18-X1
Immunoassay
Applications
in
Gene
and
Cell
Therapy
A
Market
Analysis
of
Companies
Conducting
Gene
and
Cell
Therapy
Product
Development
Anna
Nilsson,
Sebastian
Persson,
Jenny
Thorsén,
Stina
Wahlström,
Johanna
Öberg
Beställare:
Gyros
Protein
Technologies
AB
Beställarrepresentant:
Marie-Madeleine
Walz
Handledare:
Karin
Stensjö
Abstract
Gyros Protein Technologies AB are providers of immunoassay systems that can be used for the devel-opment of biopharmaceuticals, now they wish to enter the gene and cell therapy market. This thesis provides useful information in the area of gene and cell therapy and aims to provide Gyros Protein Tech-nologies AB with guidance and tools to enter the market with their platform GyrolabTM. Gene and cell
Contents
1 Introduction 1
2 Background 1
2.1 Immunoassays . . . 1
2.2 Gene and Cell Therapy . . . 1
2.3 Virus . . . 2 2.3.1 Adenovirus . . . 2 2.3.2 Adeno-associated virus . . . 3 2.3.3 Lentivirus . . . 3 3 Methods 3 3.1 Limitations . . . 4 4 Results 5 4.1 Gene and cell therapy applications . . . 5
4.1.1 Oncology . . . 5
4.1.2 Neurodegenerative diseases . . . 6
4.1.3 Ophthalmology . . . 6
4.2 Approved gene and cell therapy products in Europe and the U.S. . . 6
4.3 Gene and cell therapy companies and their products . . . 9
4.4 Gene therapy: the manufacturing process . . . 16
4.4.1 Generate a recombinant viral plasmid using homologous recombination . . . 16
4.4.2 Transfect the plasmids into a producer cell line . . . 16
4.4.3 The HEK293 cell line . . . 17
4.4.4 A safety risk with HEK293 . . . 17
4.4.5 Alternative cell lines . . . 17
4.4.6 Downstream processing of viral vectors . . . 17
4.4.7 Characterization of the final product . . . 18
4.4.8 Measure the immune response of the patient . . . 20
4.4.9 Humoral immune response . . . 22
4.4.10 Innate immune response . . . 22
4.4.11 Cellular (T cell) immune response . . . 22
4.4.12 An example of a clinical trial measuring immune response . . . 22
4.4.13 Analytes that immunoassays can detect . . . 23
4.5 Cell therapy . . . 23
4.5.1 T cell-based cell therapy . . . 24
4.5.2 Companies developing TCR and CAR T cell therapies . . . 24
4.5.3 CAR T cell therapy . . . 24
4.5.4 Extracting T cells from cell source . . . 24
4.5.5 Activating and modifying T cells . . . 25
4.5.6 Volume reduction and washing . . . 25
4.5.7 Reinfuse CAR T cells into patient . . . 25
4.5.8 Analyzing CAR T cells with ELISA . . . 26
4.5.9 The T cell receptor (TCR) . . . 27
4.5.10 Quality control . . . 28
4.6 Safety with viruses . . . 28
4.7 Assay requirements for the testing of gene and cell therapy products . . . 29
4.8 Competitive companies in the U.S. and Europe . . . 29
4.10.1 Justice and priorities . . . 31
4.10.2 Possible consequences . . . 32
4.10.3 Ethical conclusions . . . 33
5 Summary and conclusions 33 5.1 Analytes in gene therapy . . . 33
5.2 Analytes in cell therapy . . . 34
5.3 Competitive companies . . . 34
5.4 Potential customers . . . 34
5.5 Biosafety and assay requirements . . . 34
6 Acknowledgements 35 7 References 36 8 Appendix 45 8.1 Appendix 1 . . . 45
8.1.1 Project order from Gyros Protein Technologies AB . . . 45
8.2 Appendix 2 . . . 46
8.2.1 The microfluidic technology behind GyrolabTM and its applications . . . 46
8.3 Appendix 3 . . . 47
8.3.1 Enzyme-Linked Immunosorbent Assay . . . 47
8.4 Appendix 4 . . . 48
8.4.1 Contributions . . . 48
8.5 Appendix 5 . . . 49
8.5.1 Market survey . . . 49
8.6 Appendix 6 . . . 50
1
Introduction
This thesis is an order from Gyros Protein Technologies AB, see further specifications and project order in Appendix 1. The company delivers immunoassay systems for the development of biopharmaceuticals. Gyros Protein Technologies wishes to enter the gene and cell therapy market by providing their immunoas-say platform GyrolabTMand developing new ready-to-use immunoassay kits for purchase. Read more about
GyrolabTMin Appendix 2. We will present important information about the gene and cell therapy market in
order for Gyros Protein Technologies to enter the market in the best possible way. The report consists of an overview of the gene and cell therapy manufacturing process as well as the different methods for analysing the safety, potency and purity of the products. This is done in order to identify where on the market GyrolabTM
can be applied.
This thesis focuses on gene therapy using viral delivery systems and T cell-based therapy in fields of on-cology. This is because oncology is the dominating area of research in gene and cell therapy. We therefore believe this information will be useful for Gyros Protein Technologies. The current gene and cell therapy products that have been approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) are described and presented, as well as the biggest areas of research at the moment within this field. A selection of American and European companies active in the area are listed together with some of their products within clinical phase of development. In the report there are also suggestions for potential immunoassay ready-to-use kits that Gyros Protein Technologies AB could develop and deliver to gene and cell therapy companies. In the result part of the report the methods and processes of cell and gene therapy products are described and steps where enzyme-linked immunosorbent assays (ELISAs) are used have been accentuated. ELISA is an immunoassay that functions similarly to Gyros Protein Technologies’ product GyrolabTM. GyrolabTM can be applied where ELISAs are mentioned, indicating how it fits in the field.
Furthermore, there is a list of competitive companies which already provide kits that are used for mea-suring specific contaminants. We also present the safety risks of using viruses and an ethical discussion regarding the field.
2
Background
2.1
Immunoassays
Immunoassays are diagnostic tools used as quality controls and quantity specific detection of antigens and antibodies. The immunoassay technique is derived from the basic immunology concept of antigen to antibody binding (Gan et al. 2013). Enzyme-linked immunosorbent assay (ELISA) and Gyros Protein Technologies’ product GyrolabTM are both types of immunoassays. Both these immunoassays build on the method of
adding enzymes to the antibodies. When enzymes interact with added substrates they induce a reaction which can be measured and converted into quantitative amount of antigen in the tested sample (Gan et al. 2013). More detailed information about GyrolabTM and ELISA can be found in Appendix 2 and Appendix
3 respectively.
2.2
Gene and Cell Therapy
To identify where on the market and by whom the product GyrolabTM can be used, this thesis will describe
essential manufacturing processes used by companies that perform research and develops pharmaceuticals in the area. Firstly, the concept of gene and cell therapy is described.
cancer (Dudley and Rosenberg 2003). Gene therapy is the practice of inserting new genes into targeted cells. The purpose is to replace a defective gene with a healthy one or giving the cell a new property (Muños-Ruiz and Reguerio 2012).
A common way of introducing DNA into the cells in gene therapy is through vectors. There are viral vectors and non-viral vectors. Viral vectors are recombinant viral particles designed to have the gene of interest in the genome (Rodrigues et al. 2014). Non-viral vectors can be naked DNA, meaning that the DNA is non-encapsulated, or plasmid DNA. Viral vectors are more frequently used in gene therapy than non-viral vectors, despite that non-viral vectors are considered much safer. The reason why viral vectors are considered less safe is because there is a risk that they can replicate in the host cell. Non-viral vectors are still less used due to the fact that they are less efficient and more expensive than viral vectors. Therefore we have chosen to focus only on gene therapy using viral vectors in this thesis (Warnock et al. 2011).
Vectors are used in three principal ways, these are ex vivo, in vivo and in situ. Ex vivo gene therapy works by extracting the target cells and culturing them in a laboratory with a vector. Then isolation and expansion of the cells with successfully integrated genes is done. Finally the expanded cells are inserted back into the host (Muños-Ruiz and Reguerio 2012). In vivo gene therapy is in theory performed by injecting the vector into the patient’s bloodstream directly. Since the vector is designed to only administer the ge-netic material into a specific target cell with a high level of specificity, the therapeutic effects are achieved (Muños-Ruiz and Reguerio 2012). In situ gene therapy is performed by introducing the vector directly to the tissue of interest. This method offers high specificity since the vector is directly transmitted into the organ. Examples of where this method is used, is in the treatment of cystic fibrosis, tumours and muscular dystrophy (Muños-Ruiz and Reguerio 2012).
Cell therapy is the practice of using cellular material to achieve a desired effect in vitro or in vivo through their biological activities and properties (Savitz and Parsha 2015). The cells used for the treatments can be classified as either allogeneic (from donor host) or autologous (from the patient itself) (Mount et al. 2015). Modern cell based therapies also include regenerating human cells, tissues or organs (Quinley 2013). The basis of the cells used are derived from stem and progenitor cells, mature and functionally differentiated cells or engineered tissue (Bailey et al. 2015).
2.3
Virus
To better understand the use of viral vectors in gene and cell therapy we have summarized some guiding background information about the most commonly used viral vectors. These are presented in the following subsections.
As previously mentioned in section 2.1 viral vectors are more commonly used to deliver genes into the cells than non-viral vectors. Non-viral vectors such as naked DNA are considered to be much safer but they are far less efficient in delivering the transgenic genes into the nucleus of the host cells and therefore are unsufficient to use (Robbinsa and Ghivizzania 2016). There are three different types of viral vectors that have become more widely used in current gene and cell therapy studies due to their specific properties. These are adenovirus, adeno-associated virus (AAV) and lentivirus vectors and are presented below. (Sharon and Kamen 2017).
2.3.1 Adenovirus
the initiation of DNA replication. The late genes are called L1, L2, L3, L4, and L5 and they mainly encode structural proteins (Sharon and Kamen 2017).
Adenoviruses exist everywhere and most humans have been infected by at least one serotype. Adenoviruses and adenoviral vectors can infect a number of different cells, both dividing and non-dividing. The viral genome is maintained in the host cells as an episome, which means that it is not integrated into the host genome. Adenoviral vectors are mostly used in oncolytic therapy and as vaccines but can also be used for regular gene therapy, where a defect gene is replaced with a functioning one. When used in gene therapy, the adenoviral vectors are genetically modified to be non-replicating by deleting some of the “early” genes and replacing them with the transgene. The vector can then be grown in a cell line containing the deleted genes (Wold and Toth 2013).
2.3.2 Adeno-associated virus
AAV is a small single stranded DNA virus with a genome of about 5 kb. As with adenovirus there are several different AAV serotypes and AAV serotype 2 (AAV2) is the most commonly used type for viral vectors. The virus is non-pathogenic and lacks the ability to replicate on its own. To be able to replicate AAV needs helper genes, which can be provided by other viruses such as adenoviruses or herpes simplex viruses (HSV). Advantages with AAV vectors are that they can transfect several different tissues and they are able to express the transgene stably for a long time in a tissue without being attacked by the host’s immune system. This is because the AAV is non-pathogenic and therefore does not give rise to an immune response. The viral genome is not integrated into the genome but stays as episomes, just as with adenoviruses. Episomes do not co-replicate with the host’s genome and therefore the transgene expression decreases with cell replication. 87% of the AAV vectors used in gene therapy are used for treatment of hereditary disorders (Sharon and Kamen 2017).
2.3.3 Lentivirus
Lentivirus is a type of retrovirus which means that the virus uses the enzyme reverse transcriptase to copy its genome into the host cell. The genome consists of a single stranded RNA and the most common lentivirus used for gene therapy is human immunodeficiency virus 1 (HIV-1). Lentiviral vectors can infect both divid-ing and non-dividdivid-ing cells and have a very high transfection efficiency. The transduced genes are expressed indefinitely and will be passed on to daughter cells, as the virus genome integrates into the host cell genome (Sharon and Kamen 2017).
Most of the clinical trials utilizing lentiviral vectors use hematopoietic stem cells – stem cells that can differentiate into different kinds of blood cells. These are taken from the donor or from a patient and are altered using the lentiviral vector. They are altered either to treat hereditary disorders or to generate mod-ified T cells that can be used to kill cancer cells (Sharon and Kamen 2017).
The integration of the viral genome into the host can give rise to insertional mutagenesis, especially proto-oncogene activation. Although possible, trials have shown this to be very rare. There also exists lentiviral vectors with specific alterations further minimizing the risk of insertional mutagenesis. Since the viral vector is derived from HIV-1 which is a lethal virus there are extra precautions regarding safety and to meet these safety concerns several measures have been taken making sure that the viral vectors are not able to replicate (Sharon and Kamen 2017).
3
Methods
Firstly, each project member found 2-3 companies which were interesting candidates for the thesis. Not all of the questions in the specification from Gyros Protein Technologies AB (Appendix 1) could be an-swered by searching the companies’ websites. Companies were therefore contacted by email but with minor success. Since contacting companies by personal emails was unsuccessful a market survey (Appendix 4) was assembled with help from Tobias Jakobsson, Project Manager at the Department of Biological Education Centre at Uppsala University. More companies were researched and the survey was sent by email and con-tact forms to about 50 companies see Table 13 (section 4.9). Yet again it was met with low response. The collected material used to address the use of immunoassays in the areas of gene and cell therapy was hence obtained from literature publications, such as reviews, reports and books. Furthermore, researchers were contacted who contributed with helpful guidance and information about questions encountered during the work.
• Angelica Loskog, Adjunct Professor at Uppsala University at the Department of Immunology, Genetics and Pathology, Clinical Immunology
• Pontus Blomberg, Adjunct Senior Lecturer at Karolinska Institutet at the Department of Laboratory Medicine
• Sara Mangsbo, Assistant University Lecturer at Uppsala University at the Department of Pharmaceu-tical Life Sciences and Researcher at the Department of Immunology, Genetics and Pathology, Clinical Immunology
• Magnus Essand, Professor at Uppsala University at the Department of Immunology, Genetics and Pathology, Clinical Immunology
3.1
Limitations
We agreed on limitations for the thesis in case there would be time constraints with fulfilling the demands set up from our client. Limitations of the thesis study were done by setting priorities. From the beginning it was decided that some areas were given a higher priority. Areas that were given high priority was seen as important information for our client. These high prioritized areas were:
• Information about which cell lines that are being used
• Which analyses that are used in the process of gene and cell therapy
• Which analytes that might be of interest for Gyros Protein Technologies AB to design new kits for • Which companies that could be potential customers
• Safety aspects regarding e.g viruses • An ethical analysis
Some other areas were given low priorities, these were: • Information about competitive companies • Therapy areas in the spotlight
4
Results
4.1
Gene and cell therapy applications
Gene and cell therapy is no longer just in theory a promising way of treating and resolving diseases and illnesses, but is now also applicable in the medical field (Smith and Blomberg 2017). In June 2017 a database collecting clinical trials in the field of cell and gene therapy was compiled in the United Kingdom by the Cell and Gene Therapy Catapult (CGTC), listing the amount of trials and their respective covered medical area. The clinical trials database listed 59 active trials in the U.K. alone. A representation of the clinical trials and their respective medical area has been presented by the CGTC in a pie chart shown in Figure 1. The biggest area in the U.K. is cancer and tumour (oncology) therapies, with a number of 12 clinical trials which amounts up 20% of the total. Companies presented in Table 6 (section 4.3) cover a multitude of the areas presented in the CGTC pie chart in Figure 1, however companies presented in Table 6 (section 4.3) are not all from the U.K. (CGTC 2017).
Figure 1. An overview of clinical trials of cell and gene therapy and the number of active trials in the respective medical areas. The figure is modified from a figure by the Cell and Gene Therapy Catapult (2017).
4.1.1 Oncology
CAR which facilitates the targeting of cancer and malignant tumours (Rosenberg and Restifo 2015). There have been reports of patients totally recovering from advanced follicular lymphoma. The treatment was given through CD19-targeting CAR T cells together with the signal molecule interleukin-2. CD19 is a transmembrane protein present in B cells and follicular dendritic cells. This type of protein can be used as a target molecule for CAR T cells to target and eliminate B cell malignancies, such as B cell acute lymphocytic leukemia (Wang et al. 2012). More details about T cell immunotherapy are covered in section 4.5.
4.1.2 Neurodegenerative diseases
Parkinson’s Disease (PD) is a disorder where neurons which produce dopamine (dopaminergic cells) in the midbrain region substantia nigra degrade, thus diminishing the dopamine levels in this area. Some of the symptoms developed by patients diagnosed with PD include: tremors in the hands, slow movement, rigidity of the limbs and affected balance (Yasuhara et al. 2017, Ali and Morris 2015).
There have been many attempts of finding a solution to the degeneration of the dopaminergic degrad-ing and of tackldegrad-ing PD. In 2017 there were 18 studies correlated to PD and cell transplantation in the “National Institutes of Health–registered clinical trials”. In general these look very promising but there are some obstacles that must first be dealt with to obtain a viable solution to the disease (Yasuhara et al. 2017). 4.1.3 Ophthalmology
Ophthalmology is the medical area that covers the anatomy, physiology and diseases surrounding the eyeball and its orbit. Eyes have specific characteristics which makes ophthalmology very promising in gene and cell therapy since they are partly independent external organs and have blood-retinal barriers. Because of this the immune response of this area is less likely to act upon substances like cells and genes which may not occur naturally in the eye. The strategies used in the ocular diseases are gene replacement therapy, cytokine therapy and optogenetic therapy (Zhang et al. 2015).
Leber’s congenital amaurosis (LCA) is a hereditary blinding disease which shows early on in young chil-dren and worsens with age and the disease is caused by mutations of the gene RPE65. In 2007 a clinical trial consisting of three patients, suffering from LCA participated, where they were delivered RPE65 gene through a AAV vector, serotype 2 (AAV2.hRPE65v2). After the treatments the three patients showed improvement in retinal function (Maguire et al. 2008).
4.2
Approved gene and cell therapy products in Europe and the U.S.
In the U.S. the Office of Tissues and Advanced Therapies (OTAT), which is an organisation in the FDA, regulates approval of gene and cell therapy products. The 17th of May 2018 there were 18 approved products on the market (U.S. Food & Drug Administration, 2018).
Seven out of the 18 products uses Hematopoietic Progenitor Cells (HPC) from cord blood. The cells are taken from the donors umbilical cord during childbirth and are transplanted into patient in need of hematopoietic and immunologic reconstruction. One of the 18 approved products is the collection bag of these HPC cells. There are also products for several other diseases including lymphocytic leukemia, retinal dystrophy and prostate cancer. There is even one product that is made solely to improve appearance. This product is called LavivTMand is made to treat nasolabial fold wrinkles (U.S. Food & Drug Administration, 2018). The
Table 1. FDA approved cellular and gene therapy products 17th of May 2018. The indications are based on indications from the FDA(U.S. Food & Drug Administration, 2018).
Brand Description Indication
AllocordTM, ClevecordTM,
HemacordTM, DucordTM and
three more which do not have a trade name
HPC Cord Blood Transplantation of donor hematopoietic progenitor cells (HPC) for patients in need of hematopoietic and immunologic reconstruction
None yet Cord Blood Collection Unit Bags for collecting umbilical cord blood during childbirth LavivTM Autologous Fibroblast
(azficel-T) Treatment of nasolabial foldwrinkles to improve appearance
Maci® Autologous Cultured
Chondrocytes Repair of cartilage defects of theknee GintuitTM Allogeneic Cultured
Keratinocytes and Fibroblasts For application on top of asurgically created vascular wound beds in the treatment of mucogingival conditions
Imlygic® Talimogene laherparepvec For treatment of melanoma on the skin or in the lymph glands KymriahTM CAR T cells (tisagenlecleucel) T cell therapy for the treatment
of B cell precursor acute lymphocytic leukemia
LuxturnaTM Voretigene neparvovec-rzyl Gene therapy to treat patients
with a biallelic RPE65 mutation which causes retinal dystrophy Provenge® T cell therapy (sipueleucel-T) For treatment of metastatic
castrate resistant prostate cancer YescartaTM CAR T cells (Axicabtagene
ciloleucel) Autologous T cellimmunotherapy for treatment of large B cell lymphoma
Andexxa® Coagulation factor Xa
(recombinant), inactivated-zhzo For patients treated withrivaroxaban and apixaban,when reversal of anticoagulation is needed due to life-threatening or uncontrolled bleeding
Plasma Cryoprecipitate Plasma Cryoprecipitate (For
Further Manufacturing Use) Not available
Table 2. EMA approved cellular and gene therapy products 3rd of May 2018. The indications are based on indications from the European Medicines Agency.
Brand Description Status Type Indication Chondrosphere
(Spherox®) AutologousThree-dimensional chondrocyte spheroids
Approved TEP For patients suffering from cartilage knee lesions (EMA, 2018) Zalmoxis® Genetically modified
allogeneic T cells Approved CTMP Given afterhaematopoietic stem cell transplantation (HSCT) of patients with high-risk haematological malignancies to increase transplantation success and minimize risk of infection (EMA, 2018) Strimvelis® Autologous CD34+ transduced to express Human Adenosine Deaminase (ADA)
Approved GTMP For patients lacking sufficient amount ADA leading to immunodeficiency (EMA, 2018) Imlygic® Talimogene
laherparepvec Approved GTMP For treatment ofmelanoma on the skin or in the lymph glands (EMA, 2018)
Holoclar® Autologous corneal epithelial cells containing stem cells
Approved TEP Treatment for limbal stem-cell deficiency caused by burns to the eyes(EMA, 2018) Provenge® Autologous Cellular
Immunotherapy (sipueleucel-T)
Withdrawn CTMP For treatment of metastatic castrate resistant prostate cancer(EMA, 2018) Maci® Autologous Cultured
Chondrocytes Suspended TEP Repair of cartilagedefects of the knee (EMA, 2018) Glybera® Alipogene tiparvovec Discontinued GTMP For patients with
familial lipoprotein lipase deficiency (LPLD) and suffering from pancreatitis attacks despite fat restricted diet. (EMA, 2018)
ChondrocelectTM Autologous cartilage
4.3
Gene and cell therapy companies and their products
In this section we present an overview of the companies in the U.S. and Europe that are active within various fields of gene and cell therapy. These companies can be seen as potential customers to Gyros Protein Technologies AB since we believe that the companies can have usage of Gyros Protein Technologies AB platform, GyrolabTM. The companies are presented in tables that are divided with respect to the different
areas of research/applications that the companies focus on; eye-related diseases (Table 3), neuronal diseases (Table 4), CAR T and TCR therapies (Table 5), development of viral vectors for purchase (Table 6), and other multiplex areas (Table 7). One of the columns shows the companies’ products that are currently in clinical phases. For those cells that are empty in this column, no information about a specific product was found on the company website.
Table 3. An overview of companies that focus on ophthalmology (medical area that covers the anatomy and physiology surrounding the eyeball and its orbit.)
Company Focus area Therapy Products AGTC (the U.S.) Gene therapies for orphan
patients suffering with impaired vision from
Achromatopsia and X-linked Retinoschisis.
Gene CNGB3 (Phase I) Achromatopsia
XLRS (Phase I) X-Linked Retinoschisis(AGTC 2018) Horama (France) Developer of gene therapies
to treat rare ophthalmic inherited diseases.
Gene HORA-RPE65 (Phase I/II) RPE65 Retinitis pigemntosa HORA-PDE6B (Phase I/II) PDE6B Retinitis pigemntosa HORA-RLBP1(Preclinical) RLBP1 Retinal dystrophy (Horama 2018) Nightstar Therapeutics (the U.S)
Focus is on developing and commercializing novel, one-time treatments for patients suffering from rare inherited retinal diseases that would otherwise progress to blindness.
Gene NSR-REP1 (Phase III) Choroideremia NSR-RPGR (Phase I/II) X-linked Retinitis Pigmentosa (Nightstar Therapeutics 2018) Quethera (the
U.K.) Dedicated to improve thefuture treatment of common blinding eye diseases. (Quethera 2018)
Gene
In Table 4, companies which are focusing on neurological diseases are presented. In section 4.1.2 there is more information about neurological diseases.
Table 4. An overview of companies that focus on neurological diseases.
Company Focus area Therapy Products
Agilis Bio (The
U.S.) Target rare monogenicdiseases that affect the central nervous system (Agilis Bio 2018).
Gene AGIL-AADC (Phase I/II) AADC Deficiency(Agilis Bio 2018)
Company Focus area Therapy Products AveXis (the U.S.) Developing gene therapies
for rare neurological genetic diseases (AveXis 2018).
Gene AVXS-101 (Phase III) Spinal muscular atrophy (SMA) Type 1
AVXS-101 (Phase I) SMA Type 2(AveXis 2018) CombiGene
(Sweden) Developer of gene therapiesusing viral vectors and NPY-receptors to treat patients suffering from epilepsy and other neurological diseases.
Gene CG01 (Phase I/II) Epilepsy
(CombiGene 2018)
Lysogene (France,
the U.S.) Developers of genetherapies to treat neurodegenerative disorders.
Gene and viruses LYS-SAF302 (Phase II) Sanfilippo A LYS-GM101 (Preclinical) Gangliosidose (Lysogene 2018) Voyager Therapeutics (the U.S.) Focus is on severe
neurological diseases. Gene VY-AADC (Phase I)Advanced Parkinson’s Disease
(Voyager Therapeutics 2018)
In Table 5, companies which are focusing on CAR T cells and TCR to treat cancer are presented. In section 4.1.1 there is some information about oncology and in section 4.5.1 more information about T cell-based therapies is presented.
Table 5. An overview of companies that focus on immunotherapies regarding CAR T cells and TCR.
Company Focus area Therapy Products
Bluebird Bio (the
U.S.) Developing both genetherapies and cancer immunotherapies (CAR T program).
Gene & cell bb-2121 (CAR T) (Phase I/II) Multiple Myeloma cancer
Lenti-DTM (Phase II/III) Adrenoleukodystrophy BB305/LentiGlobinR(Phase
II/III) Transfusion-dependent β-thalassemia, severe sickle cell disease(Bluebird Bio 2018)
Company Focus area Therapy Products Cellectis (France) Designing next-generation
immunotherapies by gene editing CAR T cells.
Gene & cell UCART19 (Phase I) Acute lymphoma leukemia
UCART123 (Phase I) Acute myeloid leukemia, blastic plasmacytoid dendritic cell neoplasm UCART22 (Preclinical) B-acute lymphocytic leukemia(Cellectis 2018) Cell Medica (Europe, the U.S.)
Developing of CAR T cell
and TCR immunotherapies. Gene & cell CMD-003 (Phase II) Hodginlymphoma CMD-602 (Phase I) Acute lymphoma leukemia, non small cell lung cancer CMD-501 (Phase I/II) Neuroblastoma, small cell lung cancer(Cell medica 2018)
Celyad (Belgium,
the U.S) Developing CAR T cell-basedimmunotherapy against cancers.
Gene & cell CYAD-01 (Phase I/II) Different cancer forms (Celayd 2018)
Dynavax (Europe,
the U.S.) Developing immunotherapiesbased on Toll-Like Receptor (TLR) biology.
Gene & cell SD-101 + Anti IL-10 (Phase I) Multiple Malignancies AZD1419 (Phase II) Asthma SD-101 + Anti PD-1 (Phase I/II) Melanoma / Head and Neck Squamous Cell Carcinoma(Dynavax 2018) GenSight
Biologics (France) Developing novel therapiesfor patients with severe retinal neurodegenerative diseases.
Gene GS010 (Phase III) Lhon GS030 (Preclinical) Retinitis pigmentosa(GenSight 2018) Juno Therapeutics (Germany, the U.S.) Developing autologous cellular biologics by engineering T cell to treat cancer and other severe diseases.
Gene JCAR017 (Phase I) Non-Hodgkin Lymphoma JTCR016 (Phase I/II) Acute lymphoma leukemia
JTCR016 (Phase I/II) Non-Small Cell Lung Cancer, Mesothelioma(Juno
Therapeutics 2018)
Company Focus area Therapy Products MediGene AG
(Germany) Developing ofimmunotherapies with TCR technology to treat various cancer forms.
Gene & cell DC Vaccine (Phase II) Acute myeloid leukemia MDG1011 (Preclinical) Acute myeloid leukemia, myelodysplastic syndrome (MediGene AG 2018) MultiVir (the
U.S.) Developing transformingimmune gene therapies that target cancers’ most fundamental molecular defects (MultiVir 2018). Gene Poseida Therapeutics (the U.S.)
The initial applications of Poseida’s technologies will be in gene therapy and CAR-T product candidates for liver disorders and cancer, respectively.
Gene & cell P-BCMA-101 (Clinical POC) Multiple Myeloma, CAR-T Therapy(Posedia Therapeutics 2018) Tmunity Therapeutics (the U.S.) Developing immunotherapies for cancer, infectious disease and autoimmune disease.
Cell 1 Solid Tumour CAR-T Program in Clinical Development 1 Solid Tumour TCR Program in Clinical Development(Tmunity 2018) Tocagen (the
U.S.) Have developed a versatilegene therapy platform that represents a new approach in cancer immunotherapy.
Gene & cell Toca 5 (Phase III) Recurrent high-grade glioma
Toca 6 (Phase I) Metastatic solid tumours (CRC, RCC, Melanoma, Pancreatic, Lung and Breast)
Toca 7 (Phase I) Newly diagnosed high-grade glioma (Tocagen 2018)
Transgene
(France) Developing immune-targetedtherapies for the treatment of cancers and infectious
diseases.
Gene & cell TG4010 (Phase I/II) Therapeutic vaccine, non-small cell lung cancer TG4001 (Phase II) HPV positive cancers (therapeutic vaccine)
Companies providing viral vectors are presented in Table 6. The majority of the companies we found are American.
Table 6. An overview of companies that focus developing viral vectors for purchase.
Company Focus area
Askbio (the U.S.) Owns and operates a cell line manufacturing process and an extensive capsid library. They generate third generation gene vectors(Askbio 2018).
FinVector (Finland) Research and development of viral-based gene therapy prod-ucts(FinVector 2018).
Lentigen Corporation (Europe, the U.S.) Manufacturer of custom lentiviral vectors for pre-clinical and clinical applications(Lentigen Corporation 2018).
Vector BioLabs (the U.S.) Suppliers of AAV and adenoviral products (Vector BioLabs 2018).
Virovek (the U.S.) Suppliers of AAV products(Virovek 2018).
Sirion Biotech (Germany) Viral vector engineering and production; AAV, lentivirus, adenovirus(Sirion Biotech 2018).
StrideBio (the U.S.) Focusing on creating and developing novel adeno-associated viral (AAV) vector technologies(StrideBio 2018).
Companies that develop gene and cell therapy products in mixed fields are presented in Table 7.
Table 7. An overview of companies that focus on areas other than those mentioned in Table 3, 4 and 5.
Company Focus area Therapy Products
Abeona
Therapeutics (the U.S.)
Diseases in dermatology,
metabolic and hematology. Gene EB-101 (Phase III)Epidermolysis Bullosa Dystrophica
ABO-101 (Phase I/II) Sanfilippo Syndrome Type A ABO-102 (Phase I/II) Sanfilippo Syndrome Type B (Abeona Therapeutics 2018) Apceth
Biopharma (Germany)
Uses mesenchymal stem cells (MSC) and their genetic modification to for example promote tissue regeneration of diseased and damaged tissues. They have no products in clinical Phase.
Gene & cell apcethTM- 201 (Preclinical) Graft-vs-Host Disease (GvHD), inflammatory bowel disease, diabetes type I, vasculitis or chronic lung disease
apcethTM- 301 (Preclinical) Glioblastoma multiforme, other solid tumours. (Apceth Biopharma 2018)
Company Focus area Therapy Products Audentes
Therapeutics (the U.S.)
Strives to develop gene therapies for life-threatening diseases.
Gene AT132 (Phase I/II) X-Linked Myotubular Myopathy
AT342 (Phase I/II) Crigler-Najjar Syndrome (Audentes Therapeutics 2018)
Avrobio (the
U.S.) Developers of lentiviral-basedgene therapies in Fabry, Gaucher and Pompe diseases and Cystinoses.
Gene AVR-RD-01 (Phase I/II) Fabry disease(Avrobio 2018) BlueRock
Therapeutics (the U.S.)
Develop allogeneic cell therapies that aim to replace dead, damaged, or
dysfunctional cells in patients with degenerative disease.
Cell Parkinson’s disease (Preclinical) Developing dopaminergic neurons that can replace the
dopamine-secreting cells. Cardiac Diseases (Preclinical) With help from stem cells they want to replace heart muscle cells in patients who lost muscle cells after heart attacks.
(BlueRock Therapeutics 2018)
Calimmune (the
U.S.) Clinical-stage company, maindevelopment area are novel outpatient ex vivo gene therapies for hematologic diseases.
Gene CAL-1 (Phase I/II) HIV/AIDS CAL-H (Preclinical) Hemoglobinopathies (Calimmune 2018) Errant Gene Therapeutics (Europe, the U.S)
Developer of gene therapy drugs against rare diseases and disorders.
Gene Thalagen (Preclinical) Thalassemia
JW-1521 (Preclinical) Refractory prostate cancer MSKCC product
(Preclinical) Sickle cell anemi
(Errant Gene Therapeutics 2018)
Freeline Therapeutics (The U.K.)
Developer of gene therapy products against bleeding disorders like Haemophilia B. (Freeline Therapeutics 2018)
Gene
Company Focus area Therapy Products GenVec, Inc. (the
U.S.) Clinical-stage company usingtheir AdenoVerse™ gene delivery platform to develop therapeutics and vaccines.
Gene CGF166 (Phase I)
Hearing loss and vestibular dysfunction
(GenVec, Inc. 2018) Krystal Biotech
(the U.S.) Developing gene therapies totreat rare orphan dermatological disorders with their Skin TARgeted
Delivery platform, or STAR-D platform.
Gene KB103 (Phase I/II) Dystrophic EB KB105 (Research) TGM-1 Deficient ARCI KB106 (Research) Wound healing (Krystal Biotech 2018) LogicBio Therapeutics (the U.S) Developer of GeneRideTM which is used when integrating therapeutic transgenes while gene editing. (LogicBio Therapeutics 2018)
Viral vector & gene
NanoCor
Therapeutics (the U.S)
Gene therapy for
Cardiovascular Disease. Gene Carfostin(R)(NanoCor Therapeutics 2018) Orchard
Therapeutics (the U.K.)
Focus is on primary immune deficiencies and inherited metabolic disorders.
Gene Strimvelis (commercialized) OTL-102 (Clinical POC) X-linked chronic
granulomatous disease (X-CDG)
OTL-300 (Clinical POC) Beta-thalassemia (Orchard Therapeutics 2018) Renova Therapeutics (the U.S.) Developing definite treatments for congestive heart failure and type 2 diabetes.
Gene & peptide RT-100 (Phase II/III) Congestive heart failure(gene therapy)
RT-400 (Phase II)
Acute decompensated heart failure (peptide therapy) (Renova Therapeutics 2018) Solid Biosciences
(the U.S.) Focusing on solvingDuchenne muscular dystrophy. Gene SGT-001 (Clinical) Microdystrophin Gene Transfer (Solid Biosciences 2018) Spark Therapeutics (the U.S.)
Initial focus is on treating
4.4
Gene therapy: the manufacturing process
In the order from Gyros Protein Technologies AB one of the requests was to describe the general manu-facturing process of a gene therapy product. A gene therapy product normally consists of a viral vector designed with the gene of interest in the genome to be inserted into the patient. The manufacturing process of a gene therapy product can be divided into the four steps shown in Figure 2. The steps are presented in sections 4.4.1, 4.4.2, 4.4.6 and 4.4.7. In section 4.4.3, 4.4.4 and 4.4.5 the commonly used cell line HEK293 is presented, as well as the risk of replication-competent viruses and some alternative cell lines.
Figure 2. The general process of gene therapy manufacture, divided into four steps.
The manufacturing process of a gene therapy product can differ from product to product, but in this thesis the general steps used in most gene therapies are presented (Minamide et al. 2003). Hence, Gyros Protein Technologies AB will receive information that will be of importance when determining where in the process their product GyrolabTM immunoassay platform can be used. After the production, the final product is
infected into patient cells and the patient’s immune response to the treatment is evaluated. This is described in section 4.4.8.
4.4.1 Generate a recombinant viral plasmid using homologous recombination
The first step in production of a gene therapy viral vector is to generate a recombinant viral plasmid. Here, generation of adenoviral plasmids and adeno-associated viral (AAV) plasmids are described. To generate adenoviral plasmids, normally two different plasmids are used – one shuttle plasmid that contains the trans-gene and another plasmid that contains the adenoviral genome were the E1 and E3 regions have been deleted, as described in section 2.2.1. Deleting E1 and E3 ensures that the vector is unable to replicate and opens up space for the transgene to be inserted. The plasmid containing the transgene is flanked by copies of small sequences of the adenoviral genome allowing for homologous recombination between the two plasmids. This is done in Escherichia coli cells. After the homologous recombination the DNA plasmids are isolated (Minamide et al. 2003). Generation of AAV plasmids is very similar to generation of adenoviral plasmids, but there is no need for deletion of genes as the AAV already is non-replicating (Halbert et al. 2018). 4.4.2 Transfect the plasmids into a producer cell line
After the viral plasmids have been isolated they are transfected into host cells. Host cells are needed for production of viral vectors, as the virus genome is modified so that the virus cannot replicate on its own. The host cells must be able to provide trans-complementation for the virus plasmid, which means that a part of the host cell genome can complement for the missing genome region in the viral plasmid. Therefore, the host cells are taken from a producer cell line, either a cell line that has specific viral sequences in the genome for complementation or a cell line that naturally can complement for the virus genome. The host cells are transfected with the viral plasmids and viral vectors are then produced inside the host cells (Kovesdi and Hedley 2010).
When using host cells for production, residual host cell proteins (HCPs) will have to be removed in the purification and the levels of remaining HCP must be measured to guarantee product purity. The mea-surement can be performed by an immunoassay using HCP-specific antibodies – possibly by a GyrolabTM
4.4.3 The HEK293 cell line
HEK293 is a cell line widely used for producing viral vectors needed in gene therapy. The HEK293 cell line was developed 1977 by stably transforming human embryonic kidney (HEK) cells with the E1 region of adenovirus type 5 DNA. The HEK293 cell genome can trans-complement for the E1 region for production of viruses, as the cell genome contains the adenoviral E1 DNA. The E3 region however is not essential for viral replication in vitro, so no trans-complementation for it is necessary. The HEK293 cells are easy to grow in different culture media, both adherently in a serum-containing medium as well as in a suspension in a serum-free medium, and are easily transfected with exogenous DNA (Minamide et al. 2003). There are several derivatives that have been developed from the HEK293 cell line to be more efficient. 293T and 293E are two common derivatives (Stacey and Merten 2011).
4.4.4 A safety risk with HEK293
For adenoviral vector production, there is a disadvantage with the HEK293 cell line and its derivatives. HEK293 cells can produce replication-competent adenoviruses (RCA) due to homologous recombination events between the adenoviral plasmid and the adenoviral sequences in the cell genome. Therefore, since HEK293 was developed, many other adenovirus complementing cell lines have been developed with reduced homology – reduced adenoviral sequences in the cell genome (Stacey and Merten 2011).
The contamination of replication-competent viruses is a problem in large-scale clinical applications because of safety issues. The Food and Drug Administration (FDA) in the United States decided 2001 that adenoviral vector preparations must contain less than one RCA in 30 billion viral particles. The HEK293 cell line is, despite the contamination issue, still very much used for development of adenoviral vectors (Kovesdi and Hedley 2010).
4.4.5 Alternative cell lines
One of the first alternative cell lines to be developed was the 911 cell line – human embryonic retinoblast (HER) cells with adenoviral sequences incorporated in the cell genome. But the level of replication-competent adenovirus was still high. Another alternative cell line is the PER.C6 cell line. The PER.C6 cells are HER cells with the E1A- and E1B adenovirus sequences under control of a human phosphoglycerate kinase (PGK) promoter instead of the E1A promoter. The coding region for the capsid pIX protein is deleted in the cell genome. This gives the incorporated adenovirus sequence less homology to the E1-deficient adenoviral vec-tor, and therefore homologous recombination cannot occur when an appropriately adenoviral vector is used. This implicates that no RCAs should be found. The PER.C6 cell line is probably the most favourable cell line for clinical development at the moment (Kovesdi et al. 2010).
A disadvantage with the PER.C6 cell line, unlike HEK293 cells, is that PER.C6 cells currently have strict licensing costs, which makes it too expensive for many laboratories. The PER.C6 cell line is also less adapt-able to serum free culture for large scale production of viral vectors than the HEK293 cell line (Stacey and Merten 2011).
Several laboratories have developed their own cell lines that contain smaller adenoviral E1 sequences than HEK293, which reduces the replication-competent viruses due to reduced sequence homology and limited double homologous recombination crossover events. Most cell lines are based on human primary amniocyte cells, human embryonic lung (HEL) 299 cells or HeLa cells with the adenovirus E1A region incorporated in the genome with an alternative promoter. (Kovesdi and Hedley 2010)
4.4.6 Downstream processing of viral vectors
"Viral Vectors for Gene Therapy" (2011) by Segura et al. and are illustrated in Figure 3.
Figure 3. The general steps of the downstream processing of viral vectors.
The methods used for downstream processing are different in a large scale production compared to a small scale laboratory production. The different methods can be seen in Table 8 below.
Table 8. General downstream processing methods for a laboratory strategy and for a scalable strategy.
Laboratory strategy Scalable strategy Harvest Cell lysis (optional) Cell lysis (optional) Clarification Centrifugation Microfiltration Concentration Pelleting/Precipitation Ultrafiltration Purification Density gradient ultracentrifugation Chromatography 1 Polishing Density gradient ultracentrifugation Chromatography 2
The first step is to harvest the viral vector particles from the producer cell culture. If the viruses remain intracellular, as for naked viruses (e.g. adenovirus and adeno-associated virus), release of the viral particles by lysis is necessary. The lysing can be performed by different methods, such as repeated freeze-thaw cycles in a laboratory scale, or by mild pressure changes for a large scale production. Cell lysis releases the intra-cellular viruses, but also host cell DNA and proteins are released from the producer cells. Host cell DNA is often eliminated directly after the cell lysis by adding nucleases, such as BenzonaseTM (Segura et al. 2011).
Enveloped viruses (e.g. lentivirus and retrovirus) do not require cell lysis. Instead, the virus is secreted by the producer cell through a budding process where the virus take its envelope from the host cell membrane, which encloses the virus genome (Nestola et al. 2015).
In the remaining four steps of the downstream processing the product is purified from host cells, host cell pro-teins (HCPs) and reagents like BenzonaseTM. After the processing, the final product must be characterized
to ensure that it is safe and free from impurities (Segura et al. 2011). 4.4.7 Characterization of the final product
Figure 4. An example of QC testing strategy for characterization of an AAV gene therapy vector which present tests to be made of the cell harvest, of the bulk vector (after purification) and of the final product. Some of the QC tests can be made both in bulk vector testing and in final product testing. The blue arrows show where immunoassays (ELISAs) are used and thus where the GyrolabT M immunoassay platform could be used instead. The figure is modified from a figure by Wright and Zelenaia 2011.
In Figure 4 there can be seen that immunoassays, like an enzyme-linked immunosorbent assay (ELISA), can be used for three purity tests – residual bovine serum albumin (BSA), residual HEK293 (or another cell line) host cell protein, and residual BenzonaseTM (Wright and Zelenaia 2011). Bovine serum albumin (BSA) is a
protein that can be found in the product when using bovine serum in the cell culture (Zhang et al. 2010). BenzonaseTM contains nucleases and is used to digest nucleic acids in the downstream processing of viral
vectors (see section 4.4.6). The concentration of BenzonaseTM should be less than 1.0 picogram in 10 billion
vector genomes. Immunoassays can also be used for a potency test – to measure transgene expression by in vitro transduction and ELISA. This is done to evaluate the functional activity of the viral vectors (Wright and Zelenaia 2011). In these tests, the GyrolabT M immunoassay could be used instead of ELISA.
following transgene encoded protein is measured by a transgene targeting ELISA (Wright and Zelenaia 2011). Immunoassays can also be used to measure the total amount of viral particles, both functional viral par-ticles and vector-related impurities such as empty capsids, using a capsid-specific antibody. A non-specific absorbance assay can also be used (Wright and Zelenaia 2011).
The QC must contain tests for absence of microbial contamination to ensure product safety. Testing should include assays for bioburden, sterility, mycoplasma, endotoxin, general safety, and adventitious viral agents. In section 4.6 there is more detailed information of the safety testing of the viral vectors and some general safety aspects regarding use of viruses.
4.4.8 Measure the immune response of the patient
Before treatment with viral vectors, there can be pre-existing immunity to the viral vector in the patient, due to natural exposure to the virus type in question. In addition, administration of a viral vector can induce an immune response to the viral capsid, which can be a problem as vector toxicity. There are several meth-ods to evaluate the immune responses before and after administration of the viral vector that can be used for preclinical and clinical trials. GyrolabTM makes it possible to measure concentrations in small sample
volumes, which is preferable in question of patient samples. Therefore, GyrolabTM could be applied when
measuring the immune response of a patient. The immune response can be divided into three categories – the humoral (specific) immune response; the innate (non-specific) immune response; and the cellular (T cell) immune response (Calcedo et al. 2018). The three categories are explained more deeply in sections 4.4.9, 4.4.10, and 4.4.11. Immune responses can lead to a decreased vector efficacy and can disturb the vector distribution in the host (Calcedo et al. 2018).
Figure 5. The different assays used to evaluate immune responses, designed for recombinant adeno-associated virus. The immune response to viral vectors are divided into three groups and many subgroups. The blue arrows show where ELISAs are used and thus where the GyrolabTMimmunoassay could be used as an alternative assay. NAb, neutralizing
antibody; BAb, binding antibody; IFN, interferon; IgG, immunoglobulin G; IgM, immunoglobulin M; ELISpot, enzyme-linked immunospot; ELISA, enzyme-enzyme-linked immunosorbent assay. The figure is modified from a figure by Calcedo et al. 2018.
The assays need to be performed several times after the administration of viral vectors in the patient. In Table 9 below, the immune responses and assays are presented together with time points. The time points indicate how often the product GyrolabTM needs to be used in the process and thus gives an insight into
how much Gyros Protein Technologies AB can sell.
Table 9. An overview over the assays recommended to use to detect immune responses. Type of immune response, assay used, and time points to measure are shown. NAbs, neutralizing antibodies; BAbs, binding antibodies; IgG, immunoglobulin G; IgM, immunoglobulin M; ELISA, enzyme-linked immunosorbent assay; IFN, interferon; pre, before the treatment; w2, week 2 after the treatment; m2, month 2 after the treatment.
Immune response Marker(s) Assay(s) Time points Humoral NAbs In vitro NAb assay pre, w2, w4, m2, m3,
m6, m12
Humoral BAbs (IgG and IgM) ELISA pre, w2, w4, m2, m3, m6, m12
Innate Cytokines, chemokines
and growth factors Bead-based multiplexassay, or ELISA pre, 2h, 4h, 6h, 12h, 24h,48h, day 4 Cellular (T-cell) IFN-γ IFN-γ ELISA, epitope
mapping, cytokine staining
pre, w2, w4, m2, m3, m6, m12
4.4.9 Humoral immune response
The humoral (specific) immune response includes neutralizing antibodies (NAbs) and binding antibodies (BAbs) to the virus capsids. The antibody levels should be measured both before and after the vector administration. Preexisting antibodies can exist in the body due to natural infection of the specific viral vector, and a high level of them can blunt the efficacy of the treatment. NAbs can be quantified by an in vitro NAb assay using a microscope and a luminometer. BAbs, such as immunoglobulin G (IgG) and immunoglobulin M (IgM), can be measured by an ELISA using antibody-specific antibodies (Calcedo et al. 2018).
4.4.10 Innate immune response
A high innate immune response to viral vectors can be associated with severe toxicity. It is unclear if the response is triggered by the virus itself or by residual contaminants. To evaluate the innate immune response you can measure the level of inflammatory cytokines (interleukin (IL)-1, IL-2, IL-6, IL-8, IL-10, IL-18 and interferon (IFN)-α), chemokines (macrophage inflammatory protein and monocyte chemoattractant protein-1), and growth factors (vascular endothelial growth factor, tumour necrosis factor and IFN-γ-inducible protein-10). The analysis of cytokines, chemokines and growth factors can be measured using an ELISA, but is recommended to be measured by a bead-based multiplex assay (where commercially available kits exists) as it reduces the volume of serum needed and increase analytes being evaluated simultaneously, compared to an ELISA (Calcedo et al. 2018).
4.4.11 Cellular (T cell) immune response
The administration of a viral vector can give a T cell response to the virus capsid and to the transgene product. The vector can either reactivate pre-existing vector-specific T cells, existing due to natural infection of the virus, or induce a primary response in a patient where pre-existing vectors-specific T cells do not exist. The T cell responses can be quantified by the release of the cytokine IFN-γ after stimulation of vectors-specific antigens. IFN-γ is the main cytokine produced by T cells. Typically an ELISA is used. If the ELISA shows a positive response, it can be further analyzed to determine the amino acid sequence (epitope) responsible for T cell activation, called epitope mapping. The next step, when the epitope has been determined, is to characterize the T cell subset that makes the recall response, to determine if it is a CD8+ T cell response or a CD4+ T cell. This is made by intracellular cytokine staining (Calcedo et al. 2018).
4.4.12 An example of a clinical trial measuring immune response
4.4.13 Analytes that immunoassays can detect
Gyros Protein Technologies AB seek information about which potential analytes to provide new ready-to-use kits for. In this section a summary of all analytes in gene therapy that can be detected and quantified by immunoassays such as ELISA and GyrolabTM is therefore listed, see Table 10 below.
Table 10. A summary of analytes in gene therapy that can be detected by immunoassays. IgG, immunoglobulin G; IgM, immunoglobulin M; IL, interleukin.
Analyte(s)
Bovine serum albumin Producer cell protein BenzonaseTM
Transgene protein
Total amout of viral particles
Immunoglobulin G and Immunoglobulin M IL-1, IL-2, IL-6, IL-8, IL-10 and IL-18 Interferon-α
Interferon-γ-inducible protein-10 Macrophage inflammatory protein Monocyte chemoattractant protein-1 Vascular endothelial growth factor Tumour necrosis factor
4.5
Cell therapy
As previously mentioned, Gyros Protein Technologies AB are interested in areas where their product GyrolabTMcan be used. This section therefore describes the manufacturing process of a cell therapy product
and presents opportunities where GyrolabTM can be applied. As earlier presented there are numerous fields
where cell therapy can be applied. In Figure 6, an overview of the manufacturing process for a cell therapy product is presented. The process is similar for all autologous cell therapy products (Eaker et al. 2013).
Hematopoietic stem cell transplantation is by the British journal Nature, presented as a cell therapy. In stem cell transplantation, no modification of the cells is needed, therefore modifying cells is not included in all cell therapies (Figure 6 step 4) (Nature 2018).
There are several research fields concerning cell therapies that are being studied worldwide by different companies. This thesis primarily focuses on CAR T and TCR cell therapy, which are both T cell-based therapies.
4.5.1 T cell-based cell therapy
T cells play an essential role in our immune system since they are involved in preserving the immunological self-tolerance (Hori et al. 2003). Although, they are not always fast or aggressive enough to fight certain fast-growing tumours which can cause cancer (MacDonald 2016). Fortunately, the development of cell therapies against cancer by using T cell based processes for adoptive cell transfer (ACT) has recently been shown to be promising in cases of acute lymphocytic leukemia (ALL). T cell receptor (TCR) and chimeric antigen receptor (CAR) cell therapies are two of these promising cell therapies (Kaiser et al. 2015).
4.5.2 Companies developing TCR and CAR T cell therapies
The international company Cell Medica is developing a CAR T cell therapy technology that makes it possible to modify natural killer T (NKT) cells to make them target certain molecules expressed on tumours. Cell Medica also has a TCR cell therapy technology. TCRs allow T cells to recognize cancer tissues, e.g. to recognize antigens which are located on the surface of the tumour cells (Cell Medica 2018, MediGene 2018). The TCR therapies have the potential to detect a greater number of possible tumour antigens than other therapies (MediGene AG 2018). This is because TCRs are more versatile than for example CARs since TCRs can identify antigens on tumour cells from all other proteins expressed in all parts of the cell (Cell Medica 2018). German MediGene AG is another company that focuses on development of personalized T cell-based immunotherapies. As for Cell Medica, one of MediGenes main research areas is the TCR-platform. They are working on developing a library consisting of specific receptors for a variety of cancer indications (MediGene AG 2018). There are numerous companies that focuses on T cell based therapies, see Table 5 (section 4.3). 4.5.3 CAR T cell therapy
Immunotherapy which uses CD19-targeted CAR T cells have demonstrated high response rates in patients suffering from B cell malignancies (Wang and Rivière 2016, Levine et al. 2017). CD19 refers to the B cell antigen CD19 (Minton 2018). These CAR T cells are generated by extracting T cells from the blood of a patient and then modifying the T cells to express the CAR. This reprograms the T cells to target tumour cells. As for today CAR T cell therapy has shown high response rates in pediatric patients with relapsed or refractory ALL. Novartis Pharmaceuticals’ development of a CAR T cell therapy has entered phase 2 multi-site trials for this disease (NCT02435849 and NCT02228096) (Levine et al. 2017). The FDA has also given the approvement of two CD19 CAR T cell therapy products, KymyrahTM and YescartaTM, see section
4.2 (Zheng et al. 2018).
When it comes to producing CAR T cells, the production steps are many and throughout the entire process quality control testing is performed, (section 4.5.10) (Levine et al. 2017). See Figure 6 and 7 for a description of the production process of CAR T cell therapy. The process starts with extracting T cells from the patient (Wang and Rivière 2016).
4.5.4 Extracting T cells from cell source
from the leukapheresis buffer and further enrichment of lymphocytes is adopted throughout counterflow centrifugal elutriation (Figure 6 step 2). The centrifugation permits the cells to be separated by size and density while maintaining cell viability. A supplementary step that may be performed is the separation of T cell subsets at the level of CD4/CD8 composition by using specific antibody bead conjugates or markers (Levine et al. 2017).
4.5.5 Activating and modifying T cells
Activation of T cells is required to initiate and regulate immune response (Figure 6 step 4) (Pross 2017). T cell activation follows by response of T help cells (CD4) together with B cells and cytotoxic T cell (CD8) response (Soskic et al. 2014). Activation of T cells by purifying autologous antigen-presenting cells (aAPCs) from patients, it requires several additional steps making it laborious and challenging to receive a potent CAR T cell. Therefore, developing an approach to standardize activation of T cells more efficiently has been made. This was done by using beads coated with anti-CD3/anti-CD28 monoclonal antibodies. The aAPCs can then be separated from the cell culture by magnetic force. When interleukin-2 (IL-2) and aAPCs are present, T cells are able to increase logarithmically in a perfusion bioreactor under a period of a couple of weeks (Levine et al. 2017). IL-2, also known as T cell growth factor (TCGF), is observed as the controller of the immune response (Thèze 1998). Engineering aAPCs to express costimulatory ligands is another method that could be used when expanding T cells ex vivo. The aAPCs can be derived from the chronic myelogenous cell line K562 (Levine et al. 2017).
When modifying T cells (Figure 6 step 4), they are incubated for several days with a viral vector en-coding the CAR (Levine et al. 2017). The viral vectors are produced as described in section 4.4. The viral vector can be a γ-retroviral or lentiviral vector (Jin et al. 2016). The vector is then washed out by either diluting the culture or by exchanging the medium. In CAR T cell therapy, the genetic material encoding the CAR will be permanently integrated into the host genome. Lentiviral vectors like CTL019, are often used in clinical trials of CAR T cell therapy since they have a safer integration site profile than say γ-retroviral vectors. A bioreactor culture system is then used to grow large volumes of the wanted CAR T cells (Levine et al. 2017).
4.5.6 Volume reduction and washing
After the cell expansion is done (Figure 6 step 5), the cell culture can be of large volumes and must therefore be concentrated to be further infused into the patient (Levine et al. 2017). Concentration of the product can often be done by centrifugation (Eaker et al. 2013). Concentrated cells are cryopreserved in infusible medium, this benefits in preserving the CAR T cells over long periods of time (Figure 6 step 6) (Wang and Rivière 2016).
4.5.7 Reinfuse CAR T cells into patient
A summarizing figure over the product process of T cell based product is presented in Figure 7.
Figure 7. The general process of T cell-based therapy.
4.5.8 Analyzing CAR T cells with ELISA
Since Gyros Protin Technologies AB were interested in areas where their platform GyrolabTM could be
used, this section presents analytes which can be analyzed with immunoassay instruments. Enzyme-linked immunosorbent assay (ELISA), described in Appendix 3, can be used when analyzing the function of CAR T cells. In table 11, analytes which can be analyzed are presented from examples described in this section.
Table 11. Analytes that can be detected and quantified by an immunoassay to determine CAR T cell function.
Analyte Target modules Interferon-γ Interleukin-2 Interleukin-12
Tumour necrosis factor αβTCR
Kueberuwa et al. (2018) have likewise studied CAR T cell function by measuring interleukin release with ELISA. In their experiments they measured the IL-12 release in studies conducted on mice. These mice were infused with anti-CD19 CARs genetically modified to either express IL-12 secretion or not. Results of the study demonstrated that CAR T cells expressing IL-12 was effective in diminishing CD19-cells and also in attracting host immune cells to an anti-cancer immune response which led to long-time surviving among the mice. CAR T cells that did not express IL-12 secretion did not attract host immune cells in the same range. Additionally, the group measured IFN-γ with an ELISA (Kueberuwa et al. 2018).
There are risks with using viral vectors when performing cell therapies, one of them addressing insertional mutagenesis and genotoxicity. In a study from 2016, Lin et al. conducted studies to strengthen the hypoth-esis that a scaffold/matrix attachment region (S/MAR) containing non-integrating lentiviral vector (NILV) will work as an excellent vector for long-term T cell engineering with little risk of insertional mutagenesis and genotoxicity. In their experiments, they used an ELISA to determine the concentration of IFN-γ secretion from stimulated T cells, they used an anti-IFN-γ antibody (Mabtech, Nacka Strand, Sweden) to conduct the ELISA (Lin et al. 2016).
ELISA-kits have also been of usage when Sperl et al. (1995) wanted to determine the concentration range where soluble murine T cell receptor presented biological activity. This was done by manufacturing and performing four different ELISAs to quantify αβ TCR (Sperl et al. 1995).
4.5.9 The T cell receptor (TCR)
TCR cell therapy is an additional type of cell therapy similar to CAR T cell therapy discussed in ear-lier sections of this thesis. T cells express an antigen-binding molecule called the TCR (T cell receptor) (Goldsby et al. 2000). Stimulation of the TCR is triggered by the major histocompatibility complex (MHC) on antigen-presenting cells (APCs), and TCRs can only recognize antigens that are bound to the MHC molecules (Davidson et al. 2003, Brownlie and Zamoyska 2013, Goldsby et al. 2000). The binding complex antigen-MHC leads to activation of T helper cells (TH) which is a subpopulation of T cells. Another
subpop-ulation of T cells is T cytotoxic (TC) cells. The activated THcells contribute to secretion of cytokines which
leads to further activation of a series of biochemical events which represent the so-called immune response (Goldsby et al. 2000).
MHC molecules are cell-membrane proteins and can be divided into two major types; Class I and Class II molecules. The THcells become activated when they interact with the Class II complex. When TC cells
recognize Class I molecule complexes, they differentiate into a so-called cytotoxic T lymphocyte (CTL), which is an effector cell that has a vital function in eliminating any foreign cells in the body that display antigen, i.e. tumour cells or virus-infected cells. When the TC cells have differentiated into CTLs, they
recognize and kill their targets (Goldsby et al. 2000).
TCR mediated responses. The inhibitory effect of PAG also happens to depend on its ability to be tyrosine phosphorylated. Thus, PAG acts as a negative regulator of T cell activation. In response to T cell activation, PAG becomes dephosphorylated and dissociates from Csk (Davidson et al. 2003).
4.5.10 Quality control
During quality control of CAR T cell products, there is a general need of using immunoassays like GyrolabTM.
Therefore, quality control is discussed in this section. The manufacturing of CAR T cellular products and the complete therapeutic process require technical expertise and certain equipment for quality control (Kaiser et al. 2015, Piscopo et al. 2017). The quality of the cellular products must be measured throughout the whole process, by means of the process analytical techniques and the model predictive control. During model predictive control, bioengineers can use mathematical models to predict the outcomes of a treatment. Often, the current state of the process is measured and used as a basis for prediction of the outcome. For the processing of T cell cultures, spectroscopic techniques and immune biosensors can be used (Piscopo et al. 2017). Revzin et al. (2012) state that the information obtained from an analysis of leukocytes such as T and B cells can be harvested by techniques like ELISA. Thus, assays like ELISA and GyrolabTM could be
used as a complement to biosensors for immune cell analysis.
Kaiser et al. (2015) indicate that the currently available clinical manufacturing processes of therapeutic cell products can only partially meet the requirements that are set today. Quality cannot yet be ensured throughout the entire product life-cycle. There is a restricted amount of material available for quality control testing since the products are individualized and the products have limited durability. The complexity of the assays used can increase the risk of delivering unreliable results (Kaiser et al. 2015). There is a need of improving the manufacturing paradigms in order to allow CAR T cell therapy to be pursued widely. In order to confirm cell viability and immune profile changes, better quality control mechanisms are essential. The use of viral vectors poses several concerns since they insert transgenes randomly into the genome (Piscopo et al. 2017). This involves a risk of giving rise to insertional oncogenesis and thus experimental tumours in the patient (Sadelain 2004).
4.6
Safety with viruses
As mentioned in section 4.5.10, there is a general need of using immunoassays like GyrolabTM in question of
the manufacturing process of CAR T cellular products. However, this is not the only case when there is a demand for analyzing the immune response with immunoassays like GyrolabTM. The immune response needs
to be measured in certain ways with respect to different levels of analytes (e.g. viral vectors, see possible analytes as mentioned in Table 10 (section 4.4.13)). The establishment of quality control (QC) test methods is essential for the translation of research to clinical development. The current Good Manufacturing Practice (cGMP) needs to be implemented after the vector has been generated. Quality control test results must meet certain specifications concerning vector purity, safety, stability, identity and potency. ELISA is suggested as a quality control testing method for an AAV vector investigational product in the cases of measuring the pu-rity of residual HEK293, residual bovine serum albumin (BSA) and residual BenzonaseTM, see section 4.4.7.
