CAR-T-clinical-trial-review

The Global Chimeric Antigen Receptor T Cell (CAR-T) Clinical Trial Review

The Global CAR-T Therapy Clinical Trial —— A Comprehensive Review The year of 2017 is definitely one of the most important year of CAR-T field. This year, Kymriah (CTL019) of Novartis received the first ever FDA approval as a CAR-T cell therapy for children and young adults with B-cell acute lymphoblastic leukemia (ALL), and then, the Kite’s Yescarta became the second approved CAR-T therapy by FDA and the first for certain types of non-Hodgkin lymphoma (NHL). Moreover, another main force of car-t clinical research filed, Juno, has just exposed their latest progress about the importance of IL-15 level to the lethal neurotox appeared in their halted early clinical trial. As the CAR-T clinical trial progresses, scientists have figured out some causes of the lethal side effects of CAR-T therapy, which has given us more control over the safety of CAR-T therapy. CAR-T technology as a breakthrough in anti-tumor therapy is still promising. Novartis and Kite are just the tip of the iceberg in this market. Juno, cellectis, Johnson & Johnson and many other biopharmaceutical companies are all involved in this game. According to the data shown from clinicaltrials.gov, currently there are approximately 400+ completed and ongoing CAR-T clinical trials registered all over the world. Now, we are going to take a deep look into these CAR-T clinical trials to gain a better understanding of the research status quo.

Part 1 Overview of the global CAR-T clinical trials The United States is undoubtedly a bellwether in this market. The world’s first CAR-T clinical trial was conducted in the United States in 2003, and most of the pioneering research works were also initiated in USA. At present, about 140 registered clinical trials in the United States have been completed or are in progress, accounting for 35% of the total number of CAR-T clinical trials all over the world (Figure 1). As the birthplace of CAR-T technology, the United States has more than 10 years of clinical research and management experience, coupled with a relatively mature regulatory system, the number of CAR-T clinical trials grows steadily with about 5-10 clinical projects newly registered each year. 50+ 10-50 5-10 1-5 0-1 Figure 1. World CAR-T clinical trial distribution China is becoming the next leader of CAR-T Therapy clinical research. Its first trial started in 2013, ten years later than the United States, but the number of CAR-T project newly registered in China grows rapidly, especially in the past two years (Figure 2). So far for this year, the total number of registered CAR-T clinical trials, including completed and ongoing, has already reached 145 and is still growing, surpassing the United States.

China has become the country with the Number of Clinical Trials per Year: USA vs. China China largest number of registered CAR-T clinical USA trials in the world. This rapid development is 60 believed to have benefited from the lenient 50 regulatory policies and ambiguous attitudes 40 of the Chinese government towards CAR-T 30 clinical trials, which on the one hand brings 20 large capability and opportunities for the 10 development of China’s CAR-T technology, but on the other hand also brings higher risks 0 and challenges. Figure 2. Number of clinical trials per year: USA vs. China. The United States and China as two giants of CAR-T clinical research, have taken over 70% of clinical research in the world, making the rest area lagging far behind them. 10+ 2 The United Kingdom as the third largest country of 5-10 CAR-T clinical research, only has 12 registered CAR-T 3-5 trials, far less than USA and China. France and Germany 1-2 has 4 and 3 programs respectively. The number of CAR-T clinical trials of all the European counties counted 0 together may reach about 30 or more (Figure 3). Besides, Canada, Japan and Australia each runs 3 clinical trials in 12 3 their own country. Considering the fact that CAR-T is still 4 2 a cutting-edge technology full of uncertainties. It is reasonable these countries take a wait-and-see attitude 1 and hold a relatively restrict regulatory policy to CAR-T clinical program. But while remaining cautious, the Figure 3. CAR-T clinical trial opportunity to seize this market is also slipping away. distribution in Europe It seems that China as a latecomer would surpass the USA vs China United States and take the leading position. However, the depth and breadth of immunotherapy researches in the 47 12 USA still cannot be ignored. Not just CAR-T cell technology, another two similar immunotherapeutic Figure 4. Number of TCR-T & CAR-NK techniques, TCR engineered T cell (TCR-T) and chimeric clinical trials in USA and China antigen receptor NK cell, also receives considerable attention and at least 47 TCR-T or CAR-NK trials are in progress across the United States, compared with just 12 of China (Figure 4). TCR-T therapy is a technique that directly uses high affinity TCR gene to replace the origin TCR. It shows better anti-tumor effect than CAR-T therapy in the treatment of solid tumor according to recent data.

Part 2 CAR-T Cell Targets The specificity and anti-tumor performance of CAR-T cells depend largely on the choice of tumor biomarkers. An ideal tumor biomarker as anti-tumor target should be highly expressed on tumor cell surface and rarely expressed on healthy cell. However it’s quite no easy task to discover this kind of biomarker. In a more common situation, the chosen biomarker is expressed on both the tumor cell and the normal one, with a significant higher expression level on tumor cell than normal cell. so in anti-tumor therapies, whether targeted anti-tumor drugs or CAR-T cells, the accidentally injure of normal cells is inevitable. That is why we should pay close attention to the on-target-off-tumor cytotoxicity of CAR-T cell therapy. CAR-T Cell Targets - World CD33 CEA EpCAM MUC1 CD19 mesothelin 1% 1% 1% 1% BCMA GD2 CD30 GPC3 CD19/CD20 CD22 HER2 1% CD123 CD20 EGFR EGFRvIII CD19/CD22 Other CD19/CD22 CD19/CD20 2% 15% CD33 CEA EpCAM MUC1 EGFRvIII CD19 Other 2% 45% EGFR 2% CD20 2% CD123 2% HER2 2% CD22 3% GPC3 3% CD30 GD2 BCMA mesothelin 3% 4% 4% 5% Figure 5. The target distribution of world’s CAR-T clinical trials Cluster of differentiation 19 (CD19) is regarded as a star biomarker in the area of CAR-T therapy. Forty-five percent of the world’s CAR-T programs are target at CD19 molecule (Figure 5). The second position is mesothelin, accounting for just 5% of the total number, then followed by BCMA and GD2, each for 4%.

Cluster of differentiation 19 (CD19) is regarded as a star biomarker in the area of CAR-T therapy. Forty-five percent of the world’s CAR-T programs are target at CD19 molecule (Figure 5). The second position is mesothelin, accounting for just 5% of the total number, then followed by BCMA and GD2, each for 4%. 45 different biomarkers targeting numerous different types of tumor cells are used as CAR-T targets for clinical trials, including: CD19, CD20, CD22, CD30, CD33, BCMA, mesothelin, GPC3, GD2, HER2, CEA, EGFR and so on. Some of these are used in completed clinical trials and more are still under current clinical investigation (Figure 6). CD123 ROR1 GD2 CD171 CEA VEGFR2 EGFR MAGEA3 PSMA Mesetholin NY-ESO-1 FAP EGFRvIII gp100 HER2 MUC1 CEA CARs currently in CD22 EphA2 MART1 clinical trials CD19 IL13Ra2 DMF4 lo CD133 DMF5 hi BCMA hematologic CD22 GPC3 tumors CD19 CD20 CD30 TCR completed clinical trials Gene-engineered T CARs completed 1st generation GD2 lymphocytes clinical trials CAIX IL13Ra2 CD171/L1-CAM Thyroglobulin Solid tumors alpha-folateR WT1 TCR currently in 2st generation Figure 6. HBV clinical trials Mesothelin RNA HPV E6 Coggle diagram of completed MAGEA3 HER2 and ongoing TCR and CAR gene-engineered T-cell MAGEA4 immunotherapy clinical trials p53 NY-ESO-1 CD19 The human CD19 antigen is a 95 kDa transmembrane glycoprotein belonging to the immunoglobulin superfamily, which is encoded by the CD19 gene located on the short arm of chromosome 16. It is classified as a type I transmembrane protein, with a single transmembrane domain, a cytoplasmic C-terminus, and extracellular N-terminus. The CD19 antigen plays an important role in clinical oncology. In normal cells, it is the most ubiquitously expressed protein in the B lymphocyte lineage. CD19 expression is induced at the point of B lineage commitment during the differentiation of the hematopoietic stem cell, and its expression continues through preB and mature B cell differentiation until it is finally down-regulated during terminal differentiation into plasma cells. CD19 expression is maintained in B-lineage cells that have undergone neoplastic transformation, and therefore CD19 is useful in diagnosis of leukemia and lymphoma. CD19 is also expressed in a subset of acute myelogenous leukemias (AMLs). Due to the fact that B lineage leukemias and lymphomas rarely lose CD19 expression, and it is not expressed in the pluripotent stem cell, CD19 has become a potential target for a variety of immunotherapeutic agents, including immunotoxins, monoclonal antibodies and CAR-T cells. Treatment of non-Hodgkin's lymphoma (NHL) and acute lymphocytic leukemia (ALL) with anti-CD19 mAbs coupled to biological toxins has proven to be effective in vitro and in animal models, and has shown some promising results in Phase I clinical trials. CD19 molecule

Mesothelin Mesothelin molecule Mesothelin is a 40 kDa differentiation antigen whose expression in normal human tissues is limited to mesothelial cells lining the pleura, pericardium and peritoneum. However, mesothelin is highly expressed in several human cancers, including virtually all mesotheliomas and pancreatic adenocarcinomas, and approximately 70% of ovarian cancers and 50% of lung adenocarcinomas. The mesothelin gene encodes a precursor protein of 71 kDa that is processed to a 31 kDa shed protein called megakaryocyte potentiating factor (MPF) and a 40 kDa fragment, mesothelin, which is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor and a 31 kDa shed fragment named megakaryocyte-potentiating factor (MPF). Although it has been proposed that mesothelin may be involved in cell adhesion, its biological function is not known yet. Because some of the mesothelin-directed therapies may have non-overlapping toxicity with chemotherapy and immunotherapy agents and could potentially result in synergistic activity, combination clinical trials have just been initiated. BCMA B-cell maturation antigen (BCMA), also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17), in humans encoded by the TNFRSF17 gene, is a member protein of the tumor necrosis factor receptor family. Human BCMA is a 184 amino acid (aa) protein consisting of a 54 aa extracellular domain, a 23 aa transmembrane Idomain, and a 107 aa intracellular domain. BCMA expression has been found in immune organs and mature B cell lines. BCMA molecule Although some expression has been observed at the cell surface, BCMA appears to be localized to the Golgi compartment. The binding of BCMA to APRIL or BAFF has been shown to stimulate IgM production in peripheral blood B cells and increase the survival of cultured B cells, which suggests that BCMA may play an important role in B cell development, function and regulation. Besides, its function has been implicated in B-cell malignancies. BCMA is an ideal antigen for targeted immunotherapy for multiple myeloma (MM). In MM, BCMA is widely expressed on malignant plasma cells (PCs) at elevated levels. GD2 GPC3 GD2 is a disialoganglioside. Gangliosides are Glypican-3 is a protein that in humans is encoded sialic acid-containing glycosphingolipids that by the GPC3 gene. The protein encoded by this play important roles in signal transduction as gene is a member of the heparan sulfate well as cell adhesion and recognition. proteoglycan family. GD2 is a b-series ganglioside that requires the enzymes GD3 synthase It is an oncofetal protein that is expressed in the embryo and involved in and GD2 synthase to add sialic acid units onto its precursor GM2. morphogenesis and growth control during development. Its expression is Ganglioside GD2 is highly expressed on neuroectoderm-derived silenced in adult tissues. In vitro studies have shown that GPC3 induces tumors and sarcomas, including neuroblastoma, retinoblastoma, apoptosis in certain cell lines via the anchoring of the protein to the cell melanoma, small cell lung cancer, brain tumors, osteosarcoma, membrane, indicating that GPC3 may function as an inhibitor of cell rhabdomyosarcoma, Ewing's sarcoma in children and adolescents, as proliferation and a tumor suppressor in a cell line–specific manner. well as liposarcoma, fibrosarcoma, leiomyosarcoma and other soft Glypican-3 is a useful diagnostic marker for a component of hepatocellular tissue sarcomas in adults. Since GD2 expression in normal tissues is carcinoma (HCC) in human liver cancer. restricted to the brain, which is inaccessible to circulating antibodies, Glypican-3 (GPC3) was discovered as a potential and in selected peripheral nerves and melanocytes, it was deemed a serological and histochemical marker whose expression suitable target for systemic tumor immunotherapy. As a tumor antigen, is specific for HCC. The expression of GPC3 is detected GD2 has been shown to enhance tumor proliferation and invasiveness in placenta and fetal liver, but not in other normal in small cell lung cancer cells and osteosarcoma cells. GD2 provides a organs. During hepatic carcinogenesis, GPC3 has been promising clinical target for radiolabeled antibodies, bispecific reported to reappear in HCC and to be released into antibodies, chimeric antigen receptor (CAR)-modified T cells, drug serum. Its expression is also detected in melanoma. The conjugates, nanoparticles and vaccines. functions of GPC3 in cancer cells are still unclear.

CD22 CD30 molecule CD22, or cluster of differentiation-22, is a molecule belonging to the CD30 SIGLEC family of lectins. It is found on the surface of mature B cells and to a lesser extent on some immature B cells. Generally speaking, CD22 is a CD30, also known as TNFRSF8, is a cell membrane protein of the tumor regulatory molecule that prevents the over activation of the immune system necrosis factor receptor family and tumor marker. It is involved in the and the development of autoimmune diseases. CD22 is a sugar binding activation of the NF-κB pathway and the MAPKs, ultimately modulating transmembrane protein, which specifically binds sialic acid with an cell growth, proliferation, and apoptosis. This receptor is expressed by immunoglobulin (Ig) domain located at its N-terminus. The presence of Ig activated, but not by resting, T and B cells. TRAF2 and TRAF5 can interact domains makes CD22 a member of the immunoglobulin superfamily. CD22 with this receptor, and mediate the signal transduction that leads to the functions as an inhibitory receptor for B cell receptor (BCR) signaling. CD22 activation of NF-kappaB. It is a positive regulator of apoptosis, and also consists of 7 extracellular IgG-like domains and is expressed on the B-cell has been shown to limit the proliferative potential of autoreactive CD8 surface starting at the pre-B cell stage, persists on mature B cells, and is effector T cells and protect the body against autoimmunity. Two lost on plasma cells. CD22 has been validated as a successful target for alternatively spliced transcript variants of this gene encoding distinct B-cell leukemias and lymphomas using an immunotoxin approach. CD22 isoforms have been reported. antigen is broadly expressed on lung cancer cells and is a target for antibody-based therapy. CD22 molecule In addition to CD19, which is used in nearly half Table 1. Top 10 popular targets in CAR-T clinical trials. of clinical trials, other target marker molecule (United States and China) such as BCMA, mesothelin, GD2, GPC3, CD22 and CD30 are also commonly used in CAR-T Targets-USA Numbers-USA Targets-China Numbers-China clinical projects (Table 1). These markers have CD19 59 CD19 58 a common feature: they all have been used as BCMA 9 GPC3 8 anti-tumor therapy markers, and there is at least 9 EGFR 6 one specific commercialized monoclonal mesothelin 7 5 antibody discovered to target these markers for CD30 7 mesothelin 4 anti-tumor therapy use. GD2 5 CD22 4 4 EpCAM 4 EGFRvIII 4 GD2 3 CD123 4 BCMA 3 CD22 3 CD20 3 HER2 CD30 CD19/CD22 Among US trials, the 2nd-5th commonly used markers are BCMA (7%), mesothelin (7%), CD30 (5%), and GD2 (5%) (Figure7-1). CD19, CD30 and BCMA are hematologic tumor marker. Mesothelin and GD2 are more commonly used as solid tumor marker. While among Chinese trials, CD19 is still the dominant marker, the following 2nd-5th popular markers are GPC3 (6%), EGFR (4%), mesothelin (4%) and CD22 (3%). GPC3 is considered as a liver cancer associated tumor marker. The importance of GPC3 trial in China is largely due to the considerable number of patients and high morbidity of hepatocellular carcinoma in Chinese population.

CAR-T Cell Targets - USA CAR-T Cell Targets - China GPC3 PSMA ROR1 CD19 BCMA CD137 PSMA/FRa CD19 GPC3 1% 1% 1% mesothelin CD30 1% EGFR mesothelin GD2 EGFRvIII CD133 1% CD22 EpCAM cMET CD123 CD22 GD2 BCMA HER2 CD19/CD22 1% MG7 CD20 CD30 1% CD20 CEA CD33 HER2 cMET GPC3 MUC1 CD123 CEA Other PSMA ROR1 CD123 1% Multiple target CD133 CD137 2% 8% Other 1% 13% CD138 CEA CLD18 EGFRvIII CD20 MUC1 CD19 EphA2 GBM 2% 2% 41% HIV IM19 LeY LMP-1 CD19/CD22 CD19 HER2 MG7 PD-L1 2% 43% 2% PSMA/FRa Multiple target HER2 CD33 3% 2% CD22 CD30 3% 2% CD123 CD20 3% 2% EGFRvIII BCMA BCMA 4% 7% 2% GD2 mesothelin GD2 EpCAM GPC3 5% 7% 3% 3% CD22 3% EGFR 6% CD30 4% 5% mesothelin 4% Figure 7-1. CAR-T cell targets in US trials Figure 7-2. CAR-T cell targets in Chinese trials. Generally speaking, Chinese test more kinds of tumor markers than American, and more like to test solid tumor marker. Another noteworthy situation is that there are 13% multi-target programs in China, which means the Chinese tend to test more than one marker (actually 3 or more) within a single clinical trial. No such kind of multi-target program registered in US. Actually, the US scientists also run some multi-target CAR-T clinical experiments, but not like their Chinese counterparts, they often use bispecific CAR-T cell such as CD19/CD20 CARs instead of testing varieties of CAR-T cells targeting different markers in a single trial. CAR-T cell targets used in clinical trials of other countries are show in Figure 8. CD19 is still a major marker of these trials, followed by CD22, GD2, and BCMA, most are hematologic malignance markers. CAR-T Target - Other Country 16 14 12 LeY 10 CML 8 ERbB 6 BCMA 4 GD2 2 CD22 0 CD19 Figure 8. CAR-T cell targets of other country

Part 3 Indications CD19 and BCMA are two major biomarkers used for hematologic malignance therapy such as leukemia and lymphoma. Therefore, leukemia and lymphoma become two of the most commonly investigated indications of CAR-T clinical trials, both in USA and China. US has more malignant glioma and neuroblastoma projects while China has more liver cancer, lung cancer and such solid tumor projects (Figure 9). Indications of CAR-T trial- USA Solid Tumors 1 Sarcomas 1 1 Renal Cancer 1 Malignant Melanoma 1 1 Malignancies 1 Lymphomatoid Granulomatosis 1 1 Lung Cancer 1 HER2 Positive Malignancies 1 2 Glioblastoma 2 Cervical Cancer 2 2 BPDCN 2 Sarcoma Prostate Cancer 3 Plasma Cell Myeloma 3 Mesothelioma Breast Cancer 6 Liver Cancer 6 6 Cancer Pancreatic Cancer 9 Neuroblastoma 36 Malignant Glioma Multiple Myeloma 46 50 Lymphoma Leukemia 0 10 20 30 40 Indications of CAR-T trial - China Stomach Cancer 1 SLE 1 1 Nasopharyngeal Neoplasms 1 Malignant Mesothelioma 1 HIV 1 Gastric Cancer 1 Colorectal Cancer 1 Colon Cancer 1 Bladder Cancer 1 Advanced Glioma 2 Pancreatic Cancer 2 Neuroblastoma 2 Glioblastoma Multiforme Breast Cancer 3 Malignant Glioma 4 Lung Cancer 4 Multiple Myeloma Solid Tumor 7 Liver Cancer 10 Lymphoma 12 Leukemia 34 53 0 10 20 30 40 50 60 Figure 9. Indications of CAR-T clinical trials in USA and China.

As we know, CAR-T cells have demonstrated tremendous success in eradicating hematologic malignancies. However, this success has yet to be extrapolated to solid tumors. From this point of view, the CAR-T project in China is more ambitious in the choice of indications. We will wait and see, after such massive investment and effort, whether the breakthrough of car-t cell therapy against solid tumors will made in China. But one reality worth mentioning here is that almost all success of CAR-T cell therapy trials for now are still from hematologic malignancies. There would be a long way to go for the conquering of the mountain of solid tumor. CAR-T Target - Other Country 16 14 12 LeY 10 CML 8 ERbB 6 BCMA 4 GD2 2 CD22 0 CD19 Figure 10. Indications of CAR-T trials of other country. For other countries, Leukemia and lymphoma, not surprisingly, remain the most frequently investigated indications. solid tumor project is barely involved (Figure 10). Part 4 Phase of clinical trial According to the data from clinicaltrials.gov, the clinical trial phase could be concluded as recruiting, active, completed, suspended, withdrawn and unknown status. Since the United states and China own the majority of trials, here we just compare the data from these two countries. As the data shows, the United states have more active but not yet recruiting trials while less recruiting trials (Figure 11). Active but not yet recruiting status usually means these trials have already got enough volunteers, close the recruiting channels and moving forward. That is to say, the progress of US CAR-T clinical trials is a bit faster than of Chinese, and US has more completed CAR-T therapy projects as well. Although China is superior in number, most of its CAR-T project are at the beginning, 86% of the trials are still recruiting volunteers. Only 6 trials (4%) have been completed, far less than the number of US (15, 11.5%). The completion date of these newly started trials will be about 10 years later (2019~2020). Considering this, the latest clinical research progress is very much likely to be achieved in the United States. We hope it will come soon.

Nearly all of the CAR-T clinical trial from other countries are at recruiting phase, with only one trial being completed in UK and Sweden respectively (Figure 12). Figure 11. Clinical trial phase summary Figure 12. Clinical trial phase summary USA and China. other country. Part 5 Companies and institutions At the last part, we will review the companies and institutes that support and sponsor these clinical trials, which are listed in the following tables: Table 2. Sponsor company and institute of CAR-T clinical trials in United States *commercial companies are marked in blue.

Table 3. Sponsor company and institute of CAR-T clinical trials in China *commercial companies are marked in red.

More than 30 academic institutions, companies and individuals carry out CAR-T clinical trials in United States, among which the University of Pennsylvania with 31 clinical projects, becomes the largest institution in this area. The newly FDA approved CAR-T therapy CTL019 is developed by University of Pennsylvania and their cooperator, Novartis. It seems like a common practice for companies, research institutions and hospitals to co-run a CAR-T project, while companies provides financial support, academic institutions have technology, and hospitals have resource of adequate participants and medical conditions to ensure the clinical trial can be launched and run. A successful CAR-T project needs so much effort from all parties. Resources must be integrated and different organizations must work together to make things go well. Hospitals should always play as a leading actor in this kind of cooperation. After all, whether a CAR-T therapy is effective or not, the clinician and their patients have the final say. Table 4. Sponsor company and institute of CAR-T clinical trials in other countries

Part 6 Summary and outlooks Clinical trials using CAR-T cells to treat patients with cancer show modest results, but the impressive outcomes of several trials of CD19-targeted CAR-T cells in the treatment of patients with B-cell malignancies have generated an increased enthusiasm for this approach. Important lessons have been derived from clinical trials of CD19-specific CAR T cells, and ongoing clinical trials are testing CAR designs directed at novel targets involved in hematological and solid malignancies. In this Review, we have discussed these trials and the latest progress of CAR-T therapy in clinic of each country all over the world. Our purpose is to help our readers take a quick look at this research area and market, and ultimately gain an overall impression of the global conditions both academically and commercially. Copyright © 2022 Creative Biolabs. All Rights Reserved. Contact Us