Proc. Natd. Acad. Sci. USA Vol. 89, pp. 3576-3580, April 1992 Biochemistry In vitro selection and affinity maturation of from a naive combinatorial immunoglobulin library (naive immunoglobulin repertoire/fflamentous phage/random mutagenesis) HERMANN GRAM*, LORI-ANNE MARCONI, CARLOS F. BARBAS III, THOMAS A. COLLET, RICHARD A. LERNER, AND ANGRAY S. KANG The Scripps Research Institute, Departments of Molecular Biology and Chemistry, 10666 North Torrey Pines Road, La Jolla, CA 92037 Contributed by Richard A. Lerner, January 7, 1992

ABSTRACT We have used a combinatorial immunoglob- H-chain IgG for molecular cloning, and/or the high ulin library approach to obtain monoclonal antibodies from abundance of specific mRNA in the starting material (12) nonimmune adult mice, thereby establishing the principles of most likely biased the combinatorial libraries toward affinity- (i) accessing naive combinatorial libraries for prede- matured antibodies. Recently, Marks et al. (19) demonstrated termined specificities and (ii) increasing the affinity of the that active single-chain (Sc) antibodies with affinity constants selected antibody binding sites by random mutagenesis. A between 106 and 107 M-1 against a or a small number combinatorial Fab library expressing immunoglobulin Ip and it of epitopes on a protein can be obtained directly from light-chain fragments on the surface of filamentous phage was nonimmune combinatorial immunoglobulin libraries. prepared from bone marrow ofnonimmunized, adult BALB/c In its simplest form, the initial stage of the immune mice with the multivalent display vector pComb8. Phage response can be re-created in vitro by generating a combi- displaying low affinity Fabs (binding constants, 104-1O M-') natorial library of PCR-amplified immunoglobulin At and L binding to a progesterone-bovine serum albumin conjugate chains from the bone marrow of adult mice. This is a close were isolated from the library. Random mutagenesis of the approximation to the naive, unselected repertoire, since the heavy- and light-chain variable regions expressed in the mono- majority of B cells in bone marrow expressing immunoglob- valent phage display vector pComb3 was performed by error- ulin ,u chains have not been subjected to tolerance and prone PCR, and subsequently clones with improved affinity for selection and should therefore represent all the the hapten conjugate were selected. We demonstrate that combinatorial junctional diversity of immunoglobulin V re- antibodies with desirable characteristics from a nonimmune gions (20). The phagemid pComb8 facilitates the display of source may be selected and affinity maturation may be achieved multiple copies ofthe Fab fragments along the phage surface by using the twin vectors pComb8 and pComb3, thus opening permitting access to low-affinity antibodies (15, 16). Hence, the route to obtaining specific antibodies from a generic library specific VH and VL pairs could possibly be enriched from a and bypassing immunization. diverse naive repertoire. The second stage of the immune response in vivo involves The B-cell immune response to an antigen can be viewed to affinity maturation of the selected specificities by mutation occur in two stages. The initial stage generates low-affinity and selection. An efficient way to generate random mutations antibodies mostly of the IgM isotype from an existing pool of in the laboratory is by an error-prone replication mechanism, the B-cell repertoire available at the time of immunization. either by targeting the mutations to the antibody binding sites The second stage, which is driven by antigen stimulation, by error-prone PCR (21), or by passaging the phagemid produces high-affinity antibodies predominantly of the IgG Fab isotype, starting with the light-chain and heavy-chain vari- carrying the genetic information for the fragment through able region (VL and VH) genes selected in the primary an Escherichia coli mutD strain, in which the spontaneous response. The predominant mechanism for affinity matura- mutation frequency is 103-105 times higher than in a wild-type tion is hypermutation ofV region genes followed by selection strain (22). Here we describe our results using error-prone of those cells that produce antibodies of the highest affinity PCR. In subsequent communications, we will describe a (reviewed in refs. 1 and 2). Both stages in the immune complementary method using randomized complementarity- response have been accessed and extensively studied by determining region (CDR) 3 sequences of a VH region to hybridoma technology (3). generate mutants (C.F.B., unpublished data). In the present Recently, there has been much interest in using gene study, we chose progesterone as a hapten since it elicits a cloning approaches to generate and express antibodies by restricted immune response in mice (23, 24). The method combinatorial library techniques to bypass hybridoma tech- described here has three essential features: (i) the ability to nology (4-19). The main driving force for this approach is to initially access low-affinity Fabs from a naive library by the more efficiently harness the vast antibody repertoire. We and use of multivalent phage expression systems, (ii) subsequent others have successfully generated diverse high-affinity an- affinity maturation by error-prone PCR, and (iii) use of a Sc tibodies to , virus particles, and protein , construct during the maturation process to avoid a high thereby recapitulating functional molecules appearing during background of artifactual binding due to loss of the L chain. the natural immune response in animals and humans. Most of When used in concert, these methods allow selection and the molecular genetic approaches for obtaining active anti- affinity maturation of antibodies from a naive library. bodies by screening combinatorial immunoglobulin libraries were directed toward the second stage of the antibody response, since the immunization protocol, the choice of the Abbreviations: BSA, bovine serum albumin; CDR, complementar- ity-determining region; gVIII and gIII, M13 gene VIII and gene III, respectively; Sc, single chain; VL, light-chain variable region; VH, The publication costs of this article were defrayed in part by page charge heavy-chain variable region. payment. This article must therefore be hereby marked "advertisement" *Present address: Sandoz Pharma, Preclinical Research, CH-4002 in accordance with 18 U.S.C. §1734 solely to indicate this fact. Basel, Switzerland. 3576 Downloaded by guest on September 25, 2021 Biochemistry: Gram et al. Proc. Natl. Acad. Sci. USA 89 (1992) 3577 METHODS TTAGATCGGAACCAGAACCAGAAGA TTT ACC TCTAGAGACTTTC-3'), was inserted into the Xho I/Spe I RNA Isolation and cDNA Synthesis. Three nonimmunized digested truncated pComb3 vector forming the phagemid adult male (6 months old) BALB/cByJ mice (Scripps breed- ScpComb3. The internal recognition sequences for restric- ing colony) were used to prepare 5 x 107 bone marrow cells tion endonucleases Xba I (TCTAGA) and Sac I (GAGCTC) in 4% fetal calf serum in phosphate-buffered saline (PBS). To are underlined. The VH and VL segments ofthe progesterone deplete for surface IgG-positive cells, the preparation was binders were amplified by PCR using the upstream primer incubated with rat anti-mouse IgG2b (0.1 ml), goat anti- described above and the oligonucleotides 5'-ATTTGG- mouse IgG (0.1 ml), and rabbit anti-mouse IgG2b (0.1 ml) for GAAGGACTGTCTAGATGMRGAGAC-3' (M is A or C and 30 min at ambient temperature. The cells were pelleted, R is A or G) and 5'-GAGGACTAGTTACAGTTGGTGCAG- washed with PBS, and resuspended in PBS (9 ml). Rabbit CATCAG-3', respectively. The internal recognition se- complement was added (1 ml) and incubated at 370C for 30 quences for Xba I (TCTAGA) and Spe I (ACTAGT) are min. The cells were pelleted and total RNA was isolated as underlined. The VH and VL Fv PCR fragments were digested described by Kang et al. (25). Total RNA was used as a with Xho I/Xba I and Sac I/Spe I, respectively, and subse- template for cDNA synthesis for p and K chains with the quently inserted into ScpComb3. following primers: A chain, 5'-ATTGGGACT AGI TTCTGC Expression and Detection of Soluble Fabs and Sc Fv Anti- GAC AGC TGG ATT-3'; K chain, 5'-GCG CCG IC[ AiA bodies. For Fab production, the gVIII moiety in the phage- ATT AAC ACT CAT TCC TGT TGAA-3', respectively, mids encoding the progesterone binders PgA11, PgB6, and using a SuperScript kit (BRL). Briefly, 7 Ag oftotal RNA was PgF1 was excised with restriction endonucleases Spe I and mixed with 60 pmol of primer, heated to 70'C for 10 min, and EcoRI and was subsequently replaced by a synthetic linker immediately cooled on ice. Two microliters of RNase inhib- encoding a TAA stop codon (underlined). The linker was itor, 10 pl of 5 x synthesis buffer, 8 A.l of dNTP mixture (to formed by the oligonucleotides 5'-CTAGTTAACTGAG- give a final concentration of 200 AM each NTP), 5 jul of 0.1 TAAG-3' and 5'-AATTCTTACTCAGHIAA-3'. The produc- M dithiothreitol, and 1 Al of BRL SuperScript reverse tion and detection of antibody Fab fragment was performed transcriptase (200 units/,l) were added, and the reaction essentially as described (18, 26) except that the E. coli cells mixture was made up to 50 pl with diethylpyrocarbonate- were disrupted by three freeze-thaw cycles. For producing treated water. The reaction was allowed to proceed at room soluble ScFv antibody fragments, the VH-linker-VL fusions temperature for 10 min and then at 42°C for 50 min. The were excised from the ScpComb3 phagemid with Xho I and reaction was terminated by incubating at 90°C for 5 min and Spe I and subcloned into the expression vector pTAC01 (to then by placing on ice for 10 min followed by adding 1 pul of be described in detail elsewhere), a derivative of pFl260 (30). RNase H and incubating at 37°C for 20 min. PCR amplifica- It has the inducible tac promoter and the pelB leader se- tion was performed in a 100-pl reaction mixture as described, quence for secretion, and it allows in-frame fusion of the using VH1-9 and the ,i-chain primer for the H chains and inserted ScFv with a decapeptide sequence (YPYDVPDYAS) VL3-7 and the K-chain primers for the L chains (25). (31) as a tag for immunochemical detection. Expression and Naive Immunoglobulin pl/K Library Construction. The detection of the ScFv antibody fragments was as described PCR amplified ,u-chain and K-chain DNAs were cleaved as above, except that an anti-decapeptide antibody conjugated described (25). The resulting ,-chain Xho I/Spe I fragments to alkaline phosphatase was used for the ELISA. were inserted into the pComb8 phagemid vector (15) to VH/VL Targeted Mutagenesis by PCR. Equal amounts of generate a ,-chain-gene VIII (gVIII) fusion library. Trans- undigested PgF1, PgB6, and PgA11 ScpComb3 plasmids formation into E. coli XL1 blue and phage production was were mixed and serially diluted. Then 100, 10, 1, 0.1, and 0.01 carried out essentially as described (26). Subsequently, the ng of the mixture were subjected separately to 35 cycles (1 K-chain Sac I/Xba I fragments were cloned into the H-chain min at 94°C, 2 min at 50°C, 1 min at 72°C) of amplification Fd p-gVIII fusion library. under the following reaction conditions: 50 mM KCI/10 mM Panning. The panning procedure used is a modification of Tris'HCI, pH 9.0/6.5 mM MgCl2/0.5 mM MnCl2/0.01% that originally described by Parmley and Smith (27). Wells of gelatin/0.1% Triton X-100/1 mM each dCTP, dGTP, dTTP/ a microtitration plate (Costar 3690) were coated at 4°C with 0.2 mM dATP/0.1 mM dITP, using the M13 reverse sequenc- 50 ,ul of progesterone-3-(O-carboxymethyl)oxime-bovine se- ing primer 5'-AACAGCTATGACCATG-3' and a backward rum albumin (BSA) conjugate (100 ,ug/ml) (Sigma P4778) in primer complementary to the gIII moiety 5'-GACAGGAG- PBS. The wells were washed twice with water and blocked GTTGAGGCAGGT-3' at lM.100 The basic method of by completely filling with 1% (wt/vol) BSA in PBS and error-prone PCR was as originally described by Leung et al. incubating the plates at 37°C for 1 h. Blocking solution was (21). The PCRs of all template dilutions were pooled and flicked out and 50 pl of the phage library (typically 1011 treated with phenol before digestion with Xho I and Spe I. colony-forming units) in PBS/BSA (0.1%) was added to each The gel-purified and digested PCR fragments were ligated well and the plates were incubated for 2 h at 37°C. The back into the Xho I/Spe I-digested ScpComb3 plasmid. The washing steps, elution, and multiplication of the phage were ligation products were electroporated into E. coli XL1 blue done essentially as described (16). giving rise to -106 transformants. Subsequent steps of phage Colony Screening of Panned Libraries. The bacterial colo- production and panning were carried out as described above, nies were prepared essentially as described (16) and probed except that the phage were panned in the absence of BSA. for progesterone binding with a progesterone-3-(O- Affinity Determination. The binding constants of the solu- carboxymethyl)oxime-horseradish peroxidase conjugate ble antibody fragments were determined by competitive (Sigma P3659). The filters were developed with 4-chloro- ELISA (18, 32). Briefly, wells of a microtitration plate naphthol (28). (Costar 3690) were coated at 4°C with 50 ,ul of progesterone- Sc Fv-gIII Fusion. The plasmid pComb3 (16) was digested 3-(O-carboxymethyl)oxime-BSA conjugate (100 ,ug/ml) (Sig- with the restriction endonucleases Xba I and Nhe I and ma P4778) in PBS. The wells were washed twice with water religated, thus eliminating the L-chain cloning cassette. A and blocked with 1% BSA in PBS at 37°C for 1 h. Fab or ScF, synthetic DNA linker, which encodes a 15-amino acid linker supernatants were mixed with progesterone-3-(O-carboxy- sequence (29) consisting of two oligonucleotides (5'- methyl)oxime-BSA in PBS/BSA (0.1%) and incubated in the TCGAGAAAGTCTCTAGAGGTAAATCTTCTGGTTCT- wells at 370C for 2 h. The plates were washed with PBS/ GGTTCCGAATCTAAATCTACTGAGCTCAAAGTCA-3' Tween (0.05%; vol/vol), and goat anti-mouse K-chain alka- and 5'-CTAGTGACTTTGAGCTCAGTAGAT- line phosphatase conjugate (Southern Biotechnology Asso- Downloaded by guest on September 25, 2021 3578 Biochemistry: Gram et al. Proc. Natl. Acad. Sci. USA 89 (1992) ciates, B-' mingham, AL) or mouse anti-decapeptide mono- latter was isolated after the third round of selection. All three clonal antibodies conjugated to alkaline phosphatase was Fabs, produced in their soluble form, bound specifically to added and incubated (370C; 1 h). The plates were washed as progesterone-3-(O-carboxymethyl)oxime-BSA and proges- described, and substrate was added [0.1 ml ofp-nitrophenyl terone-lla-hemisuccinyl-BSA. In addition, all three Fabs phosphate at 1 mg/ml in 0.1 M Tris HCl (pH 9.4) containing displayed a significant crossreactivity against an epitope on 50 mM MgC1J. After incubation (250C; 60-180 min) the cytochrome c (Fig. 1). Their apparent binding constants for absorbance was read at 405 nm. Apparent affinities were progesterone-3-(O-carboxymethyl)oxime-BSA were deter- determined as the reciprocal of the hapten concentration mined as 4104 M-1 for PgA11 and =3 x 104 M'1 and =105 required to inhibit 50% maximal binding in a competitive M1 for PgF1 and PgB6, respectively. These binding con- ELISA. This is a close approximation to the affinity (32) and stants are much below that reported for anti-progesterone permitted the ranking of the binding activities. monoclonal antibodies with affinities of2-5 x 108 M-1. These Nucleic Acid Sequencing. The complete nucleotide se- high-affinity antibodies show little diversity in V gene usage, quences of the V regions of the H and L chains were with an exclusive restriction to the rarely found VH IX family determined from double-stranded DNA by using Sequenase and the widely used Vk1 family (23, 24). Since we established 2.0 (United States Biochemical). and screened the same naive library with the pComb3 vector, previously used to isolate rare high-affinity Fabs (18), without success for progesterone binders (H.G., unpublished re- RESULTS sults), we reasoned that the cited VH IX or Vk1 family or a Progesterone Binding Fabs from a Naive Murine Repertoire. proper combination of both was not represented in the Bone marrow depleted for surface IgG-positive cells from library. Indeed, none of our progesterone binders utilized V unprimed mice was chosen as the source for the native genes ofeither the VH IX or Vk1 family. Our clones PgB6 and antibody repertoire. Total RNA was extracted, and the PgF1 utilize the same combination of closely related VH and cDNA fragments encoding the immunoglobulin ,. Fd and K VL genes. Both of their VH genes are identical to two closely chains were amplified by PCR. A combinatorial library of 5 related but distinct germ-line genes (34, 35) with no evidence x 106 members was established by subsequently cloning the of somatic mutation, as one would expect for a naive reper- g-immunoglobulin Fd and K-chain fragments into the vector toire (Fig. 2). The true Aerm-line gene(s) for their closely pComb8, which allows fusion of the H-chain Fd fragment to related VL genes are not yet known. Since both the VH genes gVIII. Since the Fab antibody fragments are displayed at a havejoined to different diversity andjoining region segments high copy number on the phage surface (15), this vector (see Fig. 2), the clones PgB6 and PgF1 cannot be ofthe same seemed most suitable for accessing low-affinity antibodies, origin but must have been selected from two independent which are expected to be found in an unselected and cloning events, pointing toward a possible importance ofthis unprimed repertoire (2, 33). The recombinant phagemids particular combination for progesterone binding. The VL and were packaged into M13 phage particles and five rounds of VH genes used by PgAll are not closely related to the other panning on progesterone-3-(O-carboxymethyl)oxime-BSA- two clones. coated ELISA wells were performed. Phage eluted after the Thus, this part of our study demonstrated that by using a first and third round were analyzed for expression of anti- multivalent display vector, Fabs cap be isolated from naive progesterone Fabs by bacterial colony lifts. Care was taken combinatorial libraries with affinities comparable to those to exclude artifacts caused by phagemids expressing the observed for the primary immune response to haptens, such immunoglobulin u-chain Ft-gVIII fusion without a corre- as phosphocholine (33) and pitrophenol (39). Furthermore, sponding L chain. These H-chain-only phages reacted non- the combinatorial library approach can yield V genes or V specifically to unrelated antigens (BSA, horseradish perox- gene combinations that would not have been selected in vivo. idase, hen egg lysozyme), presumably due to the hydropho- Affinity Maturation by PCR-Directed Mutagenesis. To bic patch displayed on an unpaired H chain (9). mimic the process of somatic mutation, which leads to the Those colonies producing the strongest signal in the West- selection of antibodies with a higher affinity, we set out to ern blot were further examined, and three clones-PgA11, create random mutations in both the VL and VH regions and PgB6, and PgF1-were isolated for subsequent analysis. The subsequently to select antibodies with an increased affinity to first two emerged from the first round of panning, and the the hapten. To target the mutations' by error-prone PCR

100

0 o 50

0

0 FabA1l FabB6 FabFl ScB6 ScB6-1 ScB6-3 ScB6-4 antibody FIG. 1. Crossreactivity of Fabs and Sc antibodies. The reactivity to various antigens was determined by ELISA. Antigens were (from left to right) progesterone-3-(O-carboxymethyl)oxime-BSA, progesterone-lla-hemisuccinyl-BSA, horseradish peroxidase, BSA, tetanus toxoid, trypsin inhibitor, and cytochrome c. Downloaded by guest on September 25, 2021 Biochemistry: Gram et A Proc. Natl. Acad. Sci. USA 89 (1992) 3579 A CDR 1 CDR 2 CDR 3 Neg. Ref. 1 10 20 30 40 50 s0 70 s0 90 100 110 120 A11H OVKL LESGAELVRPGASVK I SCKAFGYTFT NHH I N WVKORPGOGLWIG WOGTTLTVSS <25* 36 B6H OVKLLESGGGLVOPGGSLKLSCAASGFDFS RYWS WVROAPGKGLEWIG E I NPOSST I NYTPSLKDIKF I SRDNAKNTLYLOUSKVRSEDTALYYCAR RGVKTTCFAY WTLVTVSS 0 34

.... C F1H - .------N------N-- A ...... LYG'VAMO- .3...S.A35

B6-1H...... A ...... R B6-3H.--..---.. - .------L ...... F ...... p...... p p

.-----.-.-.-.--- .. -- ...... A..H..- o EW54H - R---.- ....-A *------p....AP -P-H B CDR 1 ...... CDR 2 CDR 3 1 10 20 30 40 50 60 70 so 90 100 AIIL ELVMTOSPSSLSASLGERVDLTC RASODIGSS L WLOOEPDGTIKRLIY ATSSLDS GVPKRFSGSRSGSDYSLTISSLESEDFVDYYC LOYASSPLT OfAGTKLELK 4 37 B6L ELVLTOSPA I MSASPGEKVTMTC TASSSVSSSYLE WYOOKPGSSPOLWI Y RTSNLAS GVPPRLSGSGSGTSYSLT I SSMEAEDAATYYC HOYHRSPWT FGGGTKLE I K 11* 38

FIL - .---H-----L-----.... S...... -H...... P...... 2* 38

B6-1L...... p...... p...... p...... T -T... p...... P p

B63L- p .--- O ------.. ..p...0......

--- .... . --- ...... B63-4L P---.. R--- -..... K K S- --X-aA . H

FIG. 2. Amino acid sequences of H-chain (A) and L-chain (B) of the progesterone binders. CDR sequences are boxed. The number of nucleotide exchanges to the germ-line (neg) gene is given as well as the reference for the most homologous sequence by searching the GenBank 68.0 data base. *, True germ-line gene unknown, closest homology referenced. specifically and only to the V regions, we created a Sc ScPgB6-4, which led to multiple amino acid substitutions in Fv-gIII fusion plasmid that contained in-frame fusions of the the CDRs and framework regions of the mutants (Fig. 2). following elements: the pelB leader sequence for secretion, The affinity of the mutated Sc antibodies to progesterone- the VH and VL reading frames linked with a synthetic 3-(O-carboxymethyl)oxime-BSA as determined by compet- oligonucleotide coding for a flexible peptide (29), and the gIII itive ELISA had increased over the parent ScPgB6 antibody moiety. The use of a Sc vector further overcomes the by 30-fold for ScPgB6-1 and ='13-fold for both ScPgB6-3 and difficulties due to unwanted selection for nonspecific binding ScPgB6-4 (Fig. 3). Interestingly, the clone with the least by phage that express immunoglobulin H chains only (9). mutations exhibited the highest affinity. The crossreactivity We chose the monovalent coat protein III display system pattern for the mutant Sc antibodies did not change, except for the expression and selection ofthe mutated Sc antibodies, that ScPgB6-1 had lost most of its reactivity to cytochrome since it facilitates the enrichment for antibodies with higher c (Fig. 1). In extensively studied immune responses to affinities (16). For mutagenesis, a mixture of the three ScFv haptens (3, 33, 39), an increase in affinity by 1 order of templates was subjected to error-prone PCR amplification. magnitude could be assigned to specific single amino acid To produce DNA fragments by PCR with various numbers of substitutions, which implies that only one or two amino acid mutations, various amounts of template DNA were used for exchanges in the combining site of the Sc anti-progesterone the PCRs. The PCR products from all the reactions were antibodies may have accounted for their increased affinity to pooled before recloning them into the vector ScpComb3. A the hapten conjugate. Since a mutant with only a single amino library of mutated anti-progesterone Sc phage displaying acid exchange was not recovered, we could not identify the antibody combining sites was established and panned on critical residue(s) that caused the enhanced affinity to the progesterone-3-(O-carboxymethyl)oxime-BSA and the num- hapten conjugate. Furthermore, we did not observe amino ber ofeluted colony-forming units was taken as a measure for acid substitutions common to all three of the mutants, which the relative affinity of the displayed Sc antibodies to the 120 hapten. After the third round of panning, a 50- to 100-fold increase in the yield of eluted phagemids relative to the 100 nonmutated population was noted. Individual mutants showed a 10- to 300-fold increase in yield after panning as 80 compared to the parent clones, indicating that the mutants encoded antibody binding sites with an increased affinity. 60 The four best mutants were chosen for sequence analysis and determination of their affinity for the hapten conjugate. OR040 DNA sequencing revealed that all four mutant clones had 20 arisen from ScPgB6, with ScPgB6-1 and ScPgB6-2 being identical. The predominant type of mutation obtained by this 0- 10 1 1 7 1 03 PCR protocol was an A--G/T--)C nucleotide exchange 0-9 0-8 i 0 1 0-6 1 0-5 1 0-4 (68%), while T--,G/A--+C, G-+A/C-*T, T--A/A-s.T, G--+C/ competing antigen [M] C- G, or C--A/G--+T exchanges occurred at approximately the same frequency. DNA sequences with a higher than FIG. 3. Relative affinity of Sc anti-progesterone antibodies. The relative affinities of PgScB6 (open box), and the mutants PgScB6-1 average mutation frequency-i.e., mutational hot spots- (solid box), PgScB6-3 (open circle), and PgScB6-4 (shaded circle) were observed. We further noted that the three mutant clones were determined by inhibition ELISA using progesterone-3-(O- differed in the number of base-pair changes. The mutation carboxymethyl)oxime-BSA as competing antigen. Molarity refers to frequencies for both the VH and VL regions were found to be the progesterone moiety on the conjugate, as determined by the 1.5% for ScPgB6-1, 2.1% for ScPgB6-3, and 4.1% for supplier. Downloaded by guest on September 25, 2021 3580 Biochemistry: Gram et al. Proc. Natl. Acad. Sci. USA 89 (1992)

suggested that changing different residues can account for the 9. Ward, E. S., Gussow, D., Griffiths, A. D., Jones, P. T. & increased affinity to 3-(O-carboxymethyl)progesterone. A Winter, G. (1989) Nature (London) 341, 544-546. Ser-63 to Pro-63 substitution in the VH CDR2 is common to 10. Huse, W. D., Sastry, L., Iverson, S., Kang, A. S., Alting- the mutants ScPgB6-3 and ScPgB6-4. Although both mutants Mees, M., Burton, D. R., Benkovic, S. J. & Lerner, R. A. show a similar affinity to the hapten conjugate, we cannot yet (1989) Science 246, 1275-1281. 11. Caton, A. J. & Koprowski, H. (1990) Proc. Natl. Acad. Sci. unequivocally assess the importance of that residue for USA 87, 6450-6454. antigen binding, since multiple amino acid exchanges oc- 12. Persson, M. A., Cao, R. H. & Burton, D. R. (1991) Proc. Nati. curred in the VL and VH regions of the two mutants. Acad. Sci. USA 88, 2432-2436. 13. McCafferty, J., Griffiths, A. D., Winter, G. & Chiswell, D. J. DISCUSSION (1990) Nature (London) 348, 552-554. 14. Clackson, T., Hoogenboom, H. R., Griffiths, A. D. & Winter, In this study, we present the principle ofselection and affinity G. (1991) Nature (London) 352, 624-628. maturation of specific antibodies to a hapten from a naive 15. Kang, A. S., Barbas, C. F., Janda, K. D., Benkovic, S. J. & library. Although our in vitro selection and mutagenesis Lerner, R. A. (1991) Proc. Natl. Acad. Sci. USA 88, 4363- system is quite simple compared to the complexity of the 4366. , we note some features in common. (i) The 16. Barbas, C. F., Kang, A. S., Lerner, R. A. & Benkovic, S. J. affinity of antibodies selected from a naive combinatorial (1991) Proc. Natl. Acad. Sci. USA 88, 7978-7982. 17. Kang, A. S., Jones, T. M. & Burton, D. R. (1991) Proc. Natl. library can reach the same order of magnitude as antibodies Acad. Sci. USA 88, 11120-11123. from a primary immune response to haptens. (ii) Although 18. Burton, D. R., Barbas, C. F., Persson, M. A., Koenig, S., the mechanisms generating mutations in vivo (40) or in vitro Chanock, R. M. & Lerner, R. A. (1991) Proc. Natl. Acad. Sci. were different, we observed mutational hot spots. (iii) The USA 88, 10134-10137. increase in affinity after one round of mutation and selection 19. Marks, J. D., Hoogenboom, H. R., Bonnert, T. P., McCaf- in vitro is in the same order ofmagnitude as that observed for ferty, J., Griffiths, A. D. & Winter, G. (1991) J. Mol. Biol. 222, the transition from primary to secondary immune responses 581-597. to haptens. (iv) We could recover a mutated antibody com- 20. Decker, D. J., Boyle, N. E., Koziol, J. A. & Klinman, N. R. bining site with an altered crossreactivity, as is occasionally (1991) J. Immunol. 146, 350-361. observed in vivo (39, 41). It will be interesting to explore 21. Leung, D. W., Chen, E. & Goeddel, D. V. (1989) J. Methods whether additional cycles of random mutagenesis and selec- Cell Mol. Biol. 1, 11-15. tion could lead to a further increase in affinity, as observed, 22. Fowler, R. G., Schaaper, R. M. & Glickman, B. W. (1986) J. for in the immune response to Bacteriol. 167, 130-137. example, tertiary phenylox- 23. Deverson, E.; Berek, C., Taussig, M. & Feinstein, A. (1987) azalone (3). Eur. J. Immunol. 17, 9-13. When the combinatorial antibody approach was first de- 24. Stura, E. A., Arevalo, J. H., Feinstein, A., Heap, R. B., scribed, one wondered whether it could be used to efficiently Taussig, M. J. & Wilson, I. A. (1987) 62,511-521. tap into the vast antibody repertoire in vivo (10). While a 25. Kang, A. S., Burton, D. R. & Lerner, R. A. (1991) Methods: number of subsequent reports (11, 12, 14, 18) demonstrated Comp. Methods Enzymol. 2, 111-118. the effectiveness of this approach, the present study shows 26. Barbas, C. F. & Lerner, R. A. (1991) Methods: Comp. Meth- that we can access and evolve antibody chain combinations ods Enzymol. 2, 119-124. that may never be selected in vivo. Thus, it now seems as if 27. Parmley, S. F. & Smith, G. P. (1988) Gene 73, 305-318. it is possible to exceed the diversity of the antibody response 28. Nakane, P. K. (1968) J. Histochem. Cytochem. 16, 557-560. in vivo by molecular cloning techniques. 29. Bird, R. E., Hardman, K. D., Jacobson, J. W., Johnson, S., The ability to recapitulate some features of a primary and Kaufman, B. M., Lee, S. M., Pope, S. H., Riordan, G. S. & secondary immune response to a hapten in vitro opens the Whitlow, M. (1988) Science 242, 423-426. 30. Larimer, F. W., Mural, R. J. & Soper, T. S. (1990) Prot. Eng. road to selection ofcatalytic antibodies that cannot be readily 3, 227-231. obtained from an immune source and suggests that circum- 31. Field, J., Nikawa, J. I., Broek, D., MacDonald, B., Rodgers, venting immunization for generation of high-affinity antibod- L., Wilson, I. 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