Redirecting T cells to cancer cells using a novel bispecific antibody Receptor identification and functional analysis

(1)

I

Redirecting T cells to cancer cells using a novel bispecific antibody

Receptor identification and functional analysis

Kamna Verma

Thesis for the Master degree in Bioscience Molecular Biology and Biochemistry

60 credits

Department of Biosciences

Faculty of Mathematics and Natural Sciences UNIVERSITY OF OSLO

June 2021

(2)

II

(3)

III

Redirecting T cells to cancer cells using a novel bispecific antibody

Receptor identification and functional analysis

Kamna Verma

^1,2

1

Department of Biosciences, University of Oslo, N-0371 Oslo, Norway

2

Departement of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital, Ullernchausseen 70, 0379, Oslo, Norway

©Kamna Verma 2021

http://www.duo.uio.no/

Print: Reprosentralen, Universitet i Oslo

(4)

IV

(5)

V

Acknowledgements

The project presented in this thesis was performed in Mouldy Sioud’s laboratory at the Department of Cancer Immunology, Institute for Cancer Research at the Norwegian Radium Hospital, from January 2020 to May 2021.

First and foremost, I would like to express my sincerest gratitude to my main supervisor, Prof. Mouldy Sioud for giving me the opportunity to be a part of his group and allowing me to do master thesis under his guidance. I am thankful for his invaluable support, knowledge and enthusiasm provided to me from day one.

I am grateful to the former laboratory technician Ingrid Kjønstad for her assistance in daily laboratory work. I would also like to thank members of the research group -Anniken Olberg and Qindong Zhang for sharing their knowledge, experience and skills with me. I am thankful to the Department of Cancer Immunology, fellow students and friends for providing the great working environment and making life easy both inside and outside laboratory during this uncertain time. Further, I would like to thanks professor Pål Falnes from the Faculty of Mathematics and Natural Science, University of Oslo.

Finally, biggest thanks to my husband Vikas Gogna for supporting and believing me.

Oslo, May 2021 Kamna Verma

(6)

VI

Abstract

Current cancer treatments are struggling to eliminate entire tumors and are characterized by highly unspecific targeting, which causes adverse side effects. In contrast, antibody-based therapies could potentially be the strategy needed to ensure cancer specificity and improve patient outcome. By the use phage display technology, we have identified a number of promising single chain variable (scFv) antibody fragments that showed specific binding to cancer cells. Among the isolated scFvs, a candidate, called MS5, was converted into a human IgG1 minibody (scFv-hinge-CH2-CH3) that binds to a receptor expressed on the surface of various cancer cell lines. The objective of this thesis was to identify such receptor and to convert the MS5 scFv into "off-the-shelf"

bispecific antibody in order to redirect T cells to kill cancer cells. With respect to receptor identification, several strategies, such as immunoprecipitation and cross- linking methods, have been applied. Although further work is needed, we provide evidence that the MS5-Fc minibody interacts with heat shock proteins (hsp) 60 and 78 present on the surface of cancer cells. Interestingly, although generally localized to the cytosol, hsp 60 and 78 are also selectively expressed on the surface of most cancer cells, but not in normal cells.

To retarget T cells to kill cancer cells, a bispecific antibody in the tribody format consisting of two MS5 scFvs and a CD3 Fab fragment [(MS5)2 xCD3] was constructed and expressed in HEK-293T cells and then purified using affinity chromatography. The generated tribody specifically binds to T cells and prostate PC3 cells that express the tumor reactive receptor for MS5 scFv. The KG1a cells do not express the MS5 receptor and therefore did not bind to the tribody. Treatment of prostate PC3 cancer cells co- cultured with T cells induced interleukin 2 (IL-2) expression. Moreover, the tribody mediated lysis of PC3 cells as measured by lactase dehydrogenase (LDH) release assay.

The induction of IL-2 and LDH release required the presence of the tribody, indicating the specificity of the treatment. Collectively, these results suggest that the MS5 scFv can be used to design bispecific antibodies and the engineered prototype tribody is a promising cancer drug that merits further investigation.

(7)

VII

Abbreviations

µg/µl Micro gram/ micro liter Ab Antibody

APS Ammonium persulphate

ADCC Antibody dependent cellular cytotoxicity

ADCP Antibody dependent cell-mediated phagocytosis

BSA Bovine serum albumin

CDC Complement dependent cytotoxicity dH2O Distilled water

DMEM Dulbecco’s Modified Eagle Medium

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

DTT Dithiothreitol

DTSSP 3 3'-dithiobis(sulfosuccinimidyl propionate) DSP dithiobis(succinimidyl propionate)

oC Degrees Celsius

EDTA Ethylenediaminetetraacetic acid E.coli Escherichia coli

EMA European Medicines Agency

ER Endoplasmic reticulum

EtOH Ethanol

Fab Fragment antigen binding

FBS Fetal bovine serum

Fc Fc receptor

FDA Food and Drug Administration

(8)

VIII

FDR False discovery rate

g Gravitational force equivalent (g-force) GRP Glucose regulated protein

H2O2 Hydrogen Peroxide HRP Horseradish peroxidase HSP Heat shock protein

Ig Immunoglobin

kDa Kilo Dalton

LB Lysogeny broth

mAb Monoclonal antibody

mM/M Milli molar/ Molar mg/mL Milli gram/ milli litre

ng Nano gram

PAG Polyacrylamide gel

PAGE Polyacrylamide gel electrophoresis PBMC Peripheral blood mononuclear cell PBS Phosophate buffer saline

pg Pico gram

P/S Penicillin and streptomycin

RCF Relative centrifugal force

rpm Revolutions per minute

RPMI Roswell Park Memorial Institute Medium s.a. sine anno – without year

SDS Sodium dodecyl sulfate

T Tween

(9)

IX

TAA Tumor-Associated Antigen TBS Tris buffer saline

TEMED Tetramethyl ethylenediamine

Tris 2-amino-2-(hydroxymethyl)-propane-1,3-diol

U Units

V Volt

WB Western Blotting

ZAP Zeta-chain-associated protein

(10)

X

1 Introduction

1.1 Cancer

The human body is made up of trillions of cells organized in specialized tissues to perform specific functions. The division and differentiation of cell is highly regulated by the genetic information stored in the deoxyribonucleic acid (DNA) inside the cell’s nucleus (Hofsli, 2021; American Cancer Society 2020). With passage of time several genetic and acquired mutations get accumulated that cause alterations in the cellular DNA and deviation from this orderly developmental process. This results in uncontrolled cell growth and proliferation that gradually develops into a mass of cells with neoplastic growth in a confined area termed as a benign tumor. Further, it starts spreading to nearby or distant organs and become malignant. These malignant neoplasms are commonly known as cancer (Weinberg, 2014, p.32).

The term cancer includes more than 100 distinct types of diseases comprising defects in the regulatory processes controlling healthy cell proliferation and homeostasis (Douglas

& Weinberg, 2000). Cancer is the second leading cause of death worldwide and accounts for almost 10 million deaths in 2020 (World Health Organization, 2021). In Norway, 34,979 new cancer cases and more than 11,000 deaths were registered by 2019. Prostate, breast, lung and colorectal cancers are the four most common malignancies that account for 50% of all cancers in Norway (Cancer Registry of Norway, 2020). Factors such as age, obesity, lack of physical activity, smoking, alcohol and tobacco are the reasons behind a high number of cancer cases. Further due to the heterogeneity and complexity of this disease most of the existing treatments are not efficient enough to provide full protection against cancer. So, there is a need to develop effective and precise cancer treatments.

(18)

2

1.2 Cancer Therapies

Till now, several conventional and modern therapies are available to treat cancer (Figure 1.1). Out of them, surgery, radiotherapy and chemotherapy are the traditional and widely used treatment modalities. Before 1950, surgical removal of the solid tumor was the only preferred treatment option to cure cancer (Abbas & Rehman, 2018). Later, Marie Curie’s research in radium introduced the field of radiotherapy in medicine (Baskar et al., 2012).

Radiotherapy is based on using high energy radiations that damages the cell’s genetic material and inhibits cell division and proliferation (Abbas & Rehman, 2018). Further, at the beginning of the twentieth-century chemotherapy has started for treating cancer (DeVita & Chu, 2008). Chemotherapeutic drugs target actively dividing cancer cells and produce reactive oxygen species (ROS). These ROS damages DNA, causes cytotoxic effects and enforces apoptosis (Yang et al., 2018). With passage of time, it was realized that the available individual treatments were not efficient enough to treat and manage cancer hence, began to use in combination with other treatment modalities.

Side effects associated with traditional methods highlights the scope of modern novel cancer therapies. It includes hormonal therapy, stem cell therapy, targeted therapy and immunotherapy (Figure 1.1).

Figure 1.1. Schematic representation of the cancer therapies. The figure is adopted From Kaushik et al., 2016. An Overview of Theranostic Approaches to Cancer. Baoj Cancer Research and Therapy, Volume- 2, page 2-6.

(19)

3 Hormonal treatment controls the tumor development by limiting hormonal growth factors (Abbas & Rehman, 2018). It showed promising results in treating breast cancer and prostate cancer (American Cancer Society, 2020). Other than this, genetically modified stem cells containing customized genes with desired antitumor effects are currently being used to treat multiple myeloma, leukemia, and lymphomas (Abbas & Rehman, 2018; Chu et al., 2020). In most of the treatments normal cells also get affected along the cancer cells. In context to it, targeted therapies were developed that target only cancer specific genes, proteins, and pathways that are over expressed only in cancer cells and crucial for their growth and survival. Till now, several antibodies have been approved by FDA to use in targeted therapies and showed remarkable results in treating tumors (Ross et al., 2004).

Recently, immunotherapy is designed to boost the body's natural defence system to fight against cancer. It uses materials either made by the body or in a laboratory to improve, target, or restore immune system (Price & Sikora, 2021). Various types of cancer immunotherapies are discussed ahead in section 1.3. These treatments mainly include monoclonal antibodies and small molecule inhibitors. Till now, several antibodies have been approved by FDA (Food and Drug Administration) to use in targeted therapies and showed remarkable results in treating tumors (Jeffrey et al., 2004). With their distinct mechanisms of actions and toxicity, these agents have changed many aspects of the practice of oncology. Targeted therapies are generally better tolerated than traditional chemotherapy, but they are also associated with several adverse effects.

Other than the above-discussed remedies, precision medicine is the latest approach in which cancer treatment is tailored to the patient’s genes, proteins, and other substances (National Cancer Institute, 2021). It is based on giving right treatment to the right patient and sparing the person from unresponsive treatments (Zhang et al., 2018). Till now, we have not achieved the excepted therapy level that resists tumor relapse and decreases the mortality rate. Therefore, intensive research is required to determine the pathways and characteristics of different tumor entities for designing novel cancer therapies.

(20)

4

1.3 Various Cancer Immunotherapy Strategies

The field of cancer immunotherapy was launched in mid of the 1980s after Steven Rosenberg described the use of cytokine interleukin -2 (IL-2) for treating cancer.

(Rosenberg et al., 1985). In comparison to cancer therapies that directly kill cancer cells immnuotherapies stimulate body’s immune system directly or indirectly to attack the tumor (Kamta et al., 2017). The landscape of cancer immunotherapy is divided in two parts: passive immunotherapies and active immunotherapies (Figure 1.2).

Figure 1.2 Overview of the immunotherapies available for the cancer treatment. The figure is modified from Cisbio, 2018.

(21)

5 1.3.1 Passive immunotherapies

Passive immunotherapies target the tumor without the direct participation of the host immune cells. It involves enabling T-cells to fight the tumor without direct manipulation, typically through binding and modifying the intracellular signaling of surface receptors (Kamta et al., 2017). It includes certain monoclonal antibodies and immune checkpoint inhibitors.

a) Monoclonal Antibodies (mAbs)

Antibodies that are produced from single type of B cell clones and bind to only one type of antigen known as monoclonal antibodies (Liu, 2014). Monoclonal Ab was first time generated by Georges Köhler and Cesar Milstein using hybridoma technology (Köhler &

Milstein, 1975). Soon after the discovery of hybridomas, research into the use of mAbs to treat cancer has begun. At the start, an anti-melanoma mAb was prepared that inhibited the tumor growth in mouse model but failed to show immunogenic response in a human trial due to its murine origin (Liu, 2014) Further, recombinant DNA technology led to the development of first chimeric antibodies (partly mouse and partly human) (Marks, 2012).

Finally fully human antibodies with least immunogenicity and maximum efficiency were prepared using transgenic mouse, in vitro yeast or phage display technology. Rituximab is the first humanized anti-CD20 mAb that successfully used to treat non-Hogdkin Lymphoma.

At the start, naked or conjugated antibodies were used for treating cancer. Their mechanism of action is depicted in figure 1.3. Naked Abs do not link to any active factor but induce antitumor effects through complement activation, Ab dependent cell-mediated cytotoxicity (ADCC), Ab dependent cell-mediated phagocytosis (ADCP) (Sioud et al., 2018). Conversely, conjugated Abs carries an active factor such as radioisotope, immunotoxin, cytokine or a specific drug. These loaded Abs work as a targeting moiety to deliver cytotoxic molecules at the tumor site (John, 2005).

(22)

6

Figure1.3. The figure depicts the anti-tumor responses mediated by naked and conjugated monoclonal antibodies. Naked antibodies bind to cancer cells and cause inhibited cell growth, leading to cell death via several mechanisms such as ADCC, ADCP and CDC.

Conjugated antibodies carry the cytotoxic molecule and deliver it to tumor site to induce cell lysis. The figure is modified from Zahavi & Weiner, 2020.

Currently, Abs are used alone or in combination with other therapies and show promising results against both solid and metastatic tumors (Zahavi & Weiner, 2020). Antibodies with high target specificity and cytotoxicity are an integral part of modern therapeutics (Sugiyama et al., 2018).

b) Immune Checkpoints Inhibitors

Further, the discovery of immune checkpoint inhibitors by James P. Allison and Tasuku Honjo led to the promising immunotherapy (The Noble Prize, 2018). Immune checkpoints are the co-receptors expressed by normal cells on their surface. These are a natural brake of the immune system, so the immune system will not attack self or be hyperactivated.

(23)

7 However, some cancer cells also adopted this strategy hence “unnoticed” by Tcells and avoid an immune response against them (American Cancer Society, 2019). For example- CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) and PD- L-1 (programmed death- ligand 1) are the checkpoint proteins expressed by some cancer cells. The binding of T cell ligands to these immune checkpoints leads to T cell deactivation thus skip from T cell-mediated immune response. Therefore, agents that can block the activation of these immune checkpoints serve as promising cancer therapeutics. Ipilimumab and nivolumab are the first approved mAbs that block CTLA-4 and PD-1 respectively. Both treatments are based on the use of monoclonal antibodies that bind and block the activity of the immune checkpoints, resulting in sustained activation of T cells (Spencer et al., 2018).

1.3.2 Active Immunotherapies

Active immunotherapies work by direct engagement of the host immune system to kill cancer cells. Recent treatment modalities such as cytokines, cancer vaccines, oncolytic viruses, adoptive cell transfer and bispecific antibodies fall in this category.

Cytokines

Cytokines based treatment is the first and oldest active immunotherapy. The Infused cytokines bind to surface receptors of immune cells and induce anti-tumor responses (Cisbio, 2021). Till now, two cytokines interferon-alpha (IFN-𝝰) and interleukin 2 (IL-2) have been approved by FDA (Conlon et al., 2019; Jiang et al., 2016). Both of these agents are currently less commonly used with the development of newer agents. Other cytokines such as GM-CSF, IFN-γ, IL-7, IL-12, and IL-21 are under investigation (Conlon et al., 2019).

b) Oncolytic Viruses

Using viruses not as a vector but as a therapeutic to cure cancer leads to the development of oncolytic viruses (OVs) that target cancer cells without harming healthy cells. Herpes simplex viruses (HSV), adenoviruses, measles viruses, reoviruses and more have been engineered to form oncolytic viruses. In this approach, one of the viral genes is deleted to reduce their pathogenicity to healthy cells while preserving their ability to replicate in vivo. The deleted gene is critical for DNA synthesis and highly expressed in actively

(24)

8 dividing cancer cells. This allows OVs to replicate only inside tumor cells and release GM-CSF (colony-stimulating factor) cytokine that guides dendritic cells to induce antitumor responses (Kamta et al., 2017).

c) Cancer Vaccines

Unlike normal vaccines, tumor antigen vaccine is not preventive but therapeutic. A variety of therapeutic vaccines for example dendritic cell vaccines, whole-cell tumor vaccines, DNA or RNA-based vaccines, protein or peptide vaccines, and viral-based vaccines are being studied. Till now, only dendritic cell vaccine- Sipuleucel-T is approved to treat prostate cancer. It involves isolation, genetic manipulation, amplification and re-injection of dendritic cells that express the highly immunogenic antigen. The engineered dendritic cells can activate the immune cascade and cause cancer cell death (Kamta et al., 2017).

d) Adoptive Cell Transfer

T cells play a crucial role in the adaptive immune response. Thus, exploiting and harnessing

Tcells to induce anti-tumor responses is the basis of adoptive cell transfer therapies (Zhou et al., 2020). It includes chimeric antigen receptor (CAR)-T cells, T cell receptors (TCRs) therapy and tumour-infiltrating lymphocytes based therapies. In CAR-T cell therapy, the patient’s T cells are isolated from the blood and engineered to express CAR along with normal T cell receptors (TCRs). The engineered T cells amplified, activated and then re- injecting into the patient. The extracellular part of CAR acts like an antibody and binds to the patient’s tumor antigen, while the intracellular part act like TCR that activates the ZAP cascade and results in cytokine release to kill cancer cells (Hwang, 2020). Like CAR T cell therapy, TCRs therapy is also based on the same concept. However, the extracellular part of TCRs do not carry Ab fragment but genetically engineered to provide better specificity to the tumor antigens (Cisbio, 2021).

Unlike CAR and TCRs, no cell engineering is required in Tumour-infiltrating Lymphocytes (TILs) therapy. It is based on using T lymphocytes present at the tumor sites, capable of recognizing tumor cells but not efficient enough to kill cancer cells

(25)

9 (National Cancer Institute, 2020). Therefore the lymphocytes are harvested from the tumor, further cultured, expanded and over-activated by IL-2 before being re-injected into the patients (Foppen Geukes et al., 2015). Technically it involves injecting highly trained and supercharged lymphocytes into the tumor for maximum antitumor effects.

e) Bispecific Antibodies

In contrast to the above-discussed strategies, bispecific antibodies target both cancer cells and immune cells simultaneously so, there are lesser chances to escape from therapy and to get off-target effects. Basically it is the fusion protein derived from 2 different antibodies. As shown in Figure 1.4 through the Fab domain the antibody binds to cancer cells and T cells, whereas through the Fc part it binds to NK cells. After binding with Ab, T cells get activated and release cytokines such as perforins and granzymes to kill tumor cells (Zhou et al., 2020). Moreover, binding with NK cells through the Fc part also results in Ab dependent cellular cytotoxicity (ADCC).

Figure 1.4 Schematic representation of bispecific/mutlispecific antibody action.

Through fragment antigen binding (Fab) domain Ab binds to T cell and tumor cell and from constant region (Fc) it binds to NK cells to induce antitumor responses. The figure is modified from cisbio, 2018.

(26)

10 The cytotoxicity of T-cell recruiting antibodies mainly depends on their structural and functional properties (Sugiyama et al., 2018). For example- bsAbs targeting two different antigens expressed on the tumor cells reduced the chances to escape from therapy.

Further, it can block two pathways in parallel to impair resistance formation such as receptors tyrosine kinases and angiogenic ligands (Ivana Spasevska, 2013). Moreover, BsAb in BiTE format, BCMA/CD3 BiTE have showed appreciable results in preclinical and early studies for treating multiple myloma (Zhou et al., 2020).

Although cancer immunotherapies offer new treatment options for cancer patients, there are limitations associate with these treatments, especially when the targeted antigen is not cancer-specific. Notably, most of the targeted tumor associated antigens (TAAs) are also expressed at a low level in some normal cells. This results in off-target toxicity due to the recognition of TAAs or cross-reactive antigens expressed on normal tissues (Li & Zhao, 2017). Thus, it is important to investigate the cross-reactivity of immunotherapeutic agents before clinical use.

1.4 Antibody Structure

Antibodies or Immunoglobulin are the proteins synthesized and secreted by stimulated plasma cells (effector B lymphocytes), that identifies and neutralizes foreign invaders therefore play a vital role in innate and adaptive immune response. As shown in figure 1.5 it is a disorted Y-shaped protein composed of two identical heavy (HC) and light chains (LC), connected by disulphide bridges. Each chain has amino (NH2) end and carboxy (COOH) end and divided into variable and constant region. The LC contains one variable (VL) and one constant (CL) domain while the HC contains one variable region (VH) and three constant domains (CH1, CH2 and CH3). The variable regions are located at the NH2-terminal of heavy and light chains serve as antigen binding site known as Fab (Fragment antigen binding) or paratope. The constant region is present at the stem side of Ab known as Fc (fragment crystallizable) fragment; consist of CH2, CH3 domains (Jerne, 1973; 1985).

(27)

11

Figure 1.5 Molecular structure of the human IgG antibody. An IgG antibody contains two identical heavy (HC) and light chains (LC) held together by disulphide bonds in the hinge region. VH and VL represent the variable domains while CH1, CH2, CH3 and CL represent the constant domains of heavy and light chains respectively. Fab is the antigen binding fragment and Fc is the crystallizable\ constant fragment. CDRs stands for complementary determining regions correspond to the hypervariable sequences present in VH and VL domains. The figure is adapted from Scott-Taylor et al., 2018 (Immun Inflamm Dis. 2018 Mar; 6(1):13-33.); Rodrigo et al., 2015 (Antibodies 2015, 4(3), 259- 277)

The Fc part of Ab is highly conserved while some of the amino acid sequences in the variable region vary in different antibodies, referred as hyper variable regions or complementary determining regions (CDR1, CDR2, and CDR3) that determine its binding specificity. Further, the HC defines the class and effector function of Ab. There are five subclasses of HC and some of them have also subtypes. Based on HC differences, immnuoglobins are divided into five isotypes- IgM, IgD, IgA, IgE and IgG. IgG is the most abundant isotype present in blood serum and have further four subclasses (IgG1, IgG2, IgG3, and IgG4) (Murphy & Weaver, 2016, p.140). Out of four subclasses, IgG1 is the most common subclass with longer half-life and high potential to induce effector function.

(28)

12

1.5. Single chain variable fragment (scFv) based Antibodies

Antibody genetic engineering has led to the production of miniature version of mAbs especially Fc less scFv (single-chain fragment variable) format. In comparison to full- length Ab, these antibody fragments differ in size, design and pharmacokinetics but retain their antigen binding properties. Fv is the smallest unit of Ab molecule, vital for antigen- binding activities, consists of variable (V) domains held together by noncovalent forces.

Further, scFv antibody includes genes of VH and VL domains crosslinked via short peptide linkers of 12-15 amino acids (Ahmad et al., 2012) as shown in Figure 1.6 B.

These fragments are produced by phage display technology and expressed in E.coli expression system. Till now, several scFvs and antigen binding fragments (Fab) targeting cancer cells were discovered (Sioud, 2019). In comparison to the parental antibody, scFvs have smaller size, better tumor penetration, rapid blood clearance and reduced immunogenicity (Blanco et al., 2020). These advantages made them suitable for tumor imaging and treatment. However, the small antibody fragments exhibit short half-life therefore further work needs to be done to increase their serum half-life.

Moreover, conversion of scFvs into bivalent antibodies, multivalent antibodies or conjugated antibodies has increased serum stability and improves the pharmacokinetics parameters (Ahmad et al, 2012). For example fusion of two polypeptidic chains containing counter paired VH and VL domains connected by a short linker resulting in the formation of dimeric molecules called as diabody (55 kDa) (Blanco et al., 2020).

Additionally, combinations of scFv Ab with human IgG1 Fc domain give a scFv-Fc minibody (Figure 1.6 C) that induced Ab dependent cellular cytotoxicity and Ab dependent cell mediated phagocytosis against cancer cells (Sioud et al., 2018).

(29)

13 Figure 1.6 Represent the scFv based antibodies, produced and characterized by Sioud’s group. The MS5 scFv antibody fragment was selected from a semi-synthetic scFv antibody library by biopanning on cancer cells. The MS5 scFv was genetically fused to human IgG1 Fc domain (hinge, CH2, CH3) to produce the MS5 minibody (Sioud et al., 2018). To construct the tribody, the MS5 scFv was fused to the Fab fragment of anti-CD3 (Sioud M, unpublished results).

To retarget T cells to kill cancer cells, a bispecific antibody (tribody) in Fab format was constructed by Sioud's group (Figure 1.6 D). The most common T effector-engaging scFv targets the CD3 subunit of the TCR, whereas the other scFv targets an antigen on the cell surface of tumor (Appendix Figure B6). However, the tumor antigens to which the constructed Ab bind is not known yet. Therefore, efforts are being made to identify the Ab receptors by using the ahead cited strategies.

(30)

14

1.6 General methods for antibody receptor identification: Advantages and Disadvantages.

Identification of the receptor proteins expressed on the cancer cell surface is a key step towards the development of Ab-based targeted Immnuotherapies (Smith, 2011). For their successful identification, it is necessary to isolate them from the cancer cell surface and the work is challenging because of the need to keep the receptor proteins in intact form during their extraction and purification (Corgiat et al., 2014). Number of strategies has been adapted for the extraction, purification and characterization of receptor proteins.

1.6.1 Extraction of Surface proteins

Density centrifugation, subcellular fractionation, high salt and high pH buffers are the traditional and time consuming methods used for extraction of membrane proteins (Qoronfleh et al., 2003). Partial extractions of membrane proteins and entrapment of soluble proteins in membrane fractions are the main issues related to these extraction methods (Ohlendieck, 1996).

Moreover, extracting proteins using various kinds of denature or non-denature type detergents such as SDS, NP-40 or tritonX-100 affects the structural and functional properties of proteins (Smith, 2011). For a large polypeptide based receptor protein strong detergents are required for the extraction of all receptor components but these strong detergents cause denaturation and conformational changes in the receptor complexes hence, making their determination with mAb by immunoblot or ordinary immunoprecipitation difficult (Corgiat et al., 2014; Hirofumi & Takashi, 1987).

Moreover, the receptor complexes embedded in the lipids rafts of the plasma membrane show resistance to detergent based solubilization thus require unconventional solubilization strategies (Sot et al., 2006). Ideally, the extracting strategy should be optimized according to the type of protein need to be extracted (Corgiat et al., 2014).

Additionally, the extracted proteins are more prone to aggregation, precipitation and proteolytic degradation thus need to be stabilized exactly after solubilization. Therefore the combination of detergent and stabilizing reagent such as charged amino acids (L- arginine and L-glutamic acid) can save the extracted protein in native form (Golovanov et

(31)

15 al., 2004). Some of the detergents may also interfere with the downstream protein purification steps thus need to be removed or exchanged with other detergent (Smith, 2011).

1.6.2. Purification of Surface proteins

Following extraction and solubilization of membrane protein fractions, the receptor proteins need to be purified/captured using variety of purification techniques such as Immunoprecipitation, cross-linking and biotinylation.

a) Immunoprecipitation

Standard Immunoprecipitation is commonly used method for the isolation of a specific protein from a crude lysate using the antibody bound solid support. Usually the solid support is a magnetic bead loaded with immunoglobin (Ig) binding protein (A, G or A\G) that indirectly immobilize the Ab on the beads. This small scale affinity purification is performed in sequential steps (Figure 1.7). In standard IP, the antibody is incubated in the sub cellular fractions, followed by the addition of affinity beads to capture the receptor proteins (Figure 1.7). Alternatively, the antibody can be incubated with the beads first, followed by addition of cell lysate. After binding of the antigen antibody complex to the support, the beads are washed extensively, further the receptor proteins and antibody is eluted from the beads using suitable elution buffer. The purified proteins can be analyzed by Western blotting or can be identified by mass spectrometry using homology of enzymatic digested patterns to the primary sequence (DeCaprio & Kohl, 2017; Kaboord & Perr, 2008).

(32)

16

Figure 1.7 Shows standard immunoprecipitation procedure. It involve extraction of surface proteins, incubation with antibody, capturing Ab bound proteins using immunoglobin bound magnetic beads, washing beads and elution of Ab bound protein fractions. Same steps can be performed on cytosolic protein fraction and can be used as a control. The eluted fractions are reduced into receptor proteins and Ab fragments via DTT (Dithiothreitol) and further get separated through SDS-PAGE. The proteins can be excised and identified by mass spectrometry or verified by western blotting. The figure is modified from West R. et al., 2012 (Journal of Proteomics, 05 Jan 2012, 75(6):1966- 1972).

There are several issues related to standard immunoprecipitation. First is co-elution and co-migration of the antibody fragments along the target protein (antigen) creates problems while analyze results. This can be solved by crosslinking the immobilized Ab to the Ig binding proteins or by direct immobilization of Ab on the beads through chemical bonding between the aldehydes of beads and primary amines of Ab (Kaboord & Perr, 2008). However, under inappropriate crosslinker concentration the Ab does not bind to beads causes modification in the Ab binding site. Secondly, the nonspecific binding of the other proteins or ligands to the beads leads to false results and can be reduced by preclearing the cell lysate or by extensive washing of beads or by blocking of beads with BSA. However, removal of non-specific binding further connected to low yield of antigen. Other than this, low pH elution buffer is not universally effective to break all

(33)

17 types of Ag-Ab interactions. Unnecessarily harsh elution buffer such as SDS-PAGE loading buffer may cause co-elution of the multiple non-specific proteins results in low purity of the antigen (Otto & Lee, 1993; Thermo Scientific, 2009). Immunoprecipitation also depends upon the specificity of Ab and may bias toward protein abundance or stable protein interactions. If the interaction within the receptor-protein interaction network is too weak or labile then it is difficult to isolate or purify the receptor complexes by the standard IP assay. Further, in the absence of strict experimental controls, the IP may produce false positive results (Corgiat et al., 2014).

Optimized Immunoprecipitation

In order to overcome the above said problems the standard IP can be combined either with crosslinking or with biotinylation of surface proteins.

b) Crosslinking

The addition of chemical crosslinking to standard immunoprecipitation followed by mass spectrometry boosts the power of protein capture and identification of Ab receptors (Sinz, 2003). In this approach the Ab bound membrane proteins were crosslinked using cleavable crosslinker namely DTSSP. DTSSP (3, 3’-dithiobis [suifosuccinimidylpropionate]) is a thiol cleavable, homobifunctional (N- hydroxysuccimide ester) crosslinker with a 8-Carbon spacer arm. It is a water soluble crosslinker with molecular weight is 608.51 and spacer arm length 12Ǻ (Figure 1.8). The sulfo NHS-esters present at each side of spacer arm, react with primary amines and ɛ- amino groups of lysine side chains of protein and releases N-hydroxysulfosuccinimide (NHS) to form stable amide bonds (Sinz, 2003). Additionally, DSP is a non-sulfonated, water insoluble analog of DTSSP. It is lipophilic and membrane permeable crosslinker that was also employed in this study (Thermo Scientific, 2012)

(34)

18 Figure1.8. Chemical structure of the DTSSP crosslinker used in this study. The structure is adapted from Swaim et al., 2004 (J Am Soc Mass Spectrom 2004 May;

15(5):736-49).

The crosslinker should be added just before solubilizing proteins or lysing the cells.

Because solubilization cause changes in protein and crosslinker can’t be bound to great extent (Dorahy et al., 1995). During the solubilization step, the crosslinker stabilize labile protein interactions thus enable to identify receptor protein along with other binding partners. By using this methodology we can identify antigens for mAb by following SDS- PAGE and mass spectrometry (Hirofumi & Takashi, 1987). The DTT from the SDS sample buffer cleave the disulphide bond of the crosslinker and digest crosslinked reaction mixture into surface proteins, Ab fragments and unwanted fragments of the crosslinker that get separated through gel electrophoresis. The separated proteins can be identified using mass spectrometry and western blotting (Figure 1.9).

Figure1.9 Depicts steps involved in the crosslinking experiment used for identifying antibody receptors in this project. It involves binding of Ab to the surface proteins, crosslinking of Ab bound proteins followed by immunoprecipitation. The DTT

(35)

19 (Dithiothreitol) reduces the eluted fractions as well as the disulphide bond of the crosslinker and results in receptor proteins and Ab fractions bound to half crosslinker.

The proteins get separated through SDS-PAGE. The receptor proteins can be excised and identified by mass spectrometry or can be transferred PVDF membrane for western blott analysis. The figure is adapted from Wang et al., 2019 (Bio Protoc. 2019 Jun 5; 9(11):

e3258).

Benefits of Chemical Cross-linking (CC)

Solubilizing membrane proteins without affecting their antigenic determinants is an essential step. Most commonly used detergent for lysing or solubilizing cellular proteins is RIPA (radio-Immunoprecipitation assay) that interfere with many types of protein- protein interactions , can also benefit from the addition of covalent crosslinkers which are generally unperturbed by the RIPA reagent. Unlike IP, it is not required to prepare subcellular fractions while performing crosslinking. The cell surface proteins were crosslinked in intact form thus chances for receptor identification increases many fold.

Moreover, CC enables to determine the cellular trafficking, localization and signaling of surface receptors and their associated proteins (Corgiat et al., 2014). Intermolecular cross linking in combination with mass spectrometry (MS) is useful for mapping molecular interfaces between two proteins while intramolecular cross linking along MS is employed to map low resolution three dimensional protein structures (Sinz, 2003).

Disadvantages of Chemical Cross-linking

Crosslinking has always been an error and trial process as the diverse crosslinked products created during crosslinking reaction. Unwanted crosslinking with other highly available amino acids side chains make it difficult to identify the crosslinked target protein. In order to avoid over crosslinking, optimum cross linking reaction conditions need to be maintained for different crosslinker and proteins (Sinz, 2003). Additionally, some of the Abs are no longer able to recognize their target proteins after crosslinking. In some cases, DTT is not able to reduce the crosslinked Ab-Ag complex successfully.

Crosslinking surface proteins with membrane impermeable bis (sulfosuccinimidyl) suberate increase the apparent molecular weight of the receptor (Boudreau et al., 2012).

(36)

20 CC followed by mass spectrometric identification of surface proteins yield low resolution structure information in comparison to NMR and X-ray crystallography (Sinz, 2003).

C) Biotinylation

Recently, alternative chemical isolation method such as biotin labeling reagents has become much more popular because of the ease of affinity purification through streptavidin conjugated protein beads. In this approach either the primary amines or the N-glycoproteins on cell surface proteins get biotinylated. Further oxidation of the glycoproteins before the solubilizing protein leads to modification of only extracellular carbohydrate. More recently, biotin tagged nanobodies (Cheah & Yamada, 2017) or crosslinker that transfer biotin to the cell surface proteins is employed for the identification of receptor proteins (Remblay & Hill, 2017).

The Ab receptor identification is a challenging task because of need to keep the cellular protein intact during purification and isolation of receptor proteins. The hydrophobicity of the cell surface proteins don’t allow the surface proteins to get solubilize completely.

Further, the surface proteins are expressed in low amount but high amount of proteins are require for their proteome analysis. Additionally the structure and sub-cellular localization of the complex in the cell membrane in not known yet. Components of the receptor complex that bind with low affinity or dissociate rapidly is another problem.

Therefore, capturing these receptor complexes and unrevealing their dynamic properties still remain a challenge (Smith, 2011).

(37)

21

2 Rationale for the study

As indicated above monoclonal antibodies have become an important class of drugs for cancer. However, in many malignancies no anti-tumor antibodies or T cell-based therapy are yet available. Novel therapeutic human antibodies are therefore urgently needed.

Recently, sioud's group has identified a number of promising scFvs that showed cancer specificity. Among the selected candidates a scFv, named MS5, recognizes a receptor expressed on the surface of various cancer cell lines. To exploit the therapeutic potential of the developed MS5 scFv antibody fragment, there is a need to identify its receptor and explore its therapeutic applications.

In context to it, we wished to:

1. Identify the MS5 antibody binding receptor(s)

2. Convert the MS5 scFv into bispecific antibody and test for activity

(38)

22

3 Materials

Table 3.1 Reagents used for growth and preservation of Transformed E.coli cells.

Reagents Components Concentration

LB medium* Tryptone 10 g\L

(Autoclaved) NaCl 10 g\L

Yeast extract 5 g\L

Selection Antibiotic To desired concentration Milli-Q H2O To desired volume 50% Glycerol solution Pure Glycerol 50 ml\100 ml

dH2O 50 ml\100 ml

Table 3.2 Cell lines used in this study.

Cell line Purchased from PC-3

Human Prostate Cancer cell line

American type culture collection (ATCC)

KG1a

Human Acute myeloid Leukemia cell line

ATTC HEK-293T

Human embryonic Kidney cell line CD3⁺blood T cells

ATTC

Isolated from human peripheral blood mononuclear cells (PBMCs)

* Stock prepared in core facility

Table 3.3 Reagents used in cell methods

Type Product Reference

number

Supplier Growth

Media

DMEM, high glucose and L- glutamine

RPMI 1640 medium, L-glutamine

D5796 R8758

Sigma-Aldrich

Sigma-Aldrich Supplements Fetal Bovine Serum

Fetal Bovine Serum low IgG

F7524 F1283

ThermoFisher

Antibiotics Penicillin–Streptomycin P4333 Sigma-Aldrich

(39)

23 (10,000 Units\ml -10mg/ml)

Reagent Trypsin-EDTA (0.25%) Dimethyl sulfoxide (DMSO) ISOTON II dilutent

Lipofectamin ^TM3000

Opti-MEM^TM Reduced Serum Medium

Coulter clenz cleaning agent

T4049 D5879 4141788 11668019 31985062 8448188

Sigma-Aldrich Sigma-Aldrich Beckman Coulter Invitrogen Beckman Coulter

Table 3.4 Cell culture medium and sterile solution used in cell culturing

Type Component Amount Concentration

RPMI complete Media RPMI 1640 with L-glutamine

Fetal bovine serum (heat inactivated) Antibiotic

Penicillin-Streptomycin

450 ml 45 ml 5 ml

10%

1%

DMEM complete Media DMEM with high glucose and L-glutamine Fetal bovine serum (low IgG)

Antibiotic Penicillin- Streptomycin

450 ml 45 ml 5 ml

10%

1%

1X Phosphate Buffer Saline * (PBS), pH 7.4

NaCl KCl KH2PO4

Na2HPO4

dH2O

8 g\L 200 mg\L 245 mg\L 1.44 g\L Upto 1000 ml

137 mM 2.7 mM 1.8 mM 10 mM

* Stock prepared in core facility

(40)

24 Table 3.5 Apparatus used in this study

Product Reference

number

Supplier Cell Scraper

Cryotubes^TM 1.8 ml vials Cell culture flask 25 cm² Cell culture flask 75 cm²

Disposable Serological Pipets, 5 ml Disposable Serological Pipets, 10 ml Disposable Serological Pipets, 25 ml Disposable Inoculating loop 10 µl Dynal Magnetic Bead Separator Eppendorf tubes

Falcon tube 15 ml Falcon tube 50 ml 21 G needle

Immuno-blot PVDF membrane Lens cleaning Tissue

MicroPipets of varible volumes Micropipette tips for variable volumes Multipipette

Parafilm 1 ml Syringe

96-well microplate, flat bottom Nunclon ^TM Delta Surface

SDS-Gel Casting apparatus (Glass plates, combs, casting stand) Clamp/Retort Stand

Blotting roller

3011 377267 156367 734-2313 13-676-10H 13-676-10J 13-676-10K 078627 12321D 0030125177 62554502 625547254 304432 1704157 1115003

U90197 PM-996 H-1701 167008

Corning

Thermo Scientific Thermo Scientific VWR

Fisher Scientific Fisher Scientific Fisher Scientific Nunc^TM

Invitrogen Eppendorf Sarstedt Sarstedt

BD Microlance^TM3 BIO-RAD

VWR Eppendorf Research Sarstedt

Thermo electron Parafilm M Baxter

Thermoscientific BIO-RAD

Table 3.6 Buffers and other solutions used in Protein methods.

Buffer\Solution Component Amount Final

Concentration Affinity chromatography

Binding Buffer, pH 7.4

Elutoin Buffer, pH 2.5

Sodium Phosphate, dibasic Na2HPO4

Sodium Phosphate, monobasic NaHPO4

NaCl dH2O Glycine dH2O

12.3 g\L 1.565 g\L 8.77 g\L Upto 1L 15 g\L Upto 1000 ml

0.1 M Sodium Phosphate Buffer

0.15 M

0.2 M Glycine\HCl

(41)

25 Neutralization Buffer

pH 9

Binding Buffer Azide

Tris Base Tris-HCl dH2O

2% Azide solution Binding Buffer

103.72 g\L 22.72 g\L Upto 1L

0.5 ml\50 ml

Upto 50 ml

1 M Tris\HCl buffer

0.02%

SDS-PAGE

10X Running Buffer

1X Running Buffer

3X-Sample Loading Buffer

3X-Loading Buffer with DTT\ ß-mercapethanol

Tris-base, pH 8 Glycine

10% SDS dH2O

10X Running Buffer dH2O

Tris-HCl, pH 6.8 10% SDS

2% Bromophenol Blue 99% Glycerol

3X-Sample Loading Buffer

1 M DTT Or

ß-mercaptoethanol

30 g\L 144 g\L 6 ml\L Upto 1L 100ml Upto 1L 1.5 ml\10 ml

6 ml\10 ml 1 ml\10 ml 1.5 ml\10 ml 1 ml 100 µl\1ml Or

30 µl\1ml Add it under hood

25 mM 190 mM 1%

1X

150 mM 6% (w\v) 0.2% (w\v) 15% (v\v)

100 mM 3% (v\v)

Crosslinking Reaction 0.5% BSA in PBS

2 mM DTSSP

(crosslinking solution)

Bovine Serum Albumin (BSA)

1X-PBS DTSSP 1X-PBS

0.5 g\100 ml Upto 100 ml

1.217 mg\ml

0.5%

2 mM

(42)

26 100 mM Tris-HCl, pH

7.5 (quenching solution)

Trizma Base dH2O

HCl dH2O

1 ml 1.22 g 90 ml To adjust pH by 7.5 Upto 100 ml

100 mM

Lysis\ protein extraction buffers RIPA Buffer, pH 7.4

RIPA-Lysis Buffer (complete lysis Buffer)

Sucrose Buffer

Lysis buffer in PBS

1 M Tris-HCl, pH7.4 NaCl

0.5 M EDTA NP-40 TritonX 10 mM MgCl2

dH2O

RIPA Buffer, pH 7.4 240 mM Sodium deoxycholate (10%) Octyl-ß Glucoside (OG) Protease Inhibitor cocktail mix 100X (1:100)

Sucrose 1mM EDTA

10m Tris-HCl, pH 7.4 dH2O

NaCl

0.5 M EDTA, pH 8 Na2HPO4

(Sterile filter 1X PBS) Triton X

2.5 ml 2.19 g 0.5 ml 125 ml 2.5 ml 0.5 g Upto 500 ml 1 ml 4.16 µl\ml 6 mg\ml 10 µl\ml

21.4 g 500 µl 2.5 ml Upto 250 ml 1.46 g 0.5 ml 2.5 ml

50 mM 150 mM 1 mM 1% (v\v) 1% (v\v) 10 mM

1 mM 40 mM 1X

250 mM 0.5 M 1 M

100 mM 1 mM 10 mM 1%

(43)

27 Western Blotting

1X-Transfer Buffer

1X-Tris buffer Saline (TBS) pH 7.6

Wash Buffer

(TBS-Tween 0.01%) Blocking Buffer (5% BSA in TBS-T 0.1%)

Antibody Buffer (1% BSA in TBS-T 0.01%)

Stripping Buffer

Tris- base Glycine Methanol dH2O Tris-base NaCl dH2O 1 M HCl 1 X-TBS Tween-20 BSA 1 X-TBS Tween-20 BSA

TBS-Tween 0.01%

0.5 M Tris-HCl, pH 6.8 10% SDS

ß-mercaptoethanol dH2O

3.03 g\L 14.4 g\L 200 ml\L Upto 1 L 6.05 g\L 8.76 g\L 800 ml Upto pH 7.6 500 ml 50 µl 2.5 g Upto 50 ml 50 µl 500 mg Upto 50 ml 6.25 ml 10 ml 400 µl Upto 50 ml

25 mM 192 mM 20% (v\v)

20 mM 500 mM Upto 1000 ml 0.01%

5% (w\v) 0.1%

1% (w\v)

ELISA buffers Blocking Buffer (1% BSA in PBS) Wash Buffer

(PBS-Tween 0.01%) Coating Buffer, pH 9.6

Reagent Diluent

BSA 1X-PBS 1X-PBS Tween-20

Anhydrous Na2CO3

Anhydrous NaHCO3

dH2O BSA Tween-20 1X-TBS

500 mg Upto 50 ml 500 ml 50 µl 5.3 g 4.2 g Upto 1L 50 mg 25 µl Upto 50 ml

1%

0.01%

50 mM 50 mM

1%

0.05%

(44)

28 Stop Solution

(2 M H2SO4)

Concentrated H2SO4

dH2O

11.1 ml Upto 100 ml

2 M

Table 3.7 Recipe for separating and stacking gel solutions for SDS-PAGE.

Separating Gel Solution 7.5% SDS-

PAGE

10 % SDS- PAGE

Reagents (Volume µl) 4 Gels 8 Gels 4 Gels 8 Gels

1 M Tris-HCl, pH 8.8 10% SDS

40% Acrylamide\Bis Solution (BIO-RAD) dH2O

Ammonium persulfate (APS, BIO-RAD)

Tetramethylethylenediamine (TEMED, BIO-RAD)

7500 200 3760 8400 100 10

15000 400 7520 16800 200 20

7500 200 5000 7200 100 10

15000 400 10000 14400 200 20

Stacking Gel Solutoin For all Acrylamide percentages

Reagents (Volume µl) 4 Gels 8 Gels

1 M Tris-HCl, pH 6.8 10% SDS

40% Acrylamide\Bis Solution (BIO-RAD) dH2O

Ammonium persulfate (APS, BIO-RAD)

Tetramethylethylenediamine (TEMED, BIO-RAD)

1260 100 1200 7400 50 10

2520 200 2400 14800 100 20

Table 3.8 Silver staining reagents and their composition.

Reagent Components Amount

Fixing Reagent

Washing Solution

Sensitize Solution

Silver Stain

Developer

Ethyl alcohol (EtOH) Acetic acid

Milli-Q H2O EtOH Milli-Q H2O

Sodium thiosulfate pentahydrate Milli-Q H2O

Silver nitrate AgNO3

Milli-Q H2O

Anhydrous potassium carbonate

7.5 ml 0.125 ml Up to 25 ml 5 ml

Up to 20 ml 10 mg Up to 50 ml 50 mg Up to 25 ml 0.75 g

(45)

29 Stop Solution

Formaldehyde Sensitizer Solution Milli-Q H2O Tris-base Acetic acid Milli-Q H2O

17.5 µl 1.25 ml Upto 25 ml 2.5 g 2.5 ml Up to 50 ml

Table 3.9 Reagents used in Sandwich ELISA for IL-2 Assay

Reagent Name Stock

concentration

Working concentration

Supplier Primary

Capture antibody Secondary Detection antibody Standard

Streptavidin- HRP

Substrate

Mouse Anti-Human IL-2

Biotinylated Goat Anti-Human IL-2

Recombinant Human IL-2

Streptavidin conjugated horseradish- peroxidase

Peroxide substrate TMB (3,3′,5,5′- Tetramethylbenzidine and

Peroxide solution

480 µg\ml

6 µg\ml

75 ng\ml

1:40

dilution ratio

4 µg/ml

0.1 µg/ml

Prepare 5 serial dilutions with concentrations 1.5 ng/ml

1:1

R&D Systems

Thermofisher Scientific

(46)

30 Table 3.10 Antibodies used for Flow cytometry and Western blotting in this study.

Type Antibody Reference

number

Dlution Supplier Flow Cytometery

Primary antibody Secondary antibodies

His-Probe Mouse IgG1 mAb

Anti-mouse –IgG Fc cross absorbed FITC polyclonned in Goat

Anti-human IgG (Fc

specific) FITC polyclonned in Goat

SC-53073

31630

F9512

1:100

Santa Cruze Biotechnology Invitrogen

Sigma-Aldrich

Western Blotting Primary

antibodies

Secondary antibodies

Mouse monoclonal Ab, His tagged against Heat Shock protein-60 kDa

Mouse mAb, Heat shock protein- 70 kDa

Mouse mAb, Heat shock protein- 78 kDa (GRP78) Mouse mAb, Heat shock protein- 90 kDa

Anti mouse IgG-HRP, polyclonned in Rabbit Anti-human IgG-HRP

SC-59567

B195683

SC-166490

Ab13495

P0161

P0214

1:200

1:2000

1:100

1:1000

1:2000

1:3000

Santa Cruze Biotechnology

Santa Cruze Biotechnology Santa Cruze Biotechnology Santa Cruze Biotechnology Dako Denmark

Dako Denmark

(47)

31 Table 3.11 Reagents used in various methods

Reagent Reference

number

Supplier DNA Methods

Antibiotic Zeomicin (100 mg/ml)

Ant-zn Invivo-Gen

Cell Methods

Dimethyl sulfoxide (DMSO) Isoton II diluents

Lipofectamin ^TM 3000 Transfection Reagent

Opti-MEM^TM Reduced Serum Medium ZAP-oglobin II Lytic Reagent

Sterile water

D5879 8546719 1912585 31985062 129039 120718091

Sigma-Aldrich Beckman Coulter Invitrogen

Invitrogen

Beckman Coulter Barun

Protein Methods Imperial Blue Stain

Protein Assay Dye Reagent LDH 10X Lysis Buffer LDH Stop Solution Octyl ß-Glucopyranoside Protein G Dyna Beads

100X Protease Inhibitor Cocktail Mix γ-Globulin

24615 500-0006 1862879 18622880 00630 10003D 5871S G5009-5G

Thermofisher BIO-RAD

Thermo Scientific Thermo Scientific Sigma-Aldrich Invitrogen Cell Signalling Technology Sigma-Aldrich General

Absolute Ethanol, Isopropanol

BSA, DTT, EDTA, glycerol, MgCl2, NaCl SDS, Tris-base, Tris-HCl, Triton-X-100 Tween-20

Sodium Deoxycholate 89904

VWR

Thermo Fisher

Invitrogen

(48)

32

4 Methods

4.1 DNA methods

4.1.1. Cloning and Expression of Antibodies

The initial step of Ab preparation such as cloning was performed by Mouldy Sioud (Group leader) before starting the current project. In this study, MS5-Fc and MS5-CD3 antibodies were prepared from their transformed E.coli glycerol stocks respectively.

a) MS5-Fc Monoclonal Antibody (Minibody)

In refer to Sioud et al., 2018 a desired synthetic gene encoding human single chain variable fragment (VH-linker-VL-His tag) namely MS5 scFv and a plasmid vector pFuse human IgG1-Fc were digested by EcoR1 and BglII restriction enzymes and ligated together. During ligation the scFv MS5 gene was put into a vector in frame with the IL-2 signal sequence and the Fc domain (hinge,CH2 and CH3) of human IgG as shown in Figure 5.3a (In vivoGen, San Diego, CA, USA).

b) MS5-CD3 Bispecific Antibody (Tribody)

As indicated above, the cloning and initial expression of the tribody was done by Sioud's group. Briefly, two synthetic DNA fragments encoding for the anti-CD3 Fab fragment (one constant and one variable domain of the heavy and light chain) fused to the MS5 scFv sequence (Figure 5.6a) were made by GenScript. EcoR1/NheI restriction sites were added to 3' and 5' ends, respectively, for cloning. Additionally, c-myc and his tags were added to the N-terminus of the heavy chain for purification and detection purposes. These DNA fragments were cut with EcoR1 and NdeI restriction enzymes, gel purified and then cloned into EcoR1/NheI-cleaved pFuse-hIgG1 in frame with IL-2 signal sequence (In vivoGen, San Diego, CA, USA). After ligation, the DNA was transformed into the competent E. coli cell XL-1 Blue and then E. coli cells were on LB agar containing 50 µg/ml Zeomicin. Positive clones were selected and verified by restriction mapping and DNA sequencing. Some of the positively transformed E.coli cells were saved in 50%

glycerol at -80̊ C.

Redirecting T cells to cancer cells using a novel bispecific antibody Receptor identification and functional analysis

Redirecting T cells to cancer cells using a novel bispecific antibody

Receptor identification and functional analysis

Kamna Verma

Thesis for the Master degree in Bioscience Molecular Biology and Biochemistry

60 credits

Department of Biosciences

Faculty of Mathematics and Natural Sciences UNIVERSITY OF OSLO

Redirecting T cells to cancer cells using a novel bispecific antibody

Receptor identification and functional analysis

Kamna Verma

Department of Biosciences, University of Oslo, N-0371 Oslo, Norway

Departement of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital, Ullernchausseen 70, 0379, Oslo, Norway

Acknowledgements

Abstract

Abbreviations

Table of contents

1 Introduction

1.1 Cancer

1.2 Cancer Therapies

1.3 Various Cancer Immunotherapy Strategies

1.4 Antibody Structure

1.5. Single chain variable fragment (scFv) based Antibodies

1.6 General methods for antibody receptor identification: Advantages and Disadvantages.

Optimized Immunoprecipitation

b) Crosslinking

2 Rationale for the study

3 Materials

4 Methods

4.1 DNA methods