-Development of the
first phage display systems for the display of large proteins, particularly
50 KD heterodimeric antibody Fab fragments
-Development of the first phage displayed Human Antibody Libraries
-Cloning of the first High Affinity Human Monoclonal Antibodies using Phage
Display
-Development of Methodologies for the construction and selection of phage display
libraries of Human, Mouse, Rabbit and Chicken antibody Fab’s and scFv’s
-Development of the first Synthetic Antibody Libraries
-Development of the first Zinc Finger Display Libraries
-Development of the first Motif Grafted Antibodies
-Development of one the most potent HIV-1 neutralizing antibodies known, IgG-b12
-Development of robust methods to improve Antibody affinity- CDR Walking
-Development of the first in vitro evolved Antibodies with enhanced biological
activity, the anti-HIV-1 antibody 3B3
-Development of phage-based methods for the humanization of mouse and rabbit
Antibodies
-Development of a wide range of anti-Cancer Antibodies
-Development of novel Catalytic Antibodies and Catalytic peptides
Barbas, C. F., III; Burton,
D. R.; Scott, J.K., Silverman, G.J. Eds. (2001) Phage Display:
A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold
Spring Harbor, New York, 736 pages. Information about the manual can be found
by clicking on the title or by purchasing directly from www.cshlpress.com
or www.Amazon.com.
pComb3H
Family (Genebank AF268280) Maps
pComb3HSS
Text
pComb3HTT
Text
pAraH6HATT
Text
pMalCSS
(cytoplasmic expression) Text
pMalPSS
(periplasmic expression) Text
The
Original pComb
pComb3
Text & Map
pComb8
Text &
Map
The original pComb3 vector
was designed for phage display of Fabs which are cloned in one chain at a time
using SacI/XbaI restriction sites (for the light chain) and XhoI/SpeI restriction
sites (for the heavy chain). This vector system has also been used for the display
of a wide variety of other proteins like zinc fingers, peptides, and cDNA fragments.
Phage can be produced which express Fab or other proteins or peptides of interest
fused to the phage pIII protein for expression on the head of the phage. Soluble
Fab can be expressed by removing the gene for the pIII phage fusion protein
by SpeI/NheI digest. Sufficient soluble protein is also found in the periplasmic
space as a result of proteolysis. pComb8 is nearly identical but contains the
phage pVIII fusion protein for multi-valent expression along the sides of the
phage via fusion with pVIII.
Select References:
Barbas III, C.F. (1995)
Synthetic Human Antibodies. Nature Medicine 1,837-839.
Barbas III, C.F., Bjorling,
E., Chiodi, F., Dunlop, N., Cababa, D., Jones, T.M., Zebedee, S.L., Persson,
M.A.A., Nara, P.L., Norrby, E. and Burton, D.R. (1992) Recombinant Human Fab
fragments neutralize human type 1 immunodeficiency virus in vitro. Proc.
Natl. Acad. Sci. USA 89, 9339-9343
Barbas III, C.F., Kang,
A.S., Lerner, R.A. and Benkovic, S.J. (1991) Assembly of combinatorial antibody
libraries on phage surfaces: The gene III site. Proc. Natl. Acad. Sci. USA
88, 7978-7982
Barbas III, C.F., Crowe,
J.E., Cababa, D., Jones, T.M., Zebedee, S.L., Murphy, B.R., Chanock, R.M. and
Burton, D.R. (1992) Human Monoclonal Fab Fragments derived from a combinatorial
library bind to respiratory syncytial virus F glycoprotein and neutralize infectivity.
Proc. Natl. Acad. Sci. USA 89, 10164-10168
Barbas III, C.F., Persson, M.A.A., Koenig, S., Chanock, R.M., Burton, D.R. and
Lerner, R.A. (1992) A large array of human monoclonal antibodies to HIV-1 from
combinatorial libraries of an asymptomatic seropositive individual. Vaccines
'92: Modern Approaches to New Vaccines including Prevention of AIDS, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, New York , 9-12
Barbas III, C.F., Bain,
J.D., Hoekstra, D.M. and Lerner, R.A. (1992) Semi-synthetic combinatorial antibody
libraries: A chemical solution to the diversity problem. Proc. Natl. Acad.
Sci. USA, 89:4457-4461.
Lerner, R.A., Kang, A.S.,
Bain, J.D., Burton, D.R., Barbas III, C.F. (1992) Antibodies without immunization.
Science 258:1313-1314.
Barbas III, C.F., Amberg,
W., Somincsits, A., Jones, T.M. and Lerner, R.A. (1993) Selection of human anti-hapten
antibodies from semisynthetic libraries. Gene, 137:57-62.
Burton, D.R., Barbas III,
C.F., Persson, M.A.A., Koenig, S., Chanock, R.M. and Lerner, R.A. (1991) A large
array of human monoclonal antibodies to type 1 human immunodeficiency virus
from combinatorial libraries of asymptomatic seropositive individuals. Proc.
Natl. Acad. Sci. USA 88, 10134-10137
Barbas III, C.F., Languino,
L.R. and Smith, J.W. (1993) High Affinity Self-Reactive Human Antibodies by
Design and Selection: Targeting the Integrin Ligand Binding Site, Proc. Natl.
Acad. Sci. USA 90:10003-10007.
Smith, J.W., Hu, D., Satterthwait,
A.C., Pinz-Sweeney, S. and Barbas III, C.F. (1994) Building Synthetic Antibodies
as Adhesive Ligands for Integrins. J. Biol. Chem. 269:32788-32795.
Barbas III, C.F., and Wagner,
J. (1995) Synthetic Human Antibodies: Selecting and Evolving Functional Proteins.
Methods, A Companion to Methods in Enzymology 8, 94-103.
Other
Related Vectors
pJF3H
Text &
Map
Modified version of pComb3H
containing Jun, Fos and a cloning site for cDNA expression. Developed in the
lab of Gregg Silverman at UCSD.
References:
Barbas, C. F., III; Burton,
D. R.; Scott, J.K., Silverman, G.J. Eds. (2001) Phage Display: A Laboratory
Manual;
Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, ,
736 pages.
pIgG Text &
Map
Vector for conversion of
Fab to IgG for expression in mammalian cells.
References:
Rader, C; Popkov, M.; Neves,
J.A.; Barbas III, C.F. (2002) Integrin avb3 Targeted Therapy of Kaposi’s
Sarcoma with an In Vitro Evolved Antibody . FASEB, 16(14), 2000-2002.
Phage Display: A Laboratory
Manual
By Carlos F. Barbas III, The Scripps Research Institute;
Dennis R. Burton, The Scripps Research Institute;
Jamie K. Scott, Simon Fraser University;
Gregg J. Silverman, University of California, San Diego
2001, 736 pp., illus., index
Paperback $75 ISBN 0-87969-546-3
*Customers in Europe,
the Middle East, and Africa should check for this publication at the CSHL
Press Europe website.
Chapter 1. Filamentous
Phage Biology
Chapter 2. Phage-display Vectors
Chapter 3. Antibody Libraries
Chapter 4. Peptide Libraries
Chapter 5. Functional Domains and Scaffolds
Chapter 6. Gene Fragment Libraries and Genomic and cDNA Expression Cloning
Chapter 7. Overview: Amplification
of Antibody Genes
Chapter 8. Generation of Antibody Libraries: Immunization, RNA Preparation,
and cDNA Synthesis
Chapter 9. Generation of Antibody Libraries: PCR Amplification and Assembly
of Light- And
Heavy-chain Coding Sequences
Chapter 10. Selection from Antibody Libraries
Chapter 11. Analysis of Selected Antibodies
Chapter 12. Production and Purification of Fab and scFv
Chapter 13. Antibody Engineering
Chapter 14. Overview:
Peptide Libraries
Chapter 15. General Phage Methods
Chapter 16. Production of Peptide Libraries
Chapter 17. Screening Peptide Libraries
Chapter 18. Analysis of Phage-borne Peptides
Chapter 19. Construction and Use of pIII-displayed Peptide Libraries
Chapter 20. Construction
and Selection from Gene Fragment Phage-display Expression Libraries
Chapter 21. Construction and Selection from cDNA Phage-display Expression
Libraries
Chapter 22. In Vivo Selection of Phage-display Libraries
Chapter 23. Cell-surface Selection and Analysis of Monoclonal Antibodies from
Phage Libraries
Appendix 1. Useful Information
Appendix 2. Recipes
Appendix 3. General Procedures
Appendix 4. Cautions
Appendix 5. Suppliers
Appendix 6. Trademarks
t
chain cloning region bounded by SacI and XbaI restriction sites and a 300bp
stuffer in Fab heavy chain cloning region bound by XhoI and SpeI restriction
sites. Also, the 1600bp double stuffer (both stuffer plus the leader sequence
between the Fab light chain and heavy chain cloning regions) can be removed
by SfiI digest so that non-Fab genes of interest can also by cloned. pComb3XTT
and pComb3XLambda are only needed for the construction of chimeric Fab libraries
as described in Phage Display: A Laboratory Manual. The “TT” refers
to the human Fab to tetanus toxin from which the kappa light chain constant
region and Fd can be amplified. pComb3XLambda contains a non-functional Fab
from which the lambda light chain constant region can be amplified.
as
a control for Fab expression. Soluble Fab can be expressed by removing the gene
for the pIII phage fusion protein by SpeI/NheI digest. Sufficient soluble protein
is also found in the periplasmic space as a result of proteolysis. For expression
of non-Fab proteins, subcloning to an expression vector with tags for detection
may be necessary. We can provide arabinose inducible pAraH6HATT which contains
the human Fab to tetanus toxin and a compatible SfiI cloning region as well
as a similarly equipped Maltose- binding protein fusion vector pMal-SS (either
cytoplasmic or periplasmic). Our version of the pMal vectors, originally obtained
from New England Biolabs, are modified with the SfiI cloning cassette and SS
double stuffer for easy transfer from pComb3H.