STRUCTURAL STUDIES OF A NEW CLASS

OF IMMUNE CHECKPOINT INHIBITOR

FOR CANCER IMMUNOTHERAPY

By

NINA HARPELL

A senior thesis submitted in partial fulfillment of the requirements for the degree of

BACHELOR OF SCIENCE

UNIVERSITY OF CALIFORNIA SANTA CRUZ Chemistry and Biochemistry

APRIL 2020 ACKNOWLEDGMENT

I would like to thank my advisor, Dr. Rebecca DuBois, for providing me with the opportunity to begin this project as well as encourage my pursuit of research beyond an undergraduate degree. I would also like to thank the additional members of the Dubois Lab: Natasha

George, Ana Nunez Castrejon, Lena Meyer, John Dzimianski, Jordan Ford, and Kevin

Delgado-Cunningham. Additional thanks go to Nicholas Lorig-Roach, for his mentorship and support in my research and education.

ii Abstract

Natural Killer (NK) cells are a key component of the innate immune system that play a role in eliminating both tumors and virus-infected cells. NK cells express a deactivating receptor, KIR2DL3, which inhibits their cytotoxic function when bound by its ligand, class I

MHC. Lirilumab, an Anti-KIR antibody (mAb) in clinical trials, prevents deactivation of the

NK cell by binding to the KIR2DL3 receptor. However, the structural basis of lirilumabâĂŹs disruption of KIR2DL3 activity is unknown. To that end, the first aim of this project is to use

X-ray crystallography to determine the structure of the lirilumab-KIR2DL3 complex to reveal how the antibody blocks MHC binding to the KIR receptor. A series of expression constructs for the KIR2DL3 receptor with different tag configurations for affinity chromatography were cloned then expressed in HEK293 and CHO cells. Biolayer interferometry assays demonstrate binding of an expressed and purified KIR2DL3 receptor with a control mAb and will also be used to determine Lirilumab’s affinity. Identification of stable expression constructs, as well as structural epitopes of the KIR-mAb construct, will implement a foundation for the study of the Lirilumab mAb as a cancer therapeutic. TABLE OF CONTENTS

Page

ACKNOWLEDGMENT ...... ii

ABSTRACT ...... iii

LIST OF FIGURES ...... vi

CHAPTER

1 Introduction ...... 1

2 Background ...... 3 2.1 Cancer and Immunotherapy ...... 3 2.1.1 Immunotherapy for Cancer...... 3 2.1.2 Immune Checkpoints ...... 4 2.1.3 Immune Checkpoint Inhibitors...... 4 2.2 Characterizing Different Antibodies and Their Function...... 5 2.3 Natural Killer Cells...... 6 2.4 NK Cells KIR2DL3 Receptor and Function...... 7 2.5 Antibodies Bound to KIR2DL3 are Immune Checkpoint Inhibitors.... 7 2.6 Prior Work...... 9

3 Work Completed ...... 12 3.1 Cloning the KIR2DL3 Receptor ...... 12 3.2 Protein Expression of KIR2DL3 in HEK293 Cells ...... 14 3.3 Protein Expression of KIR2DL3 in CHO Cells ...... 18 3.4 Cloning the TRL8605 mAb...... 19 3.5 Protein Expression of TRL8605 in HEK293 and CHO Cells ...... 20 3.6 Binding studies of TRL8605 mAb and KIR2DL3 Receptor ...... 21 3.7 Cloning the Lirilumab mAb ...... 25

iv 3.8 KIR2DL3 Protein Expression in E.Coli Cells...... 25

4 Future Work ...... 28 4.1 Lirilumab Protein Expression in CHO Cells...... 28 4.2 Binding Studies of Lirilumab mAb and KIR2DL3 receptor ...... 29 4.3 Crystallization of anti-KIR mAb bound to KIR2DL3 Receptor ...... 29

5 Conclusion ...... 30

REFERENCES ...... 34

APPENDIX

A Materials ...... 36

B Detailed Methods ...... 39 B.1 Polymerase Chain Reaction (PCR) ...... 39 B.2 Restriction Digestion ...... 39 B.3 Transformation into E. coli ...... 40 B.4 SDS-PAGE, Western Blot ...... 40 B.5 Small-scale Protein Purification (His-tag purification via affinity chromatog-

raphy) ...... 40 B.6 Transfection of HEK293 Cells ...... 41 B.7 Transfection of CHO cells ...... 42

C Sequences ...... 44 C.1 Protein Sequence: TRL8605 Heavy Chain Variable Region ...... 44 C.2 Protein Sequence: TRL8605 Light Chain Variable Region ...... 44 C.3 Protein Sequence: Lirilumab SCFV...... 44 C.4 Protein Sequence: KIR2DL3 Receptor...... 45

v LIST OF FIGURES

2.1 IgG1, 2, 3, and 4 antibodies...... 6

2.2 Antibody Bound to Target Cell and FC Receptor of NK Cell...... 7

2.3 NK Cell KIR Deactivating Receptor and Tumor Cell HLA Antigen (MHC). 8

2.4 NK Cell Activation through Anti-KIR mAb blockade...... 8

2.5 Tumor volume as a function of mAb administered...... 10

2.6 Binding affinity of TRL mAbs for KIR2DL3 receptor on NK cells measured

on ELISA...... 11

3.1 KIR2DL3-cmyc-6His SDS-PAGE and Western Blot...... 17

3.2 SDS-Page of commercial His-tag KIR2DL3 Receptor...... 17

3.3 The 10-hisKIR2DL3 receptor protein on SDS-Page gel...... 18

3.4 SDS-Page gel of KIR2DL3 his-tagged receptor protein...... 19

3.5 SDS-Page gel for TRL8605 mAb produced in HEK293 and CHO cells. . . . . 21

3.6 BLI assay scheme from left to right...... 22

3.7 BLI assay of TRL8605 mAb and KIR2DL3 receptor produced in HEK293F

cells...... 23

3.8 BLI assay of the TRL8605 (CHO) mAb and KIR2DL3 receptor (CHO). . . . 24

3.9 BLI assay of KIR2DL3 receptor and TRL8605 mAb and control mAb. . . . . 24

vi 3.10 PCR products on agarose gel of Lirilumab light chain and heavy chain variable

gene fragments...... 26

3.11 Western Blot of KIR2DL3 receptor protein pellet and lysate prior to purification. 27

vii Chapter One

Introduction

Behind heart disease, cancer is the second most common cause of death in the United States.

Treatments, such as chemotherapy and radiation therapy have been developed to combat cancer by targeting rapidly dividing cancer cells. However, healthy cells that divide at similar rates are often damaged or killed. Because these treatments do not target specific cancer cells, damaging healthy cells poses dangerous risks for the patient to develop toxic side effects or death. Even when these traditional methods appear to work and shrink tumor volumes, relapse and regrowth of tumors after treatment ends is a daunting problem.

Cancer immunotherapy is a new and promising avenue for treatment that uses a patient’s own immune system to recognize and target cancer cells. Innovative therapies such as CAR

T-Cell therapy have introduced new methodologies of engineering an individual’s T-cells for targeted tumor detection and cytotoxic function [3]. Other immunotherapies are utilizing engineered mAbs to serve as checkpoint inhibitors which retain activation of T-Cells and

Natural Killer Cells in the presence of tumor cells. Ipilimumab and Avelumab are both FDA approved immune checkpoint inhibitors for the treatment of skin and lung cancer [17].

Lirilumab, a monoclonal antibody (mAb) in development by Innate Pharma and Brystol-

Meyers Squibb, is currently in clinical trials for a variety of cancer indications, both alone and in combination with other therapeutics [18]. Lirilumab is an immune checkpoint inhibitor that binds to the KIR2DL3 receptor on Natural Killer (NK) cells, activating them and as-

1 sisting their killing of tumor cells. While the Lirilumab mAb is known to bind KIR2DL3 and block ligand interaction, there is no structural information on the Lirilumab-KIR complex, which could reveal how this antibody is able to specifically target inhibitory KIRs without affecting the homologous activating KIRs (or some sentence like this).

Another company, Trellis Biosciences, has also developed a mAb, TRL8605, similar to lirilumab with higher binding affinity for the KIR receptor [19]. The structural basis for any similarities or differences between these therapeutic mAbs is unknown.Therefore, the

first goal of this project is to compare binding affinity of the TRL8605 mAb with lirilumab to verify Trellis’ claim. The second goal is to use X-Ray crystallography to determine the structures of the Anti-KIR antibodies bound to the KIR receptor.

Characterizing structural components will provide better understanding of which struc- tural epitopes are active in the complex binding site. Furthermore, understanding the mecha- nism these anti-KIR mAbs use to block interaction between the NK cell and MHC of tumor cells would provide insight into developing a mAb that can effectively target cancer cells without damaging healthy cells.

2 Chapter Two

Background

2.1 Cancer and Immunotherapy

Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body [2]. In 2015, about 90.5 million people had cancer and about 14.1 million new cases occur a year [1]. Cancer accounts for 8.8 million (roughly 15.5%) deaths each year. The most common types of cancer such as breast cancer, lung cancer, and colon cancer which are initially treated with surgery [2]. Treatment options depend on both the cancer and the patient as well as available treatment options [2]. However, most available treatment options cannot specifically target individual cancer cells. Treatments such as chemotherapy, radiation therapy, and surgery prose more risks for the patient and even death. Target-specific cancer therapies are a new avenue of cancer research that eliminate side effects of non-tumor cellular death, improving a patient’s overall health during and after cancer. Immunotherapy is a new and successful option for cancer treatment because it utilizes the immune systems ability to detect unhealthy and foreign cells.

2.1.1 Immunotherapy for Cancer

Immunotherapy is characterized as the artificial stimulation of the immune system to treat a disease, improving on the immune system’s natural ability or stimulating it when it would

3 otherwise be dormant. Cancer immunotherapy exploits the fact that cancer cells often have tumor antigens, molecules on their surface that can be detected by the immune system using antibodies or T-cell receptors [11]. This can make immunotherapy a highly targeted specific treatment. While most cancer treatments are indiscriminately toxic to all dividing cells, immunotherapy can work to reach highly specific cells based on the receptors or antigens present on the tumor cell surface [11].

2.1.2 Immune Checkpoints

Immune checkpoints are regulators of the immune system. These pathways are crucial for self-tolerance, which prevents the immune system from attacking healthy cells but lets it recognize and remove threats. Receptors on lymphocytes will bind to ligands found on cells’ surfaces, and the interaction either promotes or prevents the lymphocyte from killing the cell depending on the type of checkpoint. However, some cancers can protect themselves from attack by taking advantage of this process stimulating inhibitory immune checkpoint targets [22].

2.1.3 Immune Checkpoint Inhibitors

Checkpoint inhibitor therapy is a form of cancer immunotherapy [22]. The therapy targets immune checkpoints and blocks interaction between the lymphocyte and checkpoint protein.

Inhibitor drugs often consist of monoclonal antibodies (mAbs) that target the checkpoint, keeping the lymphocyte active in the presence of a tumor cell [22].

4 2.2 Characterizing Different Antibodies and Their Func-

tion

Antibodies play a key role in the innate immune system. An antibody, also known as an immunoglobulin is a large, Y-shaped protein produced mainly by plasma cells that is used by the immune system to neutralize pathogens such as pathogenic bacteria and viruses [13].

Each tip of the "Y" of an antibody contains a variable region (analogous to a lock) that is specific for one particular epitope (analogous to a key) on an antigen or receptor, allowing these two structures to bind together with precision. Using this binding mechanism, an antibody can tag a cell for attack by other parts of the immune system, or can neutralize its target directly [12].

Though the general structure of all antibodies is very similar, a small region at the tip of the protein is extremely variable, allowing millions of antibodies with slightly different tip structures, or antigen-binding sites, to exist [9]. This region is known as the variable region and each of these variants can bind to a different antigen.This enormous diversity of antibody paratopes on the antigen-binding fragments allows the immune system to recognize an equally wide variety of antigens.

The ability of an antibody to communicate with the other components of the immune system is mediated through its Fc region (located at the base of the "Y"), which contains a conserved glycosylation site involved in these interactions. The immunoglobulin G (IgG) class of antibodies contains four sub-classes with different Fc regions characterized by disul-

fide bonds linking both heavy chains of the immunoglobulin together. There are four IgG sub-classes (IgG1, 2, 3, and 4) in humans, named in order of their abundance in serum as seen in figure 2.1[24]. IgG1 is the most prevalent subclass and binds to the Fc receptor on phagocytic cells with high affinity. IgG4 antibodies bind to Fc receptors with intermediate to low affinity [7].

Monoclonal antibodies (mAbs) are antibodies that are made by identical immune cells

5 Figure 2.1 IgG1, 2, 3, and 4 antibodies [24] that are all clones of a unique parent cell [14]. MAbs are laboratory-produced immunoglob- ulins engineered to serve as substitute antibodies that can restore, enhance or mimic the immune system’s attack on target-specific cells such as tumor cells. They are designed to bind to antigens that are generally more numerous on the surface of tumor cells than healthy cells [20].

2.3 Natural Killer Cells

Natural killer (NK) cells are effector lymphocytes of the innate immune system that control several types of tumors and microbial infections by limiting their spread and subsequent tissue damage [25]. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, where they then enter into the circulation [8].

NK cells express an FC receptor which is an activating biochemical receptor that has high affinity to the Fc portion of IgG1 and IgG3 antibodies and low affinity to IgG4 antibodies

[25]. This allows NK cells to target cells tagged with specific IgG antibodies and lyse the cells through antibody-dependant cytotoxicity seen in figure 2.2.

6 Figure 2.2 Antibody Bound to Target Cell and FC Receptor of NK Cell [23]

2.4 NK Cells KIR2DL3 Receptor and Function

NK cells also express Killer-cell immunoglobulin-like receptors (KIRs) which are a family of type I transmembrane glycoproteins expressed on the plasma membrane [4]. KIR receptors regulate the killing function of NK cells by interacting with major histocompatibility (MHC) class I molecules, which are immune chekcpoints expressed on all nucleated cell types. Most

KIR receptors are inhibitory and their recognition of MHC molecules suppresses the cytotoxic activity of their NK cell [16]. Tumor cells commonly express MHC antigens, and when tagged with IgG1 antibodies, will elicit NK cells. The NK cytotoxic activity is inhibited when the tumor MHC antigen is bound to the KIR2DL3 receptor on NK cells as depicted in figure 2.3

[5].

2.5 Antibodies Bound to KIR2DL3 are Immune Check-

point Inhibitors

Checkpoint inhibitor therapy is a form of cancer immunotherapy. The therapy targets im- mune checkpoints, key regulators of the immune system that when stimulated can dampen the immune response to an immunologic stimulus. When the Fc receptors on NK cells in- teract with Fc regions of mAbs bound to cancer cells, the NK cell releases perforin and

7 Figure 2.3 NK Cell KIR Deactivating Receptor and Tumor Cell HLA Antigen (MHC) [18] granzyme, leading to cancer cell apoptosis [26]. Retaining NK cell activation in the presence of MHC antigens expressed on tumor cells enables successful cancer cell death. One method of ensuring NK cell function in the presence of an MHC antigen on tumor cells is to introduce an anti-KIR mAb that binds to the KIR2DL3 receptor on NK cells [10]. The Anti-KIR mAb binds to the KIR2DL3 receptor, blocking MHC, and maintaining NK cell activation seen in

figure 2.4.

Using an anti-KIR mAb to hinder deactivation of NK cells requires IgG4 subclass mAb.

IgG4 mAbs FC region bind FC receptors with low affinity, enabling NK cells to remain active without eliciting other NK cells for apoptosis [10].

Figure 2.4 NK Cell Activation through Anti-KIR mAb blockade [18]

8 2.6 Prior Work

Two mAbs were detected through high throughput screening that bound to the KIR2DL3 receptor with high affinity. The first mAb, Lirilumab, is an IgG4 subclass mAb developed by

Innate Pharma that is designed to block the interaction between KIR2DL-1,-2,-3 inhibitory receptors and their ligands [18]. Lirilumab is currently being tested in several indications and combination settings.

Lirilumab is not an effective monotherapy [6]. In a phase-2 clinical trial Lirilumab was administered to elderly patients in remission from leukemia. Results indicated a 65% mor- tality rate with 1 mg/kg of Lirlilumab. A 52% mortality rate for 0.1 mg/kg of lirilumab and a 45% mortality rate for placebo administered, no lirilumab [6], [15].

Lirilumab is more effective when administered in tandem with other mAb immunother- apies. An In vivo assessment of the therapeutic efficacy of lirilumab and elotuzumab was performed in transgenic mice expressing human KIR2DL3 for the treatment of Multiple

Myeloma [21]. The results of the phase-2 clinical trial indicated that, In vivo, as monother- apy, each mAb had some therapeutic effect while the combination of both resulted in a significantly stronger anti-tumor effect and increased survival of the mice depicted in figure

2.5.

The second mAb, TRL8605, developed by Trellis Biosciences is an Anti-KIR mAb similar to Lirilumab. Using Cell-Spot Technology Trellis has identified a high affinity anti-KIR mAb

Explain cell spot tech They obtained multiple mAb (TRL) from different healthy blood donors and were originally of IgG1 or IgG3 subtype, but cloned and expressed as IgG1 antibodies [19]. Binding studies using ELISA determined TRL8605 had the highest affinity binding to the KIR2DL3 receptor on NK cells as seen in figure 2.6[19].

9 Figure 2.5 Tumor volume as a function of mAb administered. Top left graph: no mAb administered, higher tumor volume. Top right graph: Elotuzumab (mAb) administered alone. Bottom left graph: Lirilumab administered alone. Bottom right graph: Elotuzumab and Lirilumab administered together with lower overall tumor volume. [21]

10 Figure 2.6 Binding affinity of TRL mAbs for KIR2DL3 receptor on NK cells mea- sured on ELISA (21). Absorption is measured as a function of concentration of mAb. The TRL8605 anti-KIR mAb is highlighted in green and was identified to have the highest affinity to the KIR-Receptor. [19]

11 Chapter Three

Work Completed

3.1 Cloning the KIR2DL3 Receptor

The KIR2DL3 receptor is a type 1 membrane protein with two extracellular domains respon- sible for interaction with MHC antigens and IgG class antibodies (21). To maximize results for crystallization of the KIR2DL3 receptor bound to an IgG mAb, only the extracellular domain was cloned for protein production. I created three KIR2DL3 receptor gene varia- tions with an n-terminal 10-histidine tag, a c-terminal c-myc gene and 6-histidine tag, and a tagless version of the gene. These variations were to optimize purification using affinity chromatography.

A synthetic, codon-optimized KIR2DL3 receptor gene was amplified using a polymerase chain reaction (PCR). PCR is a method of amplifying a small amount of DNA for subsequent analysis. In a thermocycler, the KIR2DL3 receptor gene is placed in a mixture containing a buffer, DNTPs, a polymerase, and primers. The thermocycler then denatures the gene DNA sequence by increasing the temperature to 96C. The thermocycler drops the temperature to 55C for primers to anneal to their template sequence. During the extension period, the thermocycler raises the temperature to 70C and the polymerase extends the primers.

This cycle repeats for two to four hours which allows for one gene sequence to be amplified exponentially.

12 To analyze my PCR products I ran agarose gel electrophoresis. An agarose gel separates

DNA based on its molecular weight. Agarose gel is a three-dimensional matrix formed of helical agarose molecules in supercoiled bundles that are aggregated into three-dimensional structures with channels and pores large enough for DNA to pass through. Electrophoresis involves running a current through a gel containing the molecules of interest. Based on their size and charge, the molecules will travel through the gel in different directions or at different speeds, allowing them to be separated from one another. By running a molecular standard in the agarose gel, the PCR products can be compared and their molecular weight can be determined.

After imaging my PCR products in the agarose gel and verifying their molecular weight, I assembled the KIR2DL3 receptor gene into two different plasmid vectors: Pet28 for bacterial cell lines and pStable for mammalian cell lines. A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. Plasmids are most commonly found as small circular, double-stranded DNA molecules in bacteria. Placing a gene inside a plasmid carries advantages such as antibiotic resistance, and uptake of the DNA by bacterial and mammalian cells is quicker.

I used Gibson Assembly to assemble the receptor into the vector. Gibson Assembly is a molecular cloning method which allows for the joining of multiple DNA fragments in a single, isothermal reaction. The three required enzyme activities are: exonuclease, DNA polymerase, and DNA ligase. The exonuclease chews back DNA from the 5’ end, thus not inhibiting polymerase activity and allowing the reaction to occur in one single process.

The resulting single-stranded regions on adjacent DNA fragments can anneal. The DNA polymerase incorporates nucleotides to fill in any gaps. The DNA ligase covalently joins the

DNA of adjacent segments, thereby removing any nicks in the DNA. The resulting product is different DNA fragments joined into one.

After gibson assembly of KIR2DL3 gene into pStable plasmid, I transformed E.Coli DH5- alpha cells with the DNA for vector amplification. A transformation is a form of horizontal

13 gene transfer in which bacteria take up foreign DNA. I allowed the DH5-alpha ells to sit with my gibson assembled vector for 20 minutes on ice. To have the DH5-alpha cells take up the vector, I shocked them in a 42C water bath which creates pores within the membrane that allow for uptake of DNA. After, I incubated the cells with nutrient dense media for an hour which enables the cells to grow exponentially. After transformation, I plated the cells and incubated them at 37C overnight. Once colonies appeared on the plate, I incubated one single colony in the media for another 24 hours for growth.

To isolate the DNA from the incubated colony in the media, I performed a transfection- grade minipreparation. A minipreparation takes advantage of the fact that plasmids are relatively small supercoiled DNA molecules and bacterial chromosomal DNA is much larger and less supercoiled. This difference in topology allows for selective precipitation of the chromosomal DNA and cellular proteins from plasmids and RNA molecules. The cells are lysed under alkaline conditions, which denatures both nucleic acids and proteins, and when the solution is neutralized by the addition of Potassium Acetate, chromosomal DNA and proteins precipitate because it is impossible for them to renature correctly (they are so large). Plasmids renature correctly and stay in solution, effectively separating them from chromosomal DNA and proteins.

3.2 Protein Expression of KIR2DL3 in HEK293 Cells

I used DNA from transfection-grade mini-prep to perform a transfection of Human Embry- onic Kidney (HEK293) cells using an Effectene transfection reagent. During a transfection, foregin DNA is uptaken by mammalian cells through either viral or non-viral methods. The transfection I performed was a non-viral lipid-mediated DNA-transfection process utilizing liposome vectors. The reagent Effectene is a non-liposomal lipid mixture that assembles micelles with an overall positive charge at physiological pH. These micelles are able to form complexes with negatively charged nucleic acids through electrostatic interactions. The

14 association of the lipid- based transfection reagent with nucleic acids results in a tightly compacted protective complex around the nucleic acids that can be internalized by endocy- tosis.

After 48 hours, the HEK293 cells were purified using cobalt affinity chromatography to obtain the purified KIR2DL3 receptor protein. This method of chromatography involves a column that contains a cobalt resin. Imidazole, a functional group in histidine, has high affinity for cobalt which enables the histidine tagged KIR2DL3 receptor to be collected in the column. Any impurities without histidine will be washed out of the column. The column is initially washed with a wash buffer to filter out any pre-existing impurities in the column.

The protein lysate is then run through the column where the his-tagged KIR2DL3 receptor is collected in the column and the remaining cellular debris is filtered out. Finally, an elution buffer with high Imidazole concentration is filtered through the column which elutes the protein out of the column.

One method of analyzing the success of the protein elution product is to determine the size of the protein eluted from the column using an SDS-PAGE gel. Polyacrylamide is a polymer consisting of acrylamide and bisacrylamide in a 19:1 ratio. The bisacrylamide crosslinks which affects the migration of protein down the gel. Proteins are separated in a gel based on their molecular size and weight. Proteins with a high molecular weight have a higher frictional coefficient and move slower than proteins with a low molecular weight down the gel. Sodium dodecyl sulfate (SDS) is an ionic detergent that binds to proteins via hydrophobic interactions. By mixing SDS with a denatured protein solution, the compound takes on a net negative charge. Once the SDS-protein solution is created, it can be placed into a well within the polyacrylamide gel. By introducing electricity into the gel, the negatively charged proteins will migrate towards the anode located on one side of the SDS-PAGE gel.

This separates proteins based on the size and can be compared to a ladder run on the gel with protein standards. Proteins are measured in kilo-Daltons (kDa) with larger proteins at the top and smaller proteins at the bottom of the gel.

15 Another method of determining if the protein of interest is within the purified protein solution is to run a Western Blot test. The Western uses antibodies that recognize and bind to a specific protein. After running an SDS-PAGE gel, the gel is transferred onto a membrane using electrophoresis. Next, a blocking buffer composed of fetal bovine serum (FBS) is added to the membrane to prevent interactions of the membrane and antibody used for detection of protein. This reduces background in the final product of the western blot, leading to clearer results, and eliminates false positives. After blocking, a solution of primary antibody diluted in either PBS or TBST wash buffer is incubated with the membrane for one hour. After rinsing the membrane to remove unbound primary antibody, the membrane is exposed to a secondary antibody which binds to the primary antibody. The secondary antibody is bound to HRP which allows the detection of the target protein by chemiluminescence.

To analyze my protein product from the cobalt column I ran an SDS-PAGE and Western

Blot. The resulting bands on both gels indicated the receptor was larger than expected as seen in figure 3.1. While the size of the extracellular domain of the KIR2DL3-cmyc-6His protein is theoretically 27 kDa, the protein gel and western revealed a positive band at

50kDa.

To verify the protein purified was the KIR2DL3-cmyc-6His construct, I compared my results to a commercial his-tagged KIR2DL3 receptor, as seen in figure 3.2, and found a band at roughly 50 kDa, indicating that there may be post-translational modifications that increases the size of the construct.

To verify that the construct was larger due to glycosylation, I deglycosylated a 10-his

KIR2DL3 protein with PNGase and ran an SDS-Page gel as seen in figure 3.3. The resulting band was at 37 kDa which is 10 kDa larger than the 10his-KIR2DL3 construct.

Because the receptor protein was still 10 kDa larger after deglycosylation, I concluded that not all of the receptor was deglycosylated due to folding and possible steric interactions between the PNGase and the position of glycans on the receptor. Comparing the glycosylated

50kDa receptor SDS-PAGE gel to the commercial gel indicates that the receptor was correctly

16 Figure 3.1 KIR2DL3-cmyc-6His SDS-PAGE (left) and Western Blot (right). The construct size is roughly 27 kDa, and observed size is 50kDa.

Figure 3.2 SDS-Page of commercial His-tag KIR2DL3 Receptor. Protein band at roughly 50 kDa. produced.

17 Figure 3.3 The 10-hisKIR2DL3 receptor protein on SDS-Page gel.Columns 1 and 3 are the receptor in HEK293T and HEK293F cells with no PNGase. Column 2 and 4 are deglycosylated receptor in HEK293T and HEK 293F cells.

3.3 Protein Expression of KIR2DL3 in CHO Cells

Chinese Hamster Ovary (CHO) cells are another mammalian cell line used to produce higher quantities of protein per cell. The KIR2DL3-cmyc-6His transfection grade DNA was trans- fected in CHO cells using electroporation. Electroporation is a method of transfection that utilizes an electrical field to increase permeability of the cellular membrane.

Electroporation is carried out in electroporators, purpose-built appliances which create an electrostatic field in a cell solution. The cell suspension is pipetted into a glass or plastic cuvette which has two aluminium electrodes on its sides. Since the cell membrane is not able to pass current (except in ion channels), it acts as an electrical capacitor. This subjects membranes to a high-voltage electric field results in their temporary breakdown, resulting in pores that are large enough to allow DNA to enter the cell.

After electroporation of the cell with the vector DNA, the cells are incubated for 48 hours

18 for protein production. To isolate the receptor protein, the receptor was purified using cobalt affinity chromatography and protein was verified using an SDS-Page gel as seen in figure 3.4 with a size of 50 kDa. The protein concentration was 1 mg per 30 mL of transfected cells.

Figure 3.4 SDS-Page gel of KIR2DL3 his-tagged receptor protein. Well 9: KIR2DL3-cmyc-6His receptor. Well 10: 10His-Kir2DL3 receptor.

3.4 Cloning the TRL8605 mAb

The Trellis Bioscience mAb identified with high affinity to the KIR2DL3 receptor was the

TRL8605 mAb. The TRL8605 heavy chain and light chain variable region gene fragments were separately amplified using PCR from a single-chain variable fragment (SCFV) gene- block. An SCFV is a fusion protein of the variable regions of the heavy and light chain of an antibody, rather than an actual fragment of the antibody. These fragments in the SCFV are linked together by a single linker peptide of roughly 10-25 amino acids. The SCFV retains the specificity of the original antibody despite the absence of the constant region of both chains. Using PCR, two sets of primers can separate the light and heavy chain genes from

19 the SCFV gene block. This enables the individual gene to be amplified and assembled into a corresponding vector plasmid.

The light chain and heavy chain variable fragments were inserted into four pMCV plas- mids containing the Lirilumab mAb IgG4 and IgG1 backbone using Gibson Assembly. The

DNA from all four reactions was then transformed in DH5-alpha cells and purified using a transfection-grade mini-prep.

3.5 Protein Expression of TRL8605 in HEK293 and CHO

Cells

To determine which cell line was better for protein expression, the TRL8605 heavy chain and light chain was transfected together into both CHO and HEK293 cell lines. The protein was then purified using immunoaffinity chromatography with protein A resin. Immunoaffinity chromatography uses the specific binding of an antibody-antigen to selectively purify the target protein. Protein A is a protein derived from bacterial cells that binds to the Fc region of IgG mAbs. A chromatography column containing protein A will bind to the TRL8605 mAb, allowing any other impurities to flow through the column. To elute the mAb, I used an elution buffer containing a 0.1 M glycine and HCl solution that dissociates most antibody- antigen binding interactions without permanently affecting protein structure.

The yield for CHO cells was 10g per 8 million cells transfected. This yield was low compared to most mAb purifications with a yield around 2 mg per 10 million cells transfected.

To confirm the size of the mAb protein, the TRL8605 mAb was run on an SDS-PAGE gel as seen in figure 3.5. The non reduced TRL8605 mAb shows up at 150 kDa on SDS-Page gels. Reducing the mAb separates both heavy and light chain fragments and shows a band at 50 kDa (heavy) and 25 kDa (light).

20 Figure 3.5 SDS-Page gel for TRL8605 mAb produced in HEK293 and CHO cells. Wells 1 and 2 are control anti-KIR mAb obtained from mice (non-reduced and reduced). Wells 3-6: TRL8605 IgG4 and IgG1 mAb expressed in CHO both non- reduced and reduced. Non-Reduced mAb appears at 150 kDa and reduced mAb appears at 50 kDa and 25 kDa. Wells 7-8: TRL8605 IgG1 mAb expressed in HEK cells (non-reduced and reduced). Wells 9-10: C-terminal and n-terminal his-tagged KIR2DL3 Receptor protein. .

3.6 Binding studies of TRL8605 mAb and KIR2DL3 Re-

ceptor

To determine whether the TRL8605 mAb had high affinity for the KIR2DL3 receptor, I conducted a binding study assay using Biolayer Interferometry (BLI). BLI is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces: a protein on the biosensor tip, and an internal reference layer such as a ligand, or protein binding partner as seen in figure 3.6. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured producing

21 a kinetic quantitative value such as the KA and KD.

Figure 3.6 BLI assay scheme from left to right.Baseline: Sensor tips are dipped into buffer. Loading: sensor tips are dipped into binding partner 1. Baseline: Binding partner 1 is dipped into buffer. Association: Binding partner 1 is dipped into binding partner 2 and rate of association (Kon) is measured. Dissociation: Sensor tips with binding partner 1 bound to partner 2 is dipped back into buffer and rate of dissociation (Koff ) is measured.

My first assay studied the affinity of TRL8605 mAb protein produced by HEK293 for the KIR2DL3 receptor expressed in HEK293 cells. In this assay I first dipped protein A and anti-Fc light sensor tips into buffer. Then, I loaded the TRL8605 mAb onto protein A and anti-Fc tips. After loading the tips with the mAb, I dipped the tips back into buffer. I observed that the mAb loaded correctly onto the tips as seen in figure 3.7. Next, I dipped the sensor tips loaded with mAb into a buffer solution containing different concentrations of the KIR2DL3 receptor. However, there was no association observed when the mAb was dipped into the KIR2DL3 receptor. This indicated that either the mAb or Receptor from

HEK293 cells was not produced correctly.

I repeated this assay using TRL8605 IgG4 mAb produced from CHO cells and the 10His-

KIR2DL3 receptor produced from CHO cells and found similar results, as seen in figure 3.8.

I used four protein A tips and loaded the TRL8605 to the tips. During the association step,

22 Figure 3.7 BLI assay of TRL8605 mAb and KIR2DL3 receptor produced in HEK293F cells. TRL8605 mAb was loaded onto protein A and anti-Fc tips. No association was observed (flat-line) when mAb was dipped into the receptor.

I dipped the mAb into varying concentrations of the 10His-KIR2DL3 receptor and observed no association. I also used two penta-His tips and loaded the 10His-KIR2DL3 receptor.

During the association step, I dipped the receptor into two different concentrations of mAb and also observed no association.

To determine which protein was folded or produced incorrectly, I used a control anti-

KIR mouse mAb in a BLI assay. I loaded different His-tag configurations of the KIR2DL3 receptor produced in CHO cells onto HIS1K tips and dipped them into high concentrations of the mouse mAb, TRL805 IgG1 mAb, and TRL8605 IgG4 mAb, as seen in figure 3.9. The association curve for the control mAb in the KIR2DL3 receptor showed that there was high association, indicating that the KIR2DL3 receptor was produced successfully and TRL8605 mAb was not correctly produced.

The TRL8605 mAb was not successfully produced indicating that there were possible mishaps in protein production, cloning, or the gene fragments. While the protein production and cloning was successfully accomplished over many trials, the most likely cause for failure

23 Figure 3.8 BLI assay of the TRL8605 (CHO) mAb and KIR2DL3 receptor (CHO) . Sensors blue, red, turquoise, and greed were loaded with TRL8605 mAb and dipped into 10His-KIR2DL3 Receptor with no association. Sensors yellow and purple were loaded with 10His-KIR2DL3 receptor and dipped into TRL8605 mAb with no asso- ciation.

Figure 3.9 BLI assay of KIR2DL3 receptor and TRL8605 mAb and control mAb. KIR2DL3 receptor showed high affinity for control mAb and no affinity for IgG TRL8605 mAb produced in HEK and CHO cells.

24 is due to the gene sequence of the heavy and light chain variable fragments. The gene fragments are based on a sequence sent to the DuBois Lab and are most likely the wrong sequence or a typo somewhere in the gene. While we have sent Trellis Bioscience information on this conclusion, we have yet to hear back with a response or new gene sequence to continue studying the TRL8605 mAb.

3.7 Cloning the Lirilumab mAb

Innate Pharma identified an anti-KIR mAb, Lirilumab, with high affinity to the KIR2DL3 receptor. The heavy chain and light chain variable gene fragments were amplified from an

SCFV gene-block using PCR. The products were verified using an agarose gel as seen in

figure 3.10. Two pCMV-VRC01 plasmids containing the IgG1 light chain, IgG1 heavy chain, and Two pStbale plasmids containing IgG4 light chain, and IgG4 heavy chain were linearized for Gibson Assembly.

The agarose gel results of linearized plasmid and gene fragments indicate successful ampli-

fication. After gibson assembly of each fragment into the corresponding pCMV-VRC01 and pStable plasmid, the DNA was transformed into DH5 cells and purified through transfection- grade mini-prep.

3.8 KIR2DL3 Protein Expression in E.Coli Cells

Previously, I expressed the KIR2DL3 receptor in both CHO and HEK293 cell lines. As cited in an article by Ryser et. al, the KIR2DL3 receptor was successfully expressed in E.Coli.

Previous attempts in the DuBois lab were unsuccessful in reproducing the receptor in the

E.Coli cell line BL21(DE3)pLysS. I attempted to re-try protein expression using a different

E.Coli cell line: T7 Express.

Using the DNA from a previous gibson assembly of the three his-tag configurations of the

25 Figure 3.10 PCR products on agarose gel of Lirilumab light chain and heavy chain variable gene fragments. The Fragments are around 300 base pairs which are observed in the gel.

KIR2DL3 receptor, I performed three separate transformations of T7 express cells. After the transformation, I ran an SDS-PAGE gel for a Western Blot test to determine if there was protein produced by the bacterial cells as seen in figure 3.11. The samples tested included the pellet of cells prior to purification and the lysate of cells prior to purification.

The Western Blot test identified wells two and three contained protein that bound to the anti-His antibody. This indicates that proteins containing a histidine tag were produced by the T7 Express cells. A SDS-PAGE gel with standards will be run to determine the size of the reactive proteins on the Western.

26 Figure 3.11 Western Blot of KIR2DL3 receptor protein pellet and lysate prior to purification. Wells 1 and 3: KIR2DL3-cmyc-6His and 10His-KIR2DL3 cellular pellet. Wells 2 and 4: KIR2DL3-cmyc-6His and 10His-KIR2DL3 lysate. Visible bands indicate protein bound to the anti-His antibody used in the Western test.

27 Chapter Four

Future Work

4.1 Lirilumab Protein Expression in CHO Cells

Lirilumab plasmids will be transfected into CHO cells using electroporation. After 48 hour incubation, the cells will be sonicated, which lyses cells open and allows DNA to be pu- rified outside of the membrane. Lirilumab protein will be purified using immunoaffinity chromatography using a protein A column. An SDS-PAGE gel will be run to determine if protein is the correct size for binding studies.

To optimize crystallization of the Lirilumab and TRL8605 mAb bound to the KIR2DL3 receptor, both IgG4 mAbs will be digested into fragment antigen-binding regions (Fabs) using papain protease. Fabs are composed of one constant and one variable domain of each of the heavy and the light chain. The variable domain contains the paratope (the antigen-binding site), comprising a set of complementarity-determining regions, at the amino terminal end of the monomer. Each arm of the “Y” thus binds an epitope on the KIR2DL3 receptor.

28 4.2 Binding Studies of Lirilumab mAb and KIR2DL3 re-

ceptor

Once Lirilumab protein is expressed, a BLI assay will be run to determine the affinity of lirilumab for the KIR2DL3 Receptor. Ultimately, a BLI assay that compares affinity of the

TRL8605 mAb bound to KIR2DL3 compared to Lirilumab mAb bound to KIR2DL3. This assay will assess Trellis Bioscience’ claim of a higher affinity anti-KIR mAb as compared to

Lirilumab.

4.3 Crystallization of anti-KIR mAb bound to KIR2DL3

Receptor

To create a binding profile of Lirilumab and TRL8605 bound to KIR2DL3, both mAbs bound to the extracellular domain of KIR2DL3 will be crystallized for x-ray crystallography. X-ray

Crystallography is a method of determining the precise positions/arrangements of atoms in a crystal where beams of X-ray strikes a crystal and causes the beam of light to diffract into many specific directions resulting in a diffraction pattern. The two-dimensional images taken at different orientations are converted into a three-dimensional model of the density of electrons within the crystal using the mathematical method of Fourier transforms, combined with chemical data known for the sample.

Obtaining a crystallographic model of both anti-KIR mAbs bound to the KIR2DL3

Receptor will help me determine if both mAbs are binding to the same position on the receptor or if they are interacting with different epitopes on the receptor. These insights are essential for understanding both Lirilumab and TRL8605 as potential immunotherapies.

29 Chapter Five

Conclusion

Assessing and comparing the affinities of the TRL8605 and Lirilumab mAb for the KIR2DL3 receptor will provide insight into which mAb has higher affinity. Trellis Bioscience claims their anti-KIR mAb has higher affinity binding to the receptor than Lirilumab which could prove to be an effective immunotherapy. There are no clinical trials assessing the TRL8605 mAb as an immunotherapy, therefore assessing its affinity for the receptor in comparison to

Lirilumab will provide insight into the unique differences of both mAbs.

Creating a structural profile of TRL8605 and Lirliumab bound to the KIR2DL3 receptor will provide visual and structural insight into the active residues that contribute to the affinities of both anti-KIR mAbs for the receptor. A crystallographic model will show which epitopes of the receptor are interacting with each anti-KIR mAb. Furthermore, this profile will determine if the anti-KIR mAbs are causing an allosteric conformational change on the

KIR2DL3 receptor. Further details such as the type of interactions will help distinguish the differences or similarities of each mAb as an immunotherapy and are necessary to produce an effective and safe treatment for cancer.

However, one challenge that faces both mAbs as immune checkpoint inhibitors is the effects of a high affinity mAb for the KIR2DL3 receptor. The KIR2DL3 receptor on NK cells function as a deactivation receptor in the presence of MHC antigens. Maintaining

NK cells activation using an anti-KIR mAb is effective for killing tumor cells. However,

30 a high affinity anti-KIR mAb may maintain the NK cells activity long after tumor cell death. This continuous activation of NK cells may cause an increase in off-target cellular death because healthy cells will not be able to bind to the KIR2DL3 receptor and deactivate the NK cell. Without a comprehensive study assessing the effects of a mAb blocking the deactivating KIR receptor on the NK cell there are still questionable risks involving both anti-KIR mAbs. Comparing affinites of anti-KIR mAbs with the affinity of MHC antigens on healthy cells could remedy this uncertainty and enhance the safety of the drug. Additional clinical studies on alternative mAb formats such as a Fab or scFv for Lirilumab and TRL8605 may also provide positive results. Both Fab and scFv mAb have shorter half-lives in the blood and would dissociate from the NK cell quicker than the current IgG4 framework of Lirilumab and TRL8605.

Both affinity studies and a binding profile of both TRL8605 and Lirilumab will provide more information on how these mAbs are interacting with the KIR2DL3 receptor and other inhibitory receptors, but do not bind to activating KIR receptors. These insights will enhance our understanding of the anti-KIR mAbs and the KIR2DL3 receptor. Furthermore, they may provide structural information for other effective immunotherapies for a broader range of treatment options against cancer.

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34 APPENDIX Appendix A

Materials

Table 1: Primer oligomers synthesized by IDT

36 37 Table 2: Western Blot buffers

Table 3: Protein Purification Buffers KIR2DL3 Protein Purification

38 Appendix B

Detailed Methods

B.1 Polymerase Chain Reaction (PCR)

Dilute primers (synthesized and shipped in dry form by IDT) to 10uM with MilliQ water, based on instructions provided by IDT and measuring concentrations via UV spectrophotom- etry analysis at 260 nm. Following Phusion R High-Fidelity DNA Polymerase PCR protocol with a PCR machine, set up a 50L PCR reaction: cDNA template, 5X Phusion R buffer,

10M forward and reverse primers, 10mmol/L deoxynucleotide triphosphates (dNTPs), MilliQ water, and Phusion High-Fidelity DNA Polymerase (add last). Using the given predicted melting temperature of each primer, select a minimum temperature for the annealing step in PCR. In this project, an annealing temperature of 58C was selected for all PCR reac- tions. Each PCR cycle consisted of a ten-second 98C denaturation step, thirty-second 58C annealing step, and a thirty-second 72C extension step. After 30 cycles, a final 10-minute

72C extension step was also run, and then PCR samples were held at 4C.

B.2 Restriction Digestion

Restriction digests were performed on plasmids and blunt-ended DNA inserts in order create sticky ends at specific restriction sites, using 10X CutSmart buffer and the respective restric- tion enzymes. Follow enzyme manufacturer’s instructions on required units of enzyme per reaction. Incubate restriction digestion reactions for at least 2 hours at 37C, or overnight to

39 ensure digestion.

B.3 Transformation into E. coli

All transformations followed protocols provided by each specific strain of E. coli competent cells, and plated onto antibiotic selection plates, including a negative control plate (trans- formation with empty ligation reaction).

B.4 SDS-PAGE, Western Blot

Instead of washing SDS-PAGE gel in MilliQ water immediately after electrophoresis, wash the SDS-PAGE gel in Transfer Buffer (see Table 2 for all Western Blot buffers). Place the gel in a Semi-Dry blot transfer system at 15V for about 15 minutes, between filter papers, and against a nitrocellulose membrane, in order to transfer the proteins from the gel to the membrane.Follow Semi-Dry blot transfer system protocols for further details. Following the SNAP i.d. R 2.0 Protein Detection System and protocols, place membrane in blot-holder and apply Blocking Buffer solution. Apply Primary Anti-Histidine antibody solution to blot, and then wash with Wash Buffer four times. Remove blot from holder, and incubate with

AmershamTM ECLTM Prime Western Blotting Detection Reagent for five minutes at room temperature. Use imager to detect chemiluminescence of histidine-tagged proteins.

B.5 Small-scale Protein Purification (His-tag purification

via affinity chromatography)

Resuspend final total 24 hour protein expression cell pellet in binding buffer (see Table A.3 for all purification buffers). Add protease inhibitor cocktail (one or two cOmplete ULTRA

Tablets, Mini, EDTA-free, EASY pack, depending on buffer volume) and vortex sample.

40 If using benzonase for DNA/RNA degradation, add 1mM MgCl, (final concentration) and

2uL benzonase to sample and mix on ice. Sonicate sample with a 1/8" tapered microtip

five times for 30 seconds at -30% amplitude, with 30 second ice-break intervals. Centrifuge the sample lysate at 4C for 30 minutes at 14000xg, and syringe filter supernatant through a 0.22-micron filter. Store a sample of the soluble fraction at 4C, and a sample of the insoluble pellet at -20C (resuspended in 1X SDS loading buffer). Centrifuge down 1004L of TALONTM Co2+ resin slurry and remove storage solution. Add 5mL binding buffer to resin and mix. Centrifuge the resin once more and remove buffer. Add washed resin to cell lysate, and incubate the mixture while rotating at 4C for at least 30 minutes, in order to fully bind the histidine-tag of the protein to the cobalt resin. Add the resin and lysate to an empty gravity-flow column (or centrifuge column), and collect the resulting flow-through,

5-7 washes (in binding buffer), and 4-6 elutions (in elution buffer). Store all samples from affinity chromatography in 4C. Note: If using DNase to degrade DNA in cell lysate (instead of benzonase), add 10mm MgCl, (final concentration) and treat the supernatant with 1004L

DNase (before syringe filtering), followed by stirring the sample at room temperature for 30 minutes.

B.6 Transfection of HEK293 Cells

Incubate the cells under their normal growth conditions (generally 37C and 5% CO2). The dishes should be 40–80% confluent on the day of transfection. The day of transfection, dilute

1 g DNA dissolved in TE buffer, pH 7 to pH 8 (minimum DNA concentration: 0.1 g/l) with the DNA-condensation buffer, Buffer EC, to a total volume of 150 l. Add 8 l Enhancer and mix by vortexing for 1 s. Incubate at room temperature (15–25C) for 2–5 min then spin down the mixture for a few seconds to remove drops from the top of the tube. Add 25 l Effectene

Transfection Reagent to the DNA-Enhancer mixture. Mix by pipetting up and down 5 times, or by vortexing for 10 s. It is not necessary to keep Effectene Reagent on ice at all times.

41 10–15 min at room temperature will not alter its stability. Incubate the samples for 5–10 min at room temperature (15–25C) to allow transfection-complex formation. While complex formation takes place, gently aspirate the growth medium from the plate, and wash cells once with 4 ml PBS. Add 4 ml fresh growth medium (can contain serum and antibiotics) to the cells. Add 1 ml growth medium (can contain serum and antibiotics) to the tube containing the transfection complexes. Mix by pipetting up and down twice, and immediately add the transfection complexes drop-wise onto the cells in the 60 mm dishes. Gently swirl the dish to ensure uniform distribution of the transfection complexes. Incubate the cells with the transfection complexes under their normal growth conditions for an appropriate time for expression of the transfected gene. The incubation time is determined by the assay and gene used.

B.7 Transfection of CHO cells

In a sterile cell culture hood, take a sample of your endotoxin-free maxiprepped DNA con- structs to nanodrop. The DNA concentration must be > 5000 ng/L in cell-culture-grade water. Redo the maxiprep if the concentration is lower.Calculate the volume of maxiprepped

DNA needed. For singular proteins, calculate the volume needed for 120 g of DNA. Count

CHO-S cells in the Berman lab using their automatic cell counter. Combine 10 L of cells with 10 L Trypan Blue, load 10 L onto a disposable hemocytometer, and insert into the cell counter. Split the cell volume evenly among several 250 mL pointy cell culture cen- trifuge tubes. Spin cells at 1600 rpm in the big benchtop centrifuge for 10 minutes at room temperature. While the cells are spinning, aliquot your DNA into Eppis, one Eppi per elec- troporation batch. Carefully aspirate off the media from the cell pellet and resuspend the cells to 2 x 108 cells/mL in room-temperature Electroporation Buffer. The cell pellets take up a lot of volume, so start by adding 30% the final volume of Electroporation Buffer. Add

400 L of resuspended cells to each electroporation batch Eppi. Mix cells very gently. Turn on

42 the MaxCyteSTX electroporation instrument, open the STXG2 software on the computer, and select Protocols CHO and Processing Assemblies OC-400 (which is the type of cuvette).

Perform electroporation: Transfer 400 L of cells with DNA from one of the Eppis into a cuvette, avoiding air bubbles. Close the cuvette and insert it into the MaxCyteSTX. Hit OK to perform the electroporation. Remove the cuvette. If you will perform another electropo- ration, hit Yes to continue. Take the cuvette back to the cell culture hood. Transfer the cells dropwise from the cuvette to a fresh 250 mL flask using P200 tips to reach into the corners of the cuvette. Try to get the cells to land in the middle of the flask and not spread out too much because they shouldn’t dry out. Put the 250 mL flask into an incubator at 37 C,

8% CO2, 85% humidity, without shaking to let the cells recover. Use the same cuvette for the other Eppi containing DNA for the same construct, following Steps 9 – 12. During step

11, transfer the second batch of electroporated cells to the same flask that the first batch of electroporated cells went into. Record the time when you put the flask back in the incubator.

Make unelectroporated control: Transfer 800 L of unelectroporated cells (without DNA) into a fresh 250 mL flask. Put the flask into the incubator. After 30 minutes of recovery at 37 C,

8% CO2, 85% humidity, without shaking, add 30 mL complete CD OptiCHO dropwise to the flasks. Make sure there are no cell clumps. Check the live cell count and viability of the electroporated cells. Some cells are always lost during centrifugation and electroporation.

Transfer the flasks to the shaker at 37 C, 8% CO2, 85% humidity, 125 rpm (as when growing up the cells). Record the time because you should feed the cells every day at around this time.

43 Appendix C

Sequences

C.1 Protein Sequence: TRL8605 Heavy Chain Variable

Region

QVQLVQSGGGVVQPGRSLRLSCAVSGFTFSSYGMHWVRQAPGKGLEWVTIISYD GSNY-

DYADSVKGRFTISRDNSKNMVYLQMNSLRADDTAVYYCAKDGFDYWGQ GTLVTVSS

C.2 Protein Sequence: TRL8605 Light Chain Variable

Region

DIVLTQSPDSLAVSLGERATINCKSSQSVLYSSNNRTYLAWFQQKSGQPPKLL IYWASTRQS-

GVADRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTTPFTFGP GTRVDFKR

C.3 Protein Sequence: Lirilumab SCFV

VGLSLGRSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSFYAISWVRQAPGQGL EWMG-

GFIPIFGAANYAQKFQGRVTITADESTSTAYMELSSLRSDDTAVYYCAR IPSGSYYYDY-

DMDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGEIVLTQSPVT LSLSPGERATLSCRASQSVS-

SYLAWYQQKPGQAPRLLIYDASNRATGIPARFS GSGSGTDFTLTISSLEPEDFAVYYC-

QQRSNWMYTFGQGTKLEIKASLVPRGS

44 C.4 Protein Sequence: KIR2DL3 Receptor

MHEGVHRKPSLLAHPGPLVKSEETVILQCWSDVRFQHFLLHREGKFKDTLHLI GEHHDGVSKAN-

FSIGPMMQDLAGTYRCYGSVTHSPYQLSAPSDPLDIVITGLY EKPSLSAQPGPTVLAGESVTLSC-

SSRSSYDMYHLSREGEAHERRFSAGPKVNG TFQADFPLGPATHGGTYRCFGSFRDSPYEWSNSS-

DPLLVSVTGNPSNSWPSPT EPSSETGNPRHLH

45