flow Cytometry

Flow experiments were performed using Beckman Coulter FC 500 flow cytometers (Brea, CA). Apoptosis assays were conducted on cells stained with fluorescein isothiocyanate (FITC) conjugated Annexin V and propidium iodide (PI) in 1X

Annexin V binding buffer (BD Biosciences, San Jose, CA). Other experiments used fluorochrome-labeled monoclonal antibodies against mouse CD3 (17A2), B220 (RA3-

6B2), CD19 (ID3), CD5 (53-7.3), CD4 (RM4-5), CD8 (53-6.7), CD45 (30-F11) (BD

Biosciences) and anti-human CD37 (K7153A) conjugated to R-Phycoerythrin (K7153A-

PE) that was provided by ImmunoGen, Inc. (Waltham, MA). Flow cytometric data was analyzed using Kaluza software (Beckman Coulter), with the exception of cell cycle experiments which were analyzed with Flowjo (Tree Star, Ashland, OR). Gating was verified with appropriate Fluorescence Minus One (FMO) controls. Quantification of

CD37 surface expression was performed using QuantiBRITE PE bead assay (BD

Biosciences) and 1:1 conjugated K7153A-PE.

Direct cytotoxicity assays

Mouse splenic B-cells were purified using EasySep kits (STEMCELL

Technologies). Cell viability was assessed by Annexin V-FITC and propidium iodide (BD

Biosciences) staining after 24 hour incubation at 37°C with 10 μg/ml anti-CD37 antibody

+/− 50 μg/ml goat anti-human Fc crosslinking antibody (Jackson ImmunoResearch,

West Grove, PA). Samples were analyzed by flow cytometry with at least 20,000 events

collected.

Results

Generation of a human CD37 transgenic mouse model of leukemia

With the ultimate goal of creating a mouse model accurately recapitulating the

characteristics of human CLL while expressing the human CD37 (hCD37) target protein,

the first steps were to characterize the hCD37 transgenic mouse (hCD37-Tg) generated

in our lab. The hCD37-Tg was created using an expression vector that contained IgVH

promoter and IgH-μ enhancer elements to achieve B-cell specific hCD37 expression

(Figure 2.1a). Presence of the transgene was confirmed by PCR using genomic DNA

(Figure 2.1b) and B-cell specific expression hCD37 expression was established using

flow cytometry performed on samples from spleen and peripheral blood (Figure 2.1c,d).

These data also demonstrated that hCD37 is not expressed on T-cells in the hCD37-Tg

mouse. However, quantification of CD37 antigen density of revealed that expression was

significantly lower than that observed on human B-cells (Figure 2.1e). CD37-specific

mAbs characteristically display high levels of direct cytotoxicity against leukemia cells in

vitro, particularly upon crosslinking with anti-Fc antibody (Zhao et al., 2007; Heider et al.,

2011; Deckert et al., 2013). To determine whether this also occurred with B-cells hCD37-

Tg mice, I purified splenic B-cells and incubated them with the anti-CD37 antibody

K7153A for 24 hours. This agent demonstrated in vitro cytotoxicity against hCD37-Tg B-

cells but did not decrease the viability of hCD37-negative B-cells from non-transgenic

littermates (Figure 2.2a,b). The observed cytotoxicity with K7153A prompted me to cross

the hCD37-Tg line with the Eμ-TCL1 mouse, an established model of IgVH unmutated

CLL which has been extensively characterized as a drug development tool (Bichi et al.,

2002; Johnson et al., 2006; Yan et al., 2006; Ramsay et al., 2008; Chen et al., 2009). B-

cells from these hCD37×TCL1 offspring retained expression of both transgenes (Figure

2.3a,b). As expected, elderly mice developed the CD5+CD19+ leukemia previously described in the original Eμ-TCL1 mouse (Bichi et al., 2002; Johnson et al., 2006). I then confirmed expression of hCD37 on this CD5+CD19+ cell population in the blood, spleen, and lymph nodes of a leukemic hCD37×TCL1 animal (Figure 2.3c).

Discussion

Herein I have described the generation of a human CD37 transgenic (hCD37-Tg) and its subsequent cross with the Eμ-TCL1 mouse to develop a model of spontaneous hCD37+ leukemia. This hCD37×TCL1 model addresses the need for improved platforms to preclinically evaluate anti-human CD37 therapeutics. Specifically, it provides the first model of a low-grade B-cell lymphoproliferative disorder that expresses human CD37.

This mouse is used extensively for key experiments described in the chapters to follow.

Existing models for evaluating anti-CD37 therapeutics are limited by their inability to reproduce the full spectrum of disease associated with B-cell malignancies. The most commonly used approach involves xenografts of human cells into immunodeficient mice, but these models fail to demonstrate the same tumor behavior or typical interactions with the tissue microenvironment (Macor et al., 2008). In CLL xenografts the injected cells largely remain in the peritoneal cavity, demonstrating a small presence of leukemic cells in the spleen and bone marrow while virtually no cells circulate in the peripheral blood

Shimoni et al., 1997; Durig et al., 2007). Models of spontaneous leukemia such as the

Eμ-TCL1 mouse produce a phenotype that more accurately portrays the compartmental distribution seen in human disease (Bichi et al., 2002; Macor et al., 2008). The leukemia developed by the Eμ-TCL1 model closely resembles the more aggressive subtype of

IgVH unmutated CLL, exhibiting similar behavior, immunodeficiencies, and epigenetic

changes as those observed in the human disease (Bichi et al., 2002; Johnson et al.,

2006; Yan et al., 2006; Ramsay et al., 2008; Chen e tal., 2009). The major disadvantage of this model, however, is that it cannot be utilized for preclinical evaluation of most therapeutic antibodies as they lack cross-reactivity with the mouse proteins. Expressing the human target protein in transgenic mice is an alternative, as when another group did so to evaluate their Fc-engineered CD37 antibody in vivo (Heider et al., 2011). However, this does not permit evaluation in the context of malignancy, where therapeutic efficacy may be dramatically altered. The hurdle for evaluating anti-CD37 therapies is overcome with the generation of our hCD37×TCL1 mouse model. In addition, the hCD37-Tg I used to generate this CLL model could be crossed with other models of spontaneous leukemia/lymphoma for testing CD37 targeted therapeutics in the context of different hematological malignancies. While the development of this mouse represents a major advance it should be noted that the expression of CD37 is lower than that observed in human CLL. This has the potential to influence response to anti-CD37 therapies tested in this model, and is further addressed in Chapter 3 where it is utilized for the preclinical evaluation of IMGN529, a CD37-specific antibody-drug conjugates.

Figure 2.1. Generation and characterization of human CD37 transgenic mouse.

A) Schematic representation of the construct used to generate the hCD37-Tg mouse. B) PCR

genotyping of founder lines with human CD37 specific primers. C) Surface expression of human

CD37 on transgenic (TG; top) and non-transgenic (NTG; bottom) splenocytes. Cells stained for

B220 and CD3 to analyze B and T lymphocytes, respectively. D) Surface expression of human

continued 25

Figure 2.1 continued

CD37 on transgenic (TG; top) and non-Ctransgenic (NTG; bottom) peripheral blood leukocytes.

Whole blood stained for CD19, CD4, CD8, and human CD37. After staining, RBCs were lysed and samples analyzed by flow cytometry. E) Quantification of human CD37 surface expression on CLL (n=10), hCD37-Tg (n=9), and NTG B-cells (n=8). ABC = Antibody Binding Capacity

Figure 2.2. Human CD37 transgenic B-cells are sensitive to anti-CD37 therapy

A) Example of flow cytometry performed on purified splenic B-cells stained with Annexin V/PI to assess viability after 24 hour treatment with 10 μg/ml anti-CD37 antibody (K7153A) and 50 μg/ml

goat anti-human Fc antibody (αFc) for crosslinking. B) Mean viability (+/- SEM) of purified splenic

B-cells from transgenic or non-transgenic spleens (n=7/group) 27

Figure 2.3. hCD37xTCL1 mice retain expression of both transgenes and develop CLL

continued 28

Figure 2.3 continued

A) Flow cytometry performed on peripheral blood from a CD37xTCL1 double transgenic mouse and a TCL1 littermate. Cells were stained with antibodies against mouse B220 to label B-cells

and human CD37. B) Western blot using protein lysates from purified B-cells obtained from

spleens of CD37xTCL1 mice or TCL1 littermates. Western blot is probed with anti-human TCL1 antibody. Lysates from C57BL/6 and leukemic TCL1 mice are included as controls in the first two lanes. C) Flow cytometry performed on samples obtained from peripheral blood, spleen, and lymph nodes of a leukemic CD37xTCL1 mouse. Cells were stained for CD5, CD19, and human

CD37. The right side of the panel demonstrates staining with K7153A-PE antibody and is gated on either CD5+CD19+ B-cells (dark gray histogram) or hCD37-negative T-cells (light gray histogram).

CHAPTER 3

A CD37-SPECIFIC ANTIBODY-DRUG CONJUGATE FOR B-CELL MALIGNANCY

IMGN529 is an antibody-drug conjugate (ADC) that consists of the cytotoxic

maytansine-derivative DM1 linked to a CD37-targeting humanized IgG1 antibody via a

stable SMCC linker (Deckert et al., 2013). This strategy seeks to combine the potent

cytotoxicity demonstrated by anti-CD37 therapeutics with the specific delivery of DM1,

which exerts anti-proliferative effects by disrupting microtubule dynamics during mitosis

(Oroudjev et al., 2010). The clinical viability of DM1-conjugated antibodies has been

demonstrated by ado-trastuzumab emtansine (T-DM1, KadcylaTM), now approved by the

U.S. Food and Drug Administration for treatment of HER2+ metastatic breast cancer

(Verma et al., 2012). Brentuximab vedotin, which is indicated for the treatment of refractory Hodgkins lymphoma and systemic anaplastic large cell lymphoma, is currently the only other ADC approved for marketing by the FDA (de Claro et al., 2012). However, given that over 20 ADCs are currently under evaluation in human trials for a wide variety of indications, this therapeutic strategy seems poised to become increasingly prominent.

While CLL was once viewed as a malignancy driven by apoptosis resistance, the importance of a proliferative component and interactions between tumor and the tissue microenvironment has become increasingly apparent (Caligaris-Cappio, 2003; Messmer et al., 2005; Soma et al., 2006; Chiorazzi et al., 2007). Current evidence suggests that a subset of transformed B-cells form proliferative centers in lymphoid tissues and are the source of the malignant cells which slowly accumulate in the peripheral blood (Soma et

al., 2006; Chiorazzi et al., 2007; Vandewoestyne et al., 2011). In vivo measurements indicate that birth rate of CLL B-cells can exceed 1% of the total malignant clone per day

(Caligaris-Cappio, 2003). However, it is still unknown whether ADCs carrying anti-mitotic payloads will have utility in CLL given that proliferation is lower than most subtypes of

NHL (Broyde et al., 2009).

Therefore, it is not surprising that the focus of IMGN529’s clinical development has been as a lymphoma therapy. My initial work with this ADC focused on its in vitro activity, but it is impossible to address the crucial question surrounding IMGN529 on the basis of these experiments because CLL B-cells do not proliferate ex vivo. Therefore, we

require an in vivo model of CLL in order to evaluate the potential utility of IMGN529. With the development of the hCD37xTCL1 model of CLL described in Chapter 3, the goal of characterizing IMGN529’s anti-proliferative effects became attainable.

Materials and Methods

Human Samples

Peripheral blood mononuclear cells (PBMCs) were obtained from normal donors or CLL patients in accordance with the Declaration of Helsinki. All subjects gave written, informed consent for their blood products to be used for research under an institutional review board approved protocol. Blood from CLL patients was collected at The Ohio

State University Wexner Medical Center (Columbus, OH). Normal cells were obtained from Red Cross partial leukocyte preparations or healthy donor blood. PBMCs were isolated by Ficoll density-gradient centrifugation (Ficoll-Paque Plus, GE Healthcare,

Uppsala, Sweden). Except when performing whole blood experiments, all CLL samples underwent negative selection of B-cells with RosetteSep (STEMCELL Technologies,

Vancouver, BC, CA) according to manufacturer’s protocol. Cells were cultured at 37°C and 5% CO2 in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum

(Sigma, St. Louis, MO), 2 mM L-glutamine (Invitrogen, Carlsbad, CA), 56 U/mL penicillin and 56 μg/ml streptomycin (Invitrogen).

Monoclonal antibody therapeutics

Therapeutics used in our studies, which include alemtuzumab, rituximab, ofatumumab, and trastuzumab, were purchased from the pharmacy at The Ohio State

University Wexner Medical Center. ImmunoGen, Inc. (Waltham, MA) provided several

humanized IgG1 reagents: IMGN529, K7153A, and a non-binding chKTI-SMCC-DM1

(IgG-DM1) control ADC.

Mice

The generation of hCD37-Tg mice and subsequent breeding to create

hCD37xTCL1 mice are described in Chapter 2. Mice were housed in microisolator cages

under controlled temperature and humidity. All animal procedures were performed in

accordance with Federal and Institutional Animal Care and Use Committee (IACUC)

requirements.

Flow Cytometry

Flow experiments were performed using Beckman Coulter FC 500 flow

cytometers (Brea, CA). Apoptosis assays were conducted on cells stained with

fluorescein isothiocyanate (FITC) conjugated Annexin V and propidium iodide (PI) in 1X

Annexin V binding buffer (BD Biosciences, San Jose, CA). Other experiments used

fluorochrome-labeled monoclonal antibodies against mouse CD3 (17A2), CD19 (ID3),

CD5 (53-7.3), CD45 (30-F11) (BD Biosciences) and anti-human CD37 (K7153A) conjugated to R-Phycoerythrin (K7153A-PE) that was provided by ImmunoGen, Inc.

(Waltham, MA). Flow cytometric data was analyzed using Kaluza software (Beckman

Coulter), with the exception of cell cycle experiments which were analyzed with Flowjo

(Tree Star, Ashland, OR). Gating was verified with appropriate Fluorescence Minus One

(FMO) controls. Absolute cell concentrations were obtained by quantitative flow cytometry using Count Bright absolute counting beads (Invitrogen).

Mouse leukemia engraftment

Splenocytes from a moribund, leukemic hCD37×TCL1 donor were isolated by

Ficoll-Paque density gradient and re-suspended in sterile PBS for injection. Healthy female hCD37-Tg mice received an intravenous lateral tail vein injection of 200 μl containing 1×107 splenocytes. Age-matched mice were randomly assigned to the

following treatment groups (n=6-7 per group): the IMGN529 ADC, its K7153A antibody

component, an IgG-DM1 ADC control, or trastuzumab as an irrelevant humanized IgG1

antibody control. Mice were monitored for disease by flow cytometry on a weekly basis.

Upon leukemia diagnosis, a 10 mg/kg dose of the appropriate treatment was

administered intraperitoneally and repeat doses were given 2 times per week for 3

weeks (70 mg/kg total). Leukemia onset was defined as when greater than 20% of

CD45+ cells in the peripheral blood consisted of CD5+CD19+ leukemic B-cells or when

splenomegaly was evident upon light palpation. To avoid unnecessary suffering, mice

were euthanized with CO2 upon reaching standard IACUC criteria for early removal (e.g.

weight loss >20%, severe lethargy, labored breathing).

Direct cytotoxicity assays

Freshly isolated CLL B-cells from patient blood was enriched using RosetteSep

(STEMCELL Technologies). Cell viability was assessed by Annexin V-FITC and

propidium iodide (BD Biosciences) staining after 24 hour incubation at 37°C with 10

μg/ml antibody +/− 50 μg/ml goat anti-human Fc crosslinking antibody (Jackson

ImmunoResearch, West Grove, PA). For cell line experiments, Raji cells were incubated

with 1 μg/ml antibody for 72 hours. Data are reported as the percentage of remaining

viable cells (those that were both Annexin V and PI negative) normalized to untreated

control. Samples were analyzed by flow cytometry and at least 20,000 events collected.

Antibody dependent cellular cytotoxicity (ADCC)

The degree of ADCC was assessed using a standard 51Cr release assay, as

described previously.10 After 30 minutes of treatment with 10 μg/ml antibody, a total of

5×104 CLL target cells labeled with 51Cr were co-incubated with NK cells obtained from healthy donors for 4 hours at 37°C in 96-well plates at effector-to-target ratios of 25:1,

6.25:1, or without effectors. Following this incubation, supernatants were harvested and chromium release was measured with a Perkin Elmer Wizard 2 gamma counter

(Waltham, MA). Maximum chromium release was determined using targets lysed with sodium dodecyl sulfate (SDS). Cytotoxicity was calculated as follows: %Specific lysis =

(Experimental 51Cr release – Spontaneous 51Cr release) ÷ (Maximum 51Cr release –

Spontaneous 51Cr release) × 100. NK cells for this assay were enriched from Red Cross partial leukocyte preparations and healthy donor blood using RosetteSep kits

(STEMCELL Technologies).

Antibody dependent cellular phagocytosis (ADCP)

Monocytes were isolated from Red Cross partial leukocyte preparations using

MACS CD14+ selection kit (Miltenyi Biotec, Auburn, CA) according to manufacturer protocols. Monocytes were cultured in 10 cm2 dishes using RPMI 1640/10% FBS containing 20 ng/ml monocyte-colony stimulating factor (M-CSF; R&D Systems,

Minneapolis, MN) to promote differentiation into monocyte-derived macrophages

(MDMs). Fresh media containing M-CSF was provided every 2 days. After 7-10 days of

incubation, adherent macrophages were harvested and labeled with Claret dye (Sigma).

CLL cells were labeled with PKH67 fluorescent dye (Sigma), then treated with 10 μg/ml

antibody for 1 hour on ice and washed twice. Labeled cells were co-incubated at an

effector-to-target ratio of 1:5 (1×106 MDMs and 5×106 CLL cells) for 30 minutes at 37°C.

Samples were fixed with 1% paraformaldehyde prior to analysis by flow cytometry.

Relative phagocytosis = (%Claret-positive MDMs becoming PKH67+ in the treatment

condition) − (%Claret-positive MDMs becoming PKH67+ in the untreated control). At

least 10,000 Claret-positive (MDM) events were collected per sample.

Complement dependent cytotoxicity (CDC)

RosetteSep purified B-cells and plasma were isolated from the blood of CLL

patients. In a final volume of 500 μl, a total of 5×105 CLL B-cells were combined with 150

μl of autologous plasma (+/− heat-inactivation for 30 minutes at 57°C),10 μg/ml antibody, and incubated for 1 hour at 37°C. Following this incubation, samples were washed once and dead cells stained with LIVE/DEAD Fixable near-IR (Invitrogen). Cells were then fixed with 1% paraformaldahyde until analysis by flow cytometry. The percentage of

lysed cells was calculated by subtracting the percentage of dead cells in the corresponding untreated control. At least 20,000 events were collected per sample.

CLL B-cell depletion in whole blood

Whole blood from CLL patients (90 μl) was incubated with 10 μg/ml antibody for

1 hour at 37°C. 27 After this treatment, blood was stained with anti-CD3 FITC (UCHT1) and CD19 PE (HIB19) from BD Biosciences. Cal-lyse (Invitrogen) was used to simultaneously lyse RBCs while fixing the leukocytes. Count Bright absolute counting beads were added just prior to analysis by flow cytometry in order to calculate absolute concentrations of cells. To adjust for differences in initial leukemia counts between patients, data was normalized as follows: Percent depletion = 100 × (1 – [concentration of cells in treated sample] ÷ [concentration of cells in untreated control]).

Cell cycle analysis

Raji cells were synchronized with 2 μg/ml aphidicolin (Sigma) for 24 hours, transferred to fresh media, and treated with 1 μg/ml antibody for an additional 24 hours.

Following incubation, 1×106 cells (re-suspended in 1 ml PBS) were fixed by addition of 3

ml absolute ethanol. After fixation for at least 24 hours, these samples were stained in a

PBS solution of 0.1% (v/v) Triton X-100, 200 μg/ml DNase-free RNase, and 30 μg/ml

propidium iodide (Sigma). The proportion of singlet cells in G1, S, and G2 were obtained

with Flowjo analysis software (Tree Star) using the Dean-Jett-Fox model and the

assumption that the G2 coefficient of variance (CV) is equal to the G1 CV.

In vivo cell proliferation assay

To assess whether IMGN529 could inhibit in vivo proliferation, we engrafted

healthy hCD37-Tg mice with CD37xTCL1 leukemia. After detection of peripheral blood

leukemia (Day 0), mice were randomized to groups that would receive either IMGN529

or IgG-DM1 control in a blinded fashion. On Day 3 and Day 5 these mice received 10

mg/kg i.p. antibody, followed by 100 μg ethynyl-2’deoxyuridine (EdU) on Day 6. Tissues

were collected 24 hours later and incorporation of EdU was detected using Click-it EdU

Alexa Fluor 647 Flow Cytometery kit (Life Technologies) according to manufacturer

recommendations. Gating for EdU positivity was determined using a control mouse

which did not receive EdU.

Statistical analysis

For Raji cell line experiments, analysis of variance (ANOVA) was performed. For

patient sample data which involved repeated measures, mixed effect models were

utilized to account for dependencies across different treatment groups. For the in vivo

study, log-rank tests were used to compare the survival probabilities between mouse

groups. Holm’s method was used to adjust multiplicities. SAS 9.3 software was used for

data analysis (SAS, Inc; Cary, NC).

Results

The CD37-targeting IMGN529 directly induces apoptosis of CLL B-cells and maintains

Fc-dependent killing by innate immune cells in vitro

Addressing whether ADCs may be capable of targeting the proliferative

component of CLL is complex and ultimately requires a suitable in vivo model. However,

I initially characterized the antibody-derived activity of the IMGN529 ADC against primary human CLL, which does not proliferate ex vivo in the absence of stimulation.

Treatment with IMGN529 or its antibody component alone (K7153A) demonstrated significant in vitro cytotoxicity against peripheral blood CLL B-cells (Figure 3.1a,b). This effect was further augmented by the addition of anti-Fc crosslinking antibody, but did not require its presence to induce cellular apoptosis. The ability for these therapies to induce apoptosis of CLL B-cells was not dependent on IgVH mutational status (Figure 3.1c).

Interestingly, B-cells isolated from healthy donor blood were less susceptible to direct killing by these antibodies (Figure 3.1d). IMGN529 and its antibody component K7153A also mediated antibody-dependent cellular cytotoxicity (ADCC) against CLL by healthy donor NK cells (Figure 3.2a). No significant difference was observed between IMGN529 and K7153A with respect to their ability to mediate ADCC. Furthermore, both agents equally promoted phagocytosis of CLL by monocyte-derived macrophages (Figure 3.2b and Figure 3.3). Neither K7153A nor IMGN529 exhibited complement-dependent cytotoxicity when CLL B-cells were incubated with autologous plasma (Figure 3.2c).

While the above experiments are informative, I sought to further evaluate these reagents in a context where the collective impact of multiple potential mechanisms could be appreciated. Therefore, I performed B-cell depletion assays in CLL patient whole blood, where various innate immune components that may contribute to the therapeutic efficacy of antibodies (complement, NK cells, monocytes, and granulocytes) are present in physiologically relevant proportions to leukemia cells. One hour treatment of CLL patient whole blood with IMGN529 or K7153A resulted in significantly greater malignant

B-cell depletion than rituximab or alemtuzumab (Figure 3.4 and Figure 3.5a). In addition,

CD37-targeted therapeutics avoided the undesirable T-cell depletion that is observed

with anti-CD52 alemtuzumab (Figure 3.5b). A limited analysis suggests that activity in whole blood does not vary on the basis of cytogenetic abnormalities, although a much larger sample size would be required to achieve greater certainty (Figure 3.5c).

Likewise, IgVH mutational status did not significantly alter activity (Figure 3.5d).

Given the lack of in vitro proliferation associated with CLL B-cells, I sought to determine whether delivery of DM1 by IMGN529 would have an impact on actively dividing transformed B-cell lines. Raji cells were treated with K7153A, IMGN529, or control antibodies for 72 hours. In contrast to what was observed with non-dividing CLL

B-cells, IMGN529 demonstrated superior cytotoxicity against Raji cells compared to its

K7153A antibody component (Figure 3.6a). DM1 is known to kill cells through disruption of microtubules, resulting in G2/M arrest and mitotic catastrophe (Oroudjev et al., 2010).

Consistent with this mechanism, cell cycle analysis of IMGN529-treated Raji cells

revealed that a substantial proportion were arrested at the G2/M checkpoint (Figure

3.6b).

IMGN529 selectively depletes leukemic B-cells in vivo and improves overall survival of mice with hCD37×TCL1 leukemia

To evaluate the therapeutic potential of IMGN529 in vivo, I engrafted splenocytes

from a leukemic hCD37×TCL1 donor into healthy hCD37-Tg recipients and monitored

these mice for disease. Upon leukemia development, mice were treated with the

IMGN529 ADC, its antibody component K7153A, an IgG-DM1 ADC control, or

trastuzumab as a non-specific humanized IgG1 control (Figure 3.7a). In contrast with my

in vitro cytotoxicity studies using human CLL cells, which showed no significant

difference between IMGN529 and its antibody component K7153A alone (see Figure

3.1b), improved overall survival was only observed in mice treated with IMGN529

(Figure 3.7b). One week of IMGN529 treatment was sufficient to eliminate peripheral

blood leukemia, while the disease continued to progress in other groups (Figure 3.7c,d).

In addition, previously detected splenomegaly disappeared with IMGN529 treatment

(Figure 3.8a). Depletion of hCD37-negative T-cells did not occur with IMGN529 (Figure

3.8b). Only a transient reduction of peripheral CD19+CD5+ B-cells was observed on day

21 after treatment with the K7153A antibody (Figure 3.7d).

IMGN529 eliminates proliferating hCD37×TCL1 leukemia cells in vivo

To better characterize the in vivo activity of IMGN529, I sought to track the effects on the proliferating subset of leukemia cells within lymphoid tissues. Mice were engrafted with hCD37×TCL1 leukemia and randomly assigned to treatment groups upon development of disease (day 0). On days 3 and 5, mice received 10 mg/kg i.p. doses of

IMGN529 or IgG-DM1 control, followed by EdU injection on day 6 to monitor in vivo cell

proliferation. At the beginning of the study, the proportions of peripheral blood

CD5+CD19+ leukemia were similar, but IMGN529 therapy eliminated most leukemia 48

hours after the second dose (Figure 3.9a). Leukemia cells were present in the blood,

spleen, and bone marrow of mice injected with IgG-DM1, but those receiving IMGN529

demonstrated significantly less tumor burden in these tissues (Figure 3.9b,c). By visual

inspection, spleens from all mice treated with IMGN529 were much smaller than those

from control mice. Although it is an underestimate of actual size difference, this is also

evident when spleen length is quantified (Figure 3.9d). Moreover, incorporation of EdU

was significantly lower among CD5+CD19+ cells remaining in the spleens of IMGN529

treated mice (Figure 3.9e).

Discussion

This chapter describes the extensive characterization of IMGN529’s in vitro activity using primary human CLL samples. I also utilized the novel hCD37xTCL1 mouse from the previous chapter to demonstrate potent in vivo efficacy in the context of murine

CLL. To my knowledge, this is the first ADC shown to effectively target the proliferative component of CLL in vivo.

My in vitro studies of IMGN529 show that conjugation of the CD37-targeting

K7153A antibody to DM1 does not diminish its ability to promote effector-mediated killing of CLL by NK cells and macrophages. Furthermore, both IMGN529 and K7153A greatly surpass rituximab in their ability to deplete malignant B-cells in CLL patient whole blood.

I also observed direct cytotoxicity against CLL in the absence of crosslinker, similar to what has been reported with another recently developed anti-CD37 therapeutic (BI

836826) but unlike the CD37-directed peptide TRU-016 (Zhao et al., 2007; Heider et al.,

2011). Interestingly, healthy donor B-cells were less sensitive to anti-CD37 induced apoptosis than human CLL B-cells. This cannot be explained by decreased expression of the target protein, as normal B-cells do not have less surface CD37 (Barrena et al.,

2005). Both murine splenic B-cells and human CLL B-cells display high degrees of spontaneous apoptosis during in vitro culture. Therefore, observed differences in direct cytotoxicity could be a result of normal human B-cells exhibiting decreased priming to undergo apoptosis, thus reducing their sensitivity to anti-CD37 therapeutics (which are expected to induce expression of pro-apoptotic mitochondrial protein BIM following

CD37 ligation (Lapalombella et al., 2012).

IMGN529 and K7153A were able to mediate similar levels of cytotoxicity against human CLL cells in vitro. This is in sharp contrast to what was observed in vivo, where

K7153A produced only modest depletion of leukemic B-cells and was unable to significantly alter the disease course in the hCD37×TCL1 model. While its antibody alone was not effective, the full IMGN529 ADC rapidly eliminated peripheral blood leukemia, reversed splenomegaly and improved overall survival. The different in vitro

and in vivo activity I observed with these agents is not entirely surprising given that CLL

cells do not proliferate in culture, which would make the delivery of anti-mitotic DM1

largely irrelevant in these short-term in vitro studies. When tested against proliferating

Raji cells in vitro, IMGN529 possessed a distinct advantage over its antibody component

with enhanced cytotoxic activity resulting in G2/M cell cycle arrest. I hypothesized that

IMGN529 eliminated dividing leukemia cells within the proliferative centers via targeted

delivery of DM1 in vivo, thus resulting in a greater therapeutic benefit than the antibody

alone. This is supported by the observation that peripheral leukemia depletion with

IMGN529 was accompanied by a rapid decrease in splenomegaly. To further investigate

the in vivo activity of IMGN529, I monitored its effects on leukemia cells within various

compartments of disease and examined those from lymphoid tissues for signs of

proliferation using EdU. In doing so, I confirmed that IMGN529 is effective at targeting

leukemia in both peripheral blood and the lymphoid compartment. EdU incorporation

was largely absent among the few CD5+CD19+ cells that remained after 2 doses,

suggesting that IMGN529 was indeed eliminating this proliferative subset of cells.

Additionally, the splenomegaly observed with IgG-DM1 controls was not present in mice

receiving IMGN529. While a small number of leukemia cells remained after 2 doses,

these displayed very little EdU incorporation. It is evident that IMGN529 is highly active

across multiple compartments of disease in this CLL mouse model. Given that the

expression of hCD37 was suboptimal compared to human CLL (Figure 2.1e), it is

unlikely that antigen density has led to an exaggerated response in the mouse model.

For this reason, delivery of the cytotoxic DM1 payload is likely even more efficient in human CLL cells.

As clinical study of IMGN529 moves forward, it would be worthwhile to explore potential combination therapies that may take advantage of the unique therapeutic properties of this ADC. Given that IMGN529 maintains the Fc-mediated effector functions of the antibody from which it was generated, combination with immunomodulatory drugs (such as lenalidomide) could prove effective. The anti-CD37 peptide TRU-016 previously demonstrated in vitro synergy with PI3K inhibitors

(Lapalombella et al., 2012). Therefore, it may also be worthwhile to explore combinations with inhibitors of PI3Kδ such as idelalisib. The newly generated hCD37×TCL1 mouse model provides a unique tool that will enable the evaluation of combination strategies for IMGN529 and other CD37-targeted therapeutics in an immunocompetent model of spontaneous CLL.

To summarize, I have demonstrated the utility for evaluation of anti-CD37 therapeutics provided by the mouse model described in Chapter 2 (which develops a transplantable CD5+CD19+hCD37+ leukemia). This model facilitated improved preclinical studies of IMGN529, elucidating its robust anti-leukemic effects in vivo. This is impressive in the context of the Eμ-TCL1 leukemia that is somewhat resistant to treatment (Johnson et al., 2006; Lucas et al., 2009; Lapalombella et al., 2012). Given the significant benefits we observed with IMGN529, despite relatively low hCD37 expression, I propose that this therapeutic could exhibit efficacy in a wide range of

CD37-positive human B-cell malignancies.

Figure 3.1. IMGN529 is directly cytotoxic to primary human CLL B-cells

continued 44

Figure 3.1 continued

A) Example of 24-hour Annexin V/PI viability assay performed on RosetteSep purified B-cells from CLL patient blood.B) Viability of freshly isolated CLL patient B-cells (n=16) following 24 hour

treatment with 10 μg/ml IMGN529 or its antibody component K7153A +/− 50 μg/ml crosslinking

antibody (αFc). Anti-HER2/neu antibody trastuzumab included as an additional negative control

for n=11 patients. Data are normalized to untreated controls. Significance is indicated by

asterisks (*p<0.05, ***p<0.0001, or NS if p>0.05). C) CLL patient B-cells with either unmutated

(UM, n=9) or mutated (M, n=5) IgVH genes assayed for viability by Annexin V/PI staining

following 24 hour treatment with 10 μg/ml IMGN529 or its antibody component K7153A +/- 50

μg/ml anti-Fc crosslinking antibody (αFc). Data are normalized to untreated control. IgVH was

considered unmutated when there was >98% homology to germline sequence. D) Healthy donor

B-cells (n=4) isolated by negative selection with Rosette-Sep. Purity was confirmed to be greater

than 90% CD19+ B-cells for all samples. Viability was assessed by Annexin V/PI staining and

flow cytometry following 24 hour treatment with 10 μg/ml IMGN529, its antibody component

K7153A, or the anti-HER2 trastuzumab as a negative humanized IgG1 control. Error bars indicate

SEM.

Figure 3.2. IMGN529 retains the Fc-mediated activities of its unconjugated precursor

continued

Figure 3.2 continued

A) NK cell mediated antibody-dependent cytotoxicity as measured by 51Cr release assay. CLL target cells (n=7) were incubated with 10 μg/ml antibody for 30 minutes, followed by incubation with healthy donor NK cells (n=8) for 4 hours. Mean specific lysis displayed for n=18 NK/CLL combinations with error bars indicating standard error of the mean (SEM). B) Induced phagocytosis of CLL cells by monocyte-derived macrophages (MDMs) following 1 hour incubation with 10 μg/ml antibody. Relative phagocytosis displayed for a total of n=10 MDM/CLL combinations (7 MDMs, 5 CLL) with error bars indicating SEM. C) Complement-dependent cytotoxicity assay for B-CLL incubated with 10 μg/ml antibody and autologous plasma (+/− heat inactivation) for 1 hour. Mean values displayed for n=9 CLL patients with error bars indicating

SEM.

Figure 3.3. Gating strategy for measuring phagocytosis of CLL B-cells

continued 48

Figure 3.3 continued

Antibody-dependent cellular phagocytosis (ADCP) of CLL cells by monocyte-derived macrophages (MDMs). Gating scheme is displayed. Initially, a singlet gate is created using FS-

Area vs. FS-Peak plot. Gate for PKH67 (CLL) vs. Claret (MDM) was verified using a sample with only Claret stained MDMs. For determination of phagocytosis, a gate is created on the singlet

Claret-positive (MDM) events. The percentage of these MDM events that are PKH67-positive is considered the percent phagocytosis. For relative phagocytosis, the %Phagocytosis in the untreated is subtracted from treated samples. Examples of each treatment group are shown.

Figure 3.4. Gating strategy for whole blood cytotoxicity assays

continued 50

Figure 3.4 continued

Whole blood CLL B-cell depletion assay. First, a singlet gate is created from the FS-Area versus FS-Peak plot, which includes both the lymphocytes and the CountBright counting beads.

Subsequent FS vs. SS gates are on either the lymphocyte population or the bead population. The

CountBright bead gate is further fefined using the SS vs. FL4 gate, as they fluoresce brightly in

the FL4 channel. The number of events obtained for each cellular event of interest (CD19+ or

CD3+ within the lymphocyte gate) and number of bead events collected were used to calculate absolute concentrations of cells using the known concentration of beads, as outlined by the manufacturer protocol. Examples of each treatment condition are shown. Data acquisition stopped at approximately 1000 bead events for each sample.

Figure 3.5. IMGN529 demonstrates exceptional cytotoxicity in CLL whole blood

continued

Figure 3.5 continued

A-B) CLL patient whole blood was treated with 10 μg/ml antibody for 1 hour at 37°C. B-cells and

T-cells were stained with anti-CD19 and CD3 antibodies respectively. Counts were obtained

using CountBright absolute counting beads and normalized to untreated control. Mean percent

depletion of B-cells A) and T-cells B) are displayed for n=23 CLL patients with error bars

indicating SEM. Greater than 97% of the CD19+ cells in these patients were confirmed to be malignant CD5+ B-cells.C) Limited analysis examining the effects of cytogenetic status on efficacy in whole blood B-cell depletion assays. Patient groups are as follows: no cytogenetic abnormalities (n=5), del13q (n=8), del17p (n=3), Trisomy 12 (n=3). Several patients for which cytogenetic status was either unavailable or with abnormalities lacking replicates are excluded. D)

Analysis examining the effects of IgVH mutational status on B-cell depletion in whole blood

assays (n=11 patients with mutated IgVH and n=8 patients unmutated IgVH). For several patients

IgVH mutational status was unavailable, and thus they were excluded. No statistically significant differences were detected (p=0.8559 for IMGN529 and p=0.8848 for K7153A). Error bars indicate

SEM.

Figure 3.6. DM1-conjugation improves killing and arrests proliferating B-cells

A) Raji cells were treated with 1 μg/ml K7153A, IMGN529, or relevant controls for 72 hours and viability assayed by Annexin V and propidium iodide staining. Data are normalized to untreated control and reported as mean with error bars indicating SEM for n=5 replicate cultures from two independent experiments. B) Cell cycle analysis of Raji cells that were synchronized with the

DNA polymerase inhibitor aphidicolin for 24 hours, released from this inhibition, and then treated with 1 μg/ml antibody for 24 hours.

Figure 3.7. IMGN529 demonstrates in vivo efficacy against hCD37+ mouse leukemia.

A) Schematic outline of the hCD37×TCL1 engraftment experiment. Healthy hCD37-Tg recipients

were injected i.v. with 1×107 splenocytes from a leukemic hCD37×TCL1 donor. Mice were

continued 55

Figure 3.7 continued randomly assigned (n=6-7/group) for treatment with 10 mg/kg IMGN529 ADC, its K7153A antibody component, IgG-DM1 ADC control, or trastuzumab control. Treatment began following leukemia development, which was defined as when >20% of CD45+ events were CD5+CD19+ B- cells. Animals received a 10 mg/kg i.p. treatment on the day of leukemia diagnosis and repeat doses twice weekly for three weeks (70 mg/kg total). B) Kaplan-Meier plot comparing the overall survival of engrafted mice. IMGN529 significantly improved survival relative to its IgG-DM1 control (p=0.042), while parent antibody K7153A had no survival benefit compared with trastuzumab control (p=0.94). Log-rank tests were used for statistical analysis. Arrow indicates day 20, when mice received their last injection. C) Representative examples of flow cytometry performed on mice 24h after receiving their third dose. Whole blood was stained for

CD45/CD19/CD5 and plots are gated on singlet CD45+ events. D) Absolute concentration of the

CD5+CD19+ B-cell population to which the leukemia belongs. Data expressed as mean +/− SEM.

Arrow indicates day 20, when mice received their last injection.

Figure 3.8. IMGN529 eliminates splenomegaly and demonstrates specificity for CD37+ cells

A) Measurement of spleen sizes in the hCD37xTCL1 leukemia engraftment study. A score was assigned to each mouse based on spleen size. Scores were given according to the following criteria: 0 = spleen not palpable, 1 = felt upon deep palpation, 2 = palpable with light touch, 3 =

spleen palpable from the diaphragm to the lower abdomen, 4 = massive splenomegaly, where

vertical growth has reached its limit and the spleen begins to wrap around the opposite side of the

mouse. Data is represented as mean spleen score +/- SEM for each treatment group. B) Mean T-

cells/µl (+/- SEM) in peripheral blood of IMGN529 mice (n=6).

Figure 3.9. IMGN529 targets proliferating mouse leukemia cells within lymphoid tissues.

A-E) Healthy hCD37-Tg mice engrafted with hCD37×TCL1 leukemia. Upon leukemia

development (day 0), mice were randomized (n=5 mice/group) to receive 10 mg/kg i.p. doses of

continued

Figure 3.9 continued

IMGN529 or IgG-DM1 control on days 3 and 5. To detect in vivo proliferation 100 μg EdU was administered on day 6 and mice were euthanized on day 7. (Mean is displayed for n=5 mice/group with error bars indicating SEM. ***p<0.0001, **p<0.01, *p<0.05) A) Percentage

CD5+CD19+ leukemia cells (of total CD45+ events) in peripheral blood on day 0 and day 7.

(***p<0.0001 for change in cell percentage). B) Percentage CD5+CD19+ leukemia cells (of total

CD45+ events) in the blood, spleen, and bone marrow on day 7. C) Examples of flow cytometry performed on peripheral blood, spleen, and bone marrow from mice treated with IMGN529 or

IgG-DM1. Plots are gated on singlet, live, CD45+ cells. D) Examples of spleens removed from mice receiving IMGN529 or IgG-DM1 (left panel) and average spleen length of all mice in the study (n=5/group; right panel). E) Percentage of EdU+ cells among CD5+CD19+ events in the spleen and bone marrow.

CHAPTER 4

DEVELOPING STRATEGIES TO ENHANCE ANTI-CD37 THERAPY

Otlertuzumab (TRU-016) has clinically validated CD37 as a target for immunotherapy (Byrd et al., 2014; Gopal et al., 2014; Pagel et al., 2014), and human trials have now begun for several novel anti-CD37 therapeutics which displayed improved preclinical activity. However, the response to immunotherapy is typically impaired when it is administered as a single agent. One strategy for improving response is to enhance Fc-mediated killing, which can be accomplished by combination with an immunomodulatory drug (or by directly altering the mAb through modifications of its Fc region). A variety of combination therapies have been used to achieve this, for example: treatment with cytokines (Roda et al., 2006), lenalidomide (Wu et al., 2008), or toll-like receptor (TLR) 7/8 agonists (Wu et al., 2008; Butchar et al., 2010).

A second approach to combination therapy with anti-CD37 therapeutics could instead focus on the direct cytotoxicity they exhibit. We previously described a mechanism that provided a likely explaination for the the cytotoxicity displayed by SMIP-

016. Upon ligation of CD37 by SMIP-016, two cytoplasmic regulatory motifs with opposing function become phosphorylated. This leads to both pro-apoptotic inactivation of Akt and (to a lesser extent) pro-survival PI3Kδ activation (Figure 1.2). Unsurprisingly, combining SMIP-016 with PI3K inhibitors led to improved in vitro cytotoxicity

(Lapalombella et al., 2012). This combination has not been explored with other CD37-

targeting therapeutics, so it has been unknown whether this effect can be replicated with

newer mAbs (particularly those which kill without need of crosslinking). In this chapter, I investigate the use of two strategies that I hypothesized would improve CD37-targeted therapy: 1) combining otlertuzumab (TRU-016) with immunomodulatory toll-like receptor

7/8 agonist, and 2) addition of PI3Kδ inhibitor to the Fc-engineered BI 836826.

Materials and Methods

Reagents

The TLR 7/8 agonist CL-075 (Invivogen, San Diego, CA) was provided by Jon

Butchar and Susheela Tridandapani as part of an ongoing collaboration. The Fc- engineered anti-CD37 antibody BI 836826 was provided by Boehringer Ingelheim

(Ingelheim am Rhein, Germany), and TRU-016 was provided by Emergent Biosolutions

(Gaithersburg, MD).

Direct cytotoxicity assays

Freshly isolated CLL B-cells from patient blood was enriched using RosetteSep

(STEMCELL Technologies). Cell viability was assessed by Annexin V-FITC and propidium iodide (BD Biosciences) staining after 24 hour incubation at 37°C with 0.1

μg/ml antibody +/− 1 μM idelalisib. Data are reported as the percentage of remaining viable cells (those that were both Annexin V and PI negative) normalized to untreated control. Samples were analyzed by flow cytometry and at least 20,000 events collected.

Antibody dependent cellular phagocytosis (ADCP)

Monocytes were isolated from Red Cross partial leukocyte preparations using

MACS CD14+ selection kit (Miltenyi Biotec, Auburn, CA) according to manufacturer

protocols. Monocytes were cultured in 10 cm2 dishes using RPMI 1640/10% FBS containing 20 ng/ml monocyte-colony stimulating factor (M-CSF; R&D Systems,