Antigen Preparation
Antibody libraries
Antibody discovery
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Antibody engineering
Antibody production
Antibody application
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Recombinant protein preparation
For antibody generation, the first step is to prepare recombinant antigen proteins. We have three expression platforms for recombinant protein expression, including mammalian cell (CHO and HEK293 serum free system), yeast and E.coli expression systems.
For human antigens, mammalian expression system is the ideal one because the refolding and posttranslational modifications, such as glycosylation, are closest to that of the native antigens. In most cases, extracellular domain of cell surface antigens, such as tumor markers, is the domain of interest for antibody generation. Recombinant protein of the extracellular domain, instead of the full surface protein (including single or multiple transmembrane and intracellular domains) need to be prepared. However, it is very challenging to express only part of a native protein partially due to the change of its native translocation ways. To overcome this problem, we have developed several novel mammalian expression vectors that enable high level expression of such difficult expression proteins.
For human antigens, mammalian expression system is the ideal one because the refolding and posttranslational modifications, such as glycosylation, are closest to that of the native antigens. In most cases, extracellular domain of cell surface antigens, such as tumor markers, is the domain of interest for antibody generation. Recombinant protein of the extracellular domain, instead of the full surface protein (including single or multiple transmembrane and intracellular domains) need to be prepared. However, it is very challenging to express only part of a native protein partially due to the change of its native translocation ways. To overcome this problem, we have developed several novel mammalian expression vectors that enable high level expression of such difficult expression proteins.
Antigen high expression cell line creation
Sometimes to make sure the antigen used for library screening is in its natural conformation, we use antigen negative and positive cell lines to screen libraries. We have a lentiviral expression system to create antigen high expression cell lines. Antigen high expression is associated with intracellular EGFP expression therefore high expression cells could be purified by Fluorescence Activated Cell Sorting (FACS).
Yeast display
Yeast display is a newly developed technology for antibody discovery. Comparing to phage display, yeast display has the advantages of eukaryotic expression, capability of utilization of FACS sorting for selective isolation of different affinity level populations, and convenient detection of antigen binding by FACS, which make yeast display a powerful tool for novel antibody discovery.
However, it is very challenging to construct a large yeast display antibody library largely due to the low transformation efficiency of yeast. We have developed a cutting-edge technology that enables to improve the yeast transformation efficiency 1000 fold higher than traditional ways, which makes it possible to construct large yeast display antibody library efficiently.
In addition to the optimization of yeast transformation efficiency, we have also developed novel yeast display vectors that can display scFv in monomer or dimmer, with unique tags and linkers for efficient scFv clone, display, detection and protection of scFv structure and function from being affected by the co-expressed AGA2 protein and tags (Illustration 1).
If the ultimate goal of the isolated scFv is to be engineered into dimmers, such as scFv-Fv or full antibody, a dimmer scFv library is advisable because high affinity monomer scFvs may not be converted to high affinity dimmers. If scFv is the ultimate antibody format for application, such as labeling for nanoparticles or developing scFv based imaging probes, a monomer scFv display library is advised to be used for scFv isolation. Sometimes antigens have only small pocket epitopes, which is more likely observed in small peptide antigens, full size scFvs are too big to fit for the small pocket epitopes, a single domain library, which contains only VH or VL, will be the ideal library to screen.
We have also developed a comprehensive library screening strategy using recombinant proteins, tag-fused proteins or cell based screening(Fig 1). The scFv affinity isolated from the library is typically in nanomolar range in monomer, and usually the affinity will increase around 10 times when engineered to dimmer, such as scFv-Fc or full antibody.
However, it is very challenging to construct a large yeast display antibody library largely due to the low transformation efficiency of yeast. We have developed a cutting-edge technology that enables to improve the yeast transformation efficiency 1000 fold higher than traditional ways, which makes it possible to construct large yeast display antibody library efficiently.
In addition to the optimization of yeast transformation efficiency, we have also developed novel yeast display vectors that can display scFv in monomer or dimmer, with unique tags and linkers for efficient scFv clone, display, detection and protection of scFv structure and function from being affected by the co-expressed AGA2 protein and tags (Illustration 1).
If the ultimate goal of the isolated scFv is to be engineered into dimmers, such as scFv-Fv or full antibody, a dimmer scFv library is advisable because high affinity monomer scFvs may not be converted to high affinity dimmers. If scFv is the ultimate antibody format for application, such as labeling for nanoparticles or developing scFv based imaging probes, a monomer scFv display library is advised to be used for scFv isolation. Sometimes antigens have only small pocket epitopes, which is more likely observed in small peptide antigens, full size scFvs are too big to fit for the small pocket epitopes, a single domain library, which contains only VH or VL, will be the ideal library to screen.
We have also developed a comprehensive library screening strategy using recombinant proteins, tag-fused proteins or cell based screening(Fig 1). The scFv affinity isolated from the library is typically in nanomolar range in monomer, and usually the affinity will increase around 10 times when engineered to dimmer, such as scFv-Fc or full antibody.
Illustration 1: Dimmer and monomer scFv display by yeast.
Fig 1: Cell based yeast display antibody library panning.
Phage display
Yeast display is challenging for Fab library construction because simultaneous expression of both VH and VL/VK at an appropriate ratio for 100% coupling between VH and VL/VK is very difficult. Therefore our Fab library was constructed using phage display. OriMAbs has developed a phage display system for Fab library construction. Two sets of phagemids, pDCK1 and pDCK1.1; as well as pDCL1 and pDCL1.1 , were designed for kappa and lambda chain Fab display respectively. Both vectors have IgG1 CH1 constant region and all the constant regions were codon-optimized for E.coli expression and all the enzymes were designed for convenient digestion and efficient cloning. Phagemid pDCK1 and pDCL1 could also be used for scFv display, in which, the enzymes sites in the linkers were also optimized to ensure the translated linker is flexible for efficient coupling between VH and VL.
Yeast display human naïve dimmer-scFv library
Using unique techniques and the novel display vector pYDS2 (display dimmer scFvs), OriMAbs has constructed a 1x1011 yeast display dimmer-scFv library from B lymphocytes isolated from 5 liters peripheral blood from 56 healthy donors. Totally, 6.9 mg total RNA was isolated and from which, 125.3 mg mRNA was purified and used for reverse transcription and VH, VK and VL amplification using a novel set of primers that are capable of rescuing all the human antibody repertoires. ScFv is displayed in dimmer on the Saccharomyces cerevisiae EBY100 strain cell surface via the interaction between AGA1 and AGA2. Among 24 clones sequenced, all the clones contain scFv genes and 83.3% have single scFv gene, 16.7% have 2 scFv genes. Flag tag is expressed in 100% clones and V5 tag is expressed in 83.3% (20/24) clones (Fig 2). Failure in V5 tag expression is due to frame shift mutation in the scFv gene that might be the real sequences in germline encoding truncated antibodies or introduced by PCR(3/24), or conformational hindrance (1/24).Usually nanomolar affinity scFvs could be isolated from this library through magnetic and flow sortings(Fig 3). This library is recommended for scFv discovery that scFv will be engineered to dimmers, such as scFv-Fc or full antibody.
Fig 2: V5 expression in 24 clones of yeast display human naive scFv library. Red: negative control; Blue: V5 tag expression
Fig 3: Flag expression in 24 clones of yeast display human naive scFv library. Red: negative control; Blue: Flag tag expression
Yeast display human naïve monomer-scFv library
A yeast display human naive monomer-scFv library was also constructed using a different yeast display vector pYDS1 and the same VH, VK and VL gene for the dimmer-scFv library construction. The library size is 1.2×10^11. This library is recommended for scFv discovery that scFv will be the ultimate format for application.
Yeast display human naïve single domain library
A yeast display human naive single domain library was also constructed using yeast display vector pYDS1 and the same VH, VK and VL gene for the monomer- or dimmer-scFv library construction. The library size is 5×10^9. This library is recommended for single domain antibody discovery for antigens with only small pocket epitopes.
Phage display human naive Fab library
We have constructed a 2×1010 Fab phage display library using vector pDCK and pDCL (kappa:lambda = 2:1) using VH and VL pools isolated from the same 5 L peripheral blood of 56 healthy donors. 12 clones evaluated showed 100% VK/VL insertion and 91.6% (11/12) VH insertion.
Antibody discovery from yeast display libraries
We have also developed a comprehensive library screening strategy using recombinant proteins, tag-fused proteins or cell based screening(Fig 1). Library panning includes 2-3 rounds of magnetic sortings followed by 2-3 rounds fine flow sortings. Sorted high affinity yeast display population can be directly used for individual clone identification using flow cytometry, or be converted to secretory scFv sublibrary and use high thoughput ELISA for individual clone identification. The identified scFvs will be expressed and purified for characterizations such as affinity measurement and interlization assay, and then go to the downstream engineering process. The scFv affinity isolated from the library is typically in nanomolar range in monomer (Fig 4), and usually the affinity will increase around 10 times when engineered to dimmer, such as scFv-Fc or full antibody.
Fig 4: Affinity measurement of sample scFvs a and b.
Antibody discovery by phage library panning
We have developed phage display library panning in both solid and liquid phases. Antigen coating directly on solid phase, such as ELISA plates or immune tubes, may distort the antigen conformation, as a result, may lose some epitopes thereby reduce the diversity of isolated antibodies, or the isolated antibody may not bind to native conformational antigen at all. To overcome this potential problem, we have developed liquid phase panning method, in which, both the antigen and antibodies are in their native conformation. The antigen binding phage population will be isolated using magnetic sorting and the isolated soluble Fab antibody will be produced in E.coli.
Antibody engineering
Single domain antibody, scFv or Fab antibodies isolated from antibody libraries usually need to be engineered for different purposes, for example, they could be engineered into scFv-Fc or full antibody for higher affinity, better stability and longer circulation time. We offer the design and engineering of antibodies in all the known format, such as scFv, dimmer scFv, Fab, F(ab')2, single domain antibody, scFv-Fc and full antibody.
Affinity maturation
Usually antibodies isolated from our libraries have nanomolar range affinity and the affinity will increase around 10 times when engineered to dimmers. So affinity maturation is usually not necessary for antibodies generated from our libraries. If even higher affinity is desirable, we have the option of affinity maturation through yeast and phage display techniques.
Yeast display has the advantage that antigen binding yeast population could be easily sorted using Fluorescence Assisted Cell Sorting (FACS) because the yeast size is big enough to be recognized by FACS. This enables yeast display more efficient than phage for affinity maturation because in additional to the antigen concentration management and stringent washing used in phage display, FACS is more efficient in direct sorting of higher affinity yeast populations from other medium to high affinity ones (Fig xx).
Yeast display has the advantage that antigen binding yeast population could be easily sorted using Fluorescence Assisted Cell Sorting (FACS) because the yeast size is big enough to be recognized by FACS. This enables yeast display more efficient than phage for affinity maturation because in additional to the antigen concentration management and stringent washing used in phage display, FACS is more efficient in direct sorting of higher affinity yeast populations from other medium to high affinity ones (Fig xx).
Fig 5: Flow sorting of high affinity yeast populations for antigen binding.
Antibody production
We have developed highly efficient expression systems for antibody expression, including E.coli system for expression of single domain antibody, scFv and Fab, in intracellular or secretory form; yeast system for secretory expression of single domain antibody, scFv and Fab; and mammlian system for secretory expression of all the fragment antibodies and full antibody. We provide service for production and purification of all antibody formats by these expression sytems.
Antibody drug conjugate
Antibody drug conjugate (ADC) is emerging as a novel technique for antibody therapy. Antibody, usually full antibody, is labeled with super toxic drugs, such as MMAE, MMAF and DM1, through cleavable or non-cleavable linkers, to deliver toxicity specifically to antigen positive cells. Released toxins (MMAE and DM1) from dead cells can also enter and kill the vicinity antigen positive cells through by-stand mechanism. We provide the service for conjugation of full antibody with these drugs and related characterizations.
Bi-specific antibody
Bi-specific antibody, one arm targeting T cell using anti-CD3 antibody, the other arm targeting tumor cells using tumor specific antibodies, works as a bridge to bring the T cells and tumor cells together and activate T cells to kill tumor. We provide service for design, construct, expression and evaluation of bi-specific antibodies.
Antibody labeling for optical imaging
Optical imaging is a convenient and useful method for in vivo antibody biodistribution study, such as the antigen expression profile study in mouse, tumor targeting evaluation in mouse model. Near infrared (NIR) dyes are often used for these studies due to their deep penetration in tissues (around 1 cm). We provide services for efficient conjugation and purification of fragment antibodies or full antibody with these dyes and related characterization.