CAR-T Cell Therapy - アズワン株式会社 · CAR-T Cell Therapy Chimeric Antigen Receptor T...
Transcript of CAR-T Cell Therapy - アズワン株式会社 · CAR-T Cell Therapy Chimeric Antigen Receptor T...
01 Sino Biological
CAR-T Introduction
CAR-T Events
CAR-T Cell Therapy
Chimeric Antigen Receptor T cell therapy, or CAR-T cell therapy, has been widely used in the field of cancer immunotherapy and clinical application.
The basic procedures for CAR-T cell therapy start with the collection and extraction of T cells from the tumor patient’s peripheral blood. The T cells are then genetically engineered in vitro to make them express chimeric antigen receptors (CARs) that can recognize specific tumor-associated antigens and activate self-proliferation and cytotoxicity. Finally, CAR-T cells are expanded and reinfused into the patient, achieving the effect of recognizing and fighting the tumor.
In 2012, Emily Whitehead became the first child in the world to receive an experimental CAR-T therapy.In August 2017, Novartis received FDA approval for a CAR-T cell therapy, KymriahTM.In October 2017, Kite Pharma received FDA approval for a CAR-T cell therapy, YescartaTM.
1. Bloodstream collection
2. Isolation and activation of T cells
3. T cell engineering
4. CAR-T cell expansion
5. Pretreatment and CAR-T cell infusion
CAR-T Cell Therapy Procedures
Sino Biological 02
Solid Tumors Target Proteins
Solid tumor-associated antigens are expressed at low levels in normal tissues. Meanwhile, solid tumors have heterogeneity. Even within the same tumor region from the same patient, tumor cells might express different tumor-associated antigen. Therefore, choosing a target is a significant challenge for CAR-T cell therapy against solid tumors.
Table 1 Products List of Solid Tumors Targets
Binding of Biotinylated Human ErbB2 (Cat. No. 10004-H08H-B) to ErbB2 Antibody (Herceptin®) in a functional ELISA
Binding of Biotinylated Human GPC3 (Cat. No. 10777-H08H-B) and Unconjugated Human GPC3 Protein (Cat. No. 10777-H08H) to bFGF Protein in a functional ELISA
Binding of two PD-1 antibody drugs to Human PD-1 (Cat. No. 10377-H08H) in a functional ELISA
0.1 1 10 100 1000 10000
0.0
0.5
1.0
1.5
2.0
2.5
Abs
.(OD
450n
m-B
lank
)
Herceptin Conc.ng/mL
Human ErbB2 Protein(Cat:10004-H08H-B;Lot:LC11JU2205)
ErbB2 antibody Conc. (ng/mL)
Ad
s. (O
D45
0nm
-Bla
nk)
Human ErbB2 ProteinErbB2/Biotin
0.1 1 10 100 1000
0
1
2
3
4
Abs.
(OD4
50nm
-Bla
nk)
FGFbasic Conc.ng/mL
GPC3-His/Biotin(Cat:10088-H08H-B;Lot:HP11JL2609) GPC3-His(Cat:10088-H08H;Lot:MA05SE0101)
FGFbasic Conc. (ng/mL)
Ad
s.(O
D45
0nm
-Bla
nk)
GPC3-His/BiotinGPC3-HisGPC3-His/BiotinGPC3-His
0.01 0.1 1 10 100 1000 10000-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Abs.
(OD4
50nm
-Bla
nk)
PD1-His Conc.ng/mL
Keytruda Opdivo
PD1-His Conc. (ng/mL)
Ad
s. (O
D45
0nm
-Bla
nk)
MAb Drug AMAb Drug BMAb Drug BMAb Drug C
Sino Biological can provide all biotinylated proteins for targets listed above.
Molecule Other Name Host Tumor Type
CEACAM5 CEA Human cells A variety of solid tumors
MET c-MET Human cells Breast cancer
EGFR Human cells/Insect cells Colorectal cancer, Advanced EGFR positive solid tumors
EGFRvIII Insect cells Glioblastoma
EPCAM CD326 Human cells Hepatoma, Gastric cancer
EPHA2 Human cells Malignant glioma
ERBB2 HER2 Human cells A variety of solid tumors
GPC3 Glypican-3 Human cells Hepatocellular carcinoma, Squamous cell lung cancer
MSLN Mesothelin Human cells Mesothelioma, Pancreatic cancer, Ovarian cancer
Muc1 CA15-3 Human cells Hepatocellular carcinoma, Non-small cell lung cancer, Pancreatic cancer, Breast cancer
PDCD1 PD-1 Human cells Advanced lung cancer, Advanced liver cancer, Advanced gastric cancer
CD274 PD-L1 Human cells Glioblastoma
KDR VEGFR2 Human cells Metastatic carcinoma, Metastatic melanoma, Renal cancer
IL13RA2 Human cells Glioma
FOLH1 PSMA Human cells Prostate cancer
FAP Human cells Malignant pleural mesothelioma
CA9 CAIX Human cells Metastatic renal cell carcinoma
FOLR1 FR a Human cells Ovarian cancer
L1CAM CD171 Human cells Ovarian cancer, Neuroblastoma
ROR1 Human cells Non-small cell lung cancer, Breast cancer, Leukemia
03 Sino Biological
Hematologic Malignancies Target ProteinsCD19 is the most widely used hematologic malignancy target in CAR-T cell therapy and good therapeutic effects have been achieved in clinical trials of hematological malignancies such as B-ALL, CLL, FL, DLBCL and MCL. In addition to CD19, molecules such as CD20, CD22, CD30, IL3RA are also hot targets for hematologic malignancies research, especially for CD19-negative hematologic malignancies. Sino Biological has a variety of hematologic malignancies target proteins, which can be used to detect CAR expression on T cells.
Binding of Biotinylated Human CD30 (Cat. No. 1077-H08H-B) and Unconjugated Human CD30 (Cat. No. 1077-H08H) to CD30L in a functional ELISA
Binding of Human CD20 (Cat. No. 11007-H34E) to Anti-CD20 Mab Drug A in a functional ELISA
Table 2 Products List of Hematologic Malignancies Targets
Table 3 Selected References Using Sino Biological Proteins in CAR-T Research
Binding of human IL1R3 (Cat No: 10121-H03H) in lyophilized batch and liquid batch to IL1B in a functional ELISA
0.1 1 10 100 1000 10000
0
1
2
3
4
5
Abs.
(OD4
50nm
-Bla
nk)
Fc-CD30L Conc.ng/mL
CD30(Cat:10777-H08H;Lot:MB10OC1812) CD30/Biotin(Cat:10777-H08H-B;Lot:HP10NO1803)
Fc-CD30L Conc. (ng/mL)
Ad
s.(O
D45
0mm
-Bla
nk)
CD30CD30/Biotin
0.1 1 10 100 1000 10000
0
1
2
3
4
5
Abs.
(OD
450n
m-B
lank
)
IL1B-His/Biotin Conc.ng/mL
IL1R3-Fch (Cat:10121-H03H; Lot;ML03AU0401) IL1R3-Fch (Cat:10121-H03H; Lot;LC09AP0704)
IL1B-His/Biotin Conc. (ng/mL)
Ad
s.(O
D45
0mm
-Bla
nk)
IL1R3-Fch Lyophilized BatchIL1R3-Fch Liquid Batch
100 1000 10000 100000
0
1
2
3
4
5
SCT400 Conc.ng/mL
Abs.
(OD4
50nm
-Bla
nk)
CD20(Cat:11007-H34E;Lot:ME11AU0702)
MAb Drug A Conc. (ng/mL)
Ad
s.(O
D45
0mm
-Bla
nk)
CD20
Molecule Other Name Host Tumor Type
TNFRSF17 BCMA Human cells Multiple myeloma
IL3RA CD123 Human cells Myeloid malignancies
SDC1 CD138 Human cells Multiple myeloma
CD19 Human cells Leukemia, Lymphoma, Multiple myeloma
MS4A1 CD20 E. coli Leukemia, Lymphoma, Multiple myeloma
CD22 Siglec-2 Human cells B-cell lymphoma
TNFRSF8 CD30 Human cells Lymphoma
CD33 Siglec-3 Human cells Myeloid malignancies
CD38 Human cells Acute myeloid leukemia, Multiple myeloma
CD5 Leu-1 Human cells T cell acute lymphoblastic lymphoma
NCAM1 CD56 Human cells Acute myeloid leukemia
ULBP1 NKG2D ligand 1 Human cells A variety of hematological malignancies
ULBP2 NKG2D ligand 2 Human cells A variety of hematological malignancies
IL1RAP IL1R3 Human cells Chronic myeloid leukemia
ROR1 Human cells Non-small cell lung cancer, Breast cancer, Leukemia
Sino Biological can provide all biotinylated proteins for targets listed above.
Molecule Tag Type Application Magazine Year
CD123 His tag Detection of cell surface CAR expression (NK cell) by flow cytometry Human gene therapy 2017
CEACAM5 His tag Detection of cell surface CAR expression by flow cytometry Molecular Therapy 2017
EGFR His tag Detection of cell surface CAR expression by flow cytometry US 20140242701 A1 2014
Sino Biological 04
T cell Activation
T cell Activation and Expansion
Before T cell engineering, T cells which are collected and isolated from the patient’s peripheral blood needto be activated. CD3, CD28 antibodies are commonly used for T cell activation in vitro.
Human CD3 antibody (Cat. No. 10977-H001) induces CD4+ T cell proliferation and IFN-gamma secretion.
Human CD28 antibody (Cat. No. 11524-H001) induces IL2 secretion of the CD4+ T cells. In the presence of 2 μg/mL of CD3 antibody, the IC50 of CD28 antibody is typically 0.2560 μg/mL.
Table 4 Sino Biological T Cell Activation Antibodies
Sino Biological is capable of manufacturing GMP-level antibodies which will be on sale soon.
Target Purity (by HPLC) Endotoxin (EU/mg) Cat. No.
Human CD3 (humanized OKT3) >95% <0.5 10977-H001
Human CD28 >95% <0.5 11524-H001
Antibody Conc. (ug/mL)
0.015630.0
0.2
0.4
0.6
0.031250.0625 0.125 0.25 0.5 1 2 4 8
IL-2 (O
D Value
)
Antibody Conc. (ug/mL) Antibody Conc. (ug/mL)
Proliferation (O
D Value
)
IFN
-gam
ma
(OD
Val
ue)
0.10.0 0.0
0.01 0.1 1
0.1
0.2
0.2
0.3
0.4
0.4
0.5
0.6
0.6
0.8
1.0
1.20.7
A B
1 5 10
CD4+ T Cells CD4+ T Cells
05 Sino Biological
T cell Expansion
Sino Biological High-Quality Cytokines
SEC-HPLC: ≥95%Animal free
* Sino Biological is capable of manufacturing GMP-level cytokines which will be on sale soon.
SDS-PAGE: ≥95%Validated bioactivity
Endotoxin < 0.01 EU/μgLot-to-lot consistency
1E-4 1E-3 0.01 0.1 1 10 100-20
0
20
40
60
80
100
Prol
ifera
tion,
%
IL2 Conc.(ng/mL)
MY09MY0601 LC11JL0706
1E-3 0.01 0.1 1 10 100 1000-1000
0
1000
2000
3000
4000
5000
6000
Prol
ifera
tion,
%
IL7 Conc.(ng/mL)
ME08NO1901 LC11FE1308
0.01 0.1 1 10 100 1000 10000
0
2000
4000
6000
8000
10000
12000
Proli
ferati
on,%
IL15 Conc.(ng/mL)0.01 0.1 1 10
0
10
20
30
40
50 ME08JL1001 LC09AU0305
secr
etio
n of
IFN-
γ;
ng/m
L;
IL21 conc;;ng/mL;
Table 5 High-Quality Cytokines
Cat. No. Molecule Cat. No. Molecule
11848-HNAE IL2 10584-HNAE IL21
10015-HNAH GMCSF 10602-HNAE TNFa
10360-HNCE IL15 10605-HNAE EGF
10395-HNAE IL6 11725-HNAS IFN-gamma
11821-HNAE IL7 11846-HNAE IL4
CT011-H08H IL12
IL2 induces the expansion of CTLL2 mouse T lymphocytes.
IL15 induces the proliferation of MO7e megakaryocyte lymphocytes.
IL17 induces the proliferation of human peripheral blood lymphocytes activated by CD3 antibody.
IL21 induces IFN-gamma secretion in NK-92 human natural killer cells.
Human IL2
IL2 Conc. (ng/mL)
IL15 Conc. (ng/mL)
IL7 Conc. (ng/mL)
IL21 Conc. (ng/mL)
Proliferation%
Proliferation%
Proliferation%
Proliferation%
Human IL15
Human IL7
Human IL21
Liquid formLyophilized form
Liquid formLyophilized form
Liquid formLyophilized form
Sino Biological 06
Cytokine ELISA Kits
Cytokines detection is one of the main ways to indicate CAR-T cell potency and it can also be used to study tumor immunosuppressive microenvironment. More than 90% ELISA Kits from SBI use recombinant proteins expressed in eukaryotic systems as controls. All ELISA Kits strictly follow eight QC standards including precision, recovery, range, the limit of detection (LOD), stability, natural sample test, cross-reactivity and interference to guarantee valid and reliable results.
Table 6 T Cell Related Cytokine ELISA Kits (Part of the List)
8 Stringent QC Test Indicators: Ensure high quality ELISA Kits
Molecule Cat. No. Sample Limit of Detection (pg/mL) Range (pg/mL)
TNF KIT10602 PBMC supernatant 11.96 23.44-1500
IFNG KIT11725A PBMC supernatant 6.5 13.28-850
IL4 KIT11846 PBMC supernatant 2.54 10.94-700
IL6 KIT10395A Serum, PBMC supernatant 0.69 3.13-200
IL10 KIT10947 PBMC supernatant 14.37 23.44-1500
CSF2 KIT10015 PBMC supernatant 2.04 7.81-500
CXCL9 KIT10888 Serum 11.96 23.44-1500
CCL2 KIT10134 Serum, Urine 1.9 3.91-250
IL13 KIT10369 PBMC supernatant 0.59 4.69-300
IL7 KIT11821 PBMC supernatant 1.89 6.25-400
IL17A KIT12047 PBMC supernatant 0.58 1.88-120
CRP KIT11250 Serum 12.8 46.88-3000
To evaluate the extent of the ELISA kit results close to actual values.
A high or low recovery results in false positive or negative result affecting the accuracy of the measured value.
Recovery
To ensure the stability of the ELISA kit for long-term storage.
Better stability of the ELISA kit means longer storage time.
Stability
Make sure ELISA kit can detect natural samples and determine its applicable types of samples.
If the types of natural sample are not confirmed, a false positive or negative result may be obtained.
Natural sampletest
To guarantee if similar results can be obtained when the same samples are repeatedly measured.
Poor precision of the kit will result in poor reproducibility when the same sample is repeatedly measured.
Precision
To evaluate the specificity of the ELISA kit and determine the binding ability of other molecules to the paired antibodies.
High cross-reactivity stands for low specificity.
Cross-reactivity
To determine the sensitivity of the ELISA kit.
Lower limit of detection stands for higher sensitivity.
The limit ofdetection (LOD)
To determine the effects of other molecules to the interaction of antigens with paired antibodies.
High interference stands for low accuracy of the measured value.
Interference
To assess the accuracy of the ELISA kit.
High or low results of linear test will produce false positive or negative data.
Range
07 Sino Biological
T Cell Flow Cytometry (FACS) Antibodies
Flow cytometry is an important way to determine the potency and components of CAR-T cell, and the qualityof FACS antibodies directly affects the success of experiments. Sino Biological develops a variety of T cellrelated FACS antibodies. Most of these antibodies were immunized by recombinant proteins expressed ineukaryotic systems, and they also have high specificity, high affinity and excellent S/N ratio, avoiding effectively the impact of FACS antibodies on the experiments.
Using CD4 antibody (Cat. No. 10400-MM08F) and CD8 antibody (Cat. No. 10980-MM28-F) to detect the expression of CD4 and CD8 on the surface of human PBMC by flow cytometry.
Table 7 T Cell FACS Antibodies (Part of the List)
Anti-CD4Anti-CD4
FITC CD4
PE CD3
Anti-CD8Anti-CD8
FITC CD8aPE CD3
Molecule Antibody Name Conjugate Antibody Type Target Species
CD3E CD3e / CD3 epsilon Antibody APC, PerCP, FITC, PE Mouse MAb Human
CD4 CD4 Antibody APC, PerCP, FITC, PE Mouse MAb Human
CD8A CD8/CD8 alpha/Leu-2 Antibody APC, FITC Mouse MAb Human
IL-18R IL18R1 Antibody APC, FITC, PE Mouse MAb Human
CD26 DPP4 / CD26 Antibody PE, APC, FITC Mouse MAb Human
TNF-α TNF-alpha / TNFA / TNFSF2 Antibody FITC Mouse MAb Human
TNF-α TNF-alpha / TNFA / TNFSF2 Antibody APC, FITC, PE Rabbit MAb Human
IFN-gamma Interferon Gamma/IFN gamma/IFNG Antibody APC, PerCP, FITC, PE Rabbit MAb Human
CD45RA CD45RA Antibody FITC, PE Mouse MAb Human
CD62L CD62L / L-Selectin / SELL Antibody APC, PerCP, PE Mouse MAb Human
CD38 CD38/ADPRC1 Antibody APC, PerCP, FITC, PE Mouse MAb Human
CD25 IL2RA/IL-2RA/CD25 Antibody FITC, PE Mouse MAb Human
CD25 IL2RA / IL-2RA / CD25 Antibody APC, PerCP, FITC, PE Rabbit MAb Human
CD127 IL7R / IL-7R / CD127 Antibody APC, PerCP, FITC, PE Rabbit MAb Human
CD73 CD73 / NT5E Antibody FITC, PE Mouse MAb Human
Sino Biological 08
Sino Biological Annexin V/7-AAD Apoptosis Detection Kits List
Annexin V/7-AAD Apoptosis Detection KitsProduced in house Quality guarantee Bulk in stock Deliver in 24h
Detection Kit Principle
Reliable Sino Biological Apoptosis Detection Kits
Annexin V combined with 7-AAD/PI staining method is easy to use, more time-saving, and capable of producing stable and reliable results. It’s the most ideal method to detect cell apoptosis now. Tips: 7-AAD/PI staining method cannot distinguish between apoptosis and necrosis. It’s recommended that more than two methods should be employed to comprehensively judge the apoptosis stage.
Can accurately distinguish early and late apoptosis compared with leading competitor
Jurkat Hela
Best S/N ratio, easy for setting gates to analyze, and more reliable results
Sino Biological Annexin V-FITC
S/N=89
S/N=93
Competitor A Annexin V-Alexa flour 488
S/N=65
S/N=50
S/N=39 Competitor A Annexin V-PE
Sino Biological Annexin V-PE
Competitor B Annexin V-PE
Product Name Cat. No. Size
Annexin V-FITC/7-AAD Apoptosis Detection Kit APK10448-F 20 Tests
Annexin V-FPE/7-AAD Apoptosis Detection Kit APK10448-P 100 Tests
100 100
100 100
101 101
102 102
103 103
104 104
Sino Biological Competitor A
101 101102 102103 103104 104
Annexin V-FITC Annexin V-FITC
7-A
DD
7-A
DD
100 100
100 100
101 101
102 102
103 103
104 104
Sino Biological Competitor A
101 101102 102103 103104 104
Annexin V-FITC Annexin V-Alexa 488
7-A
DD
7-A
DD
磷脂酰丝氨酸
正常细胞细胞核
Ca2+
Ca2+Ca2+
Annexin V
FITC或PE等荧光标记物
细胞核
Ca2+
Ca2+Ca2+
细胞核
7-AAD/PI
Annexin V -7-AAD -
凋亡早期Annexin V +7-AAD -
凋亡晚期Annexin V +7-AAD +
FITC/PE fluorescent Conjugates 7-AAD/PI
Annexin V
Phosphatidylserine
Normal cellsAnnexin V -7-AAD -
Early apoptosisAnnexin V +7-AAD -
Nucleus Nucleus NucleusLate apoptosisAnnexin V +7-AAD +
09 Sino Biological
Supernuclease (Equivalent to Benzonase® Nuclease)
Supernuclease (Cat. No. SSNP01) is equivalent to Benzonase® Nuclease, can be used to remove DNAimpurities in the process of lentiviral vector amplification. The HPLC purity of Supernuclease is over 99%. TheDNA degradation rate is comparable with leading brands.
Purity by SDS-PAGE HPLC Detection CurveDNA Degradation Rate
116.0
66.2
45.0
35.0
25.0
18.4
14.4
Plasmid M SuperNuclease Competitor 1 Competitor 2 DNase Ⅰ
5 1 0.2 5 1 0.2 5 1 0.2 5 1 0.2
1
74bp
2 3 4 5 6 7 8 9 10 11 12 13 14
(U) 0
0
0
100
50
150
200
250
mAU
5 10 15 20 25 min
11.1
93
13.4
75
16.0
47
Transfection Reagent
Lentiviral Vector Amplification
Lentiviral vectors are widely used in CAR-T cell therapy for transducing CAR into T cells. Sino Biological’sSinofection® Transfection Reagent (Cat. No. STF02) can be applied to efficiently transfect lentiviral vectors,and the TCID50 of the obtained recombinant lentiviruses samples can reach over 107.
Recombinant lentiviral vectors were transfected into adherent HEK293T cells by Sinofection® Transfection Reagent. After 72 hours Fluorescent images were taken to detect the expression intensity of EGFP protein.
Recombinant lentiviral vectors (expressing EGFP protein) transfected by Sinofection® were retransfected into HEK293T cells. After 72 hours Fluorescent images were taken to detect the expression intensity of EGFP protein.
Sino Biological 10
Sino Biological Inc.
Advantages
Best Monoclonal Antibody Tools Worldwide
Centralized Protein & Antibody Expression Lab Worldwide
Leading Pre-Clinical CRO for Biologics Worldwide
Established in 2007, Sino Biological is a global biotechnology company manufacturing high quality researchreagents for basic, translational, and clinical research. Sino Biological laboratories consist of over 80,000 squaremeters, producing cDNAs, recombinant proteins, antibodies and antibody-based kits that are used to studycellular function and human diseases.
Sino Biological scientists are experienced in recombinant protein expression and purification from bacterial,yeast, insect and mammalian system. The scientists have also developed technologies to produce andpurify a comprehensive collection of mouse monoclonal antibodies, rabbit polyclonal antibodies, and rabbitmonoclonal antibodies. Sino Biological offers integrated service from gene cloning to protein expression,protein purification, and immunization for polyclonal or monoclonal antibody generation. We have providedCRO services to produce targets proteins, antibodies, and optimized ELISA tools to meet the needs of thebiopharmaceutical industry, including the top 10 pharmaceutical companies.
Sino Biological product sales are supported through a network of distributors in North American, Europeanand Asian markets. We are proud that our products are helping scientists to solve significant problems in lifescience research. Every product has to pass rigorous QC test standards to ensure consistent high quality. Wevalidate our product application and guarantee its performance as specified. We are developing tools andtechnologies to provide the global scientific community with the best-in-class product to accelerate scientificdiscovery and innovation.
Maximum 200-Fold Enhanced ELISA sensitivityLeading 2nd Generation Rabbit mAb TechnologyNo Products From Origin Entrusted Manufacturer
60+ Highly Centralized Cell Culture Bioreactors 6000+ Proteins Bulks in Stock 2000+ New Products Released Per Year 30-100 Grams Custom-Tailored mAb Production in Weeks
One-stop Service from Discovery to ProductionRecord Speed from DNA to Purified mAbsServing Top-10 global Pharmaceutical/Biotech Companies