Proc. Natd. Acad. Sci. USA

Vol. 89, pp. 3576-3580, April 1992 Biochemistry

In vitro selection and affinity maturation of antibodies 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, S. KANG

AND ANGRAY

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

H-chain IgG isotype for molecular cloning, and/or the high abundance of specific mRNA in the starting material (12) most likely biased the combinatorial libraries toward affinitymatured antibodies. Recently, Marks et al. (19) demonstrated that active single-chain (Sc) antibodies with affinity constants between 106 and 107 M-1 against a hapten or a small number of epitopes on a protein can be obtained directly from nonimmune combinatorial immunoglobulin libraries. In its simplest form, the initial stage of the immune response can be re-created in vitro by generating a combinatorial library of PCR-amplified immunoglobulin At and L chains from the bone marrow of adult mice. This is a close approximation to the naive, unselected repertoire, since the majority of B cells in bone marrow expressing immunoglobulin ,u chains have not been subjected to tolerance and antigen selection and should therefore represent all the combinatorial junctional diversity of immunoglobulin V regions (20). The phagemid pComb8 facilitates the display of multiple copies of the Fab fragments along the phage surface permitting access to low-affinity antibodies (15, 16). Hence, specific VH and VL pairs could possibly be enriched from a diverse naive repertoire. The second stage of the immune response in vivo involves affinity maturation of the selected specificities by mutation and selection. An efficient way to generate random mutations in the laboratory is by an error-prone replication mechanism, either by targeting the mutations to the antibody binding sites by error-prone PCR (21), or by passaging the phagemid carrying the genetic information for the Fab fragment through an Escherichia coli mutD strain, in which the spontaneous mutation frequency is 103-105 times higher than in a wild-type strain (22). Here we describe our results using error-prone PCR. In subsequent communications, we will describe a complementary method using randomized complementaritydetermining region (CDR) 3 sequences of a VH region to generate mutants (C.F.B., unpublished data). In the present study, we chose progesterone as a hapten since it elicits a restricted immune response in mice (23, 24). The method described here has three essential features: (i) the ability to initially access low-affinity Fabs from a naive library by the use of multivalent phage expression systems, (ii) subsequent affinity maturation by error-prone PCR, and (iii) use of a Sc construct during the maturation process to avoid a high background of artifactual binding due to loss of the L chain. When used in concert, these methods allow selection and affinity maturation of antibodies from a naive library.

We have used a combinatorial immunoglobABSTRACT ulin library approach to obtain monoclonal antibodies from nonimmune adult mice, thereby establishing the principles of (i) accessing naive combinatorial antibody libraries for predetermined specificities and (ii) increasing the affinity of the selected antibody binding sites by random mutagenesis. A combinatorial Fab library expressing immunoglobulin Ip and it light-chain fragments on the surface of filamentous phage was prepared from bone marrow of nonimmunized, adult BALB/c mice with the multivalent display vector pComb8. Phage displaying low affinity Fabs (binding constants, 104-1O M-') binding to a progesterone-bovine serum albumin conjugate were isolated from the library. Random mutagenesis of the heavy- and light-chain variable regions expressed in the monovalent phage display vector pComb3 was performed by errorprone PCR, and subsequently clones with improved affinity for the hapten conjugate were selected. We demonstrate that antibodies with desirable characteristics from a nonimmune source may be selected and affinity maturation may be achieved by using the twin vectors pComb8 and pComb3, thus opening the route to obtaining specific antibodies from a generic library and bypassing immunization. The B-cell immune response to an antigen can be viewed to occur in two stages. The initial stage generates low-affinity antibodies mostly of the IgM isotype from an existing pool of the B-cell repertoire available at the time of immunization. The second stage, which is driven by antigen stimulation, produces high-affinity antibodies predominantly of the IgG isotype, starting with the light-chain and heavy-chain variable region (VL and VH) genes selected in the primary response. The predominant mechanism for affinity maturation is hypermutation of V region genes followed by selection of those cells that produce antibodies of the highest affinity (reviewed in refs. 1 and 2). Both stages in the immune response have been accessed and extensively studied by hybridoma technology (3). Recently, there has been much interest in using gene cloning approaches to generate and express antibodies by combinatorial library techniques to bypass hybridoma technology (4-19). The main driving force for this approach is to more efficiently harness the vast antibody repertoire. We and others have successfully generated diverse high-affinity antibodies to haptens, virus particles, and protein antigens, thereby recapitulating functional molecules appearing during the natural immune response in animals and humans. Most of the molecular genetic approaches for obtaining active antibodies 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, complementarity-determining region; gVIII and gIII, M13 gene VIII and gene III, respectively; Sc, single chain; VL, light-chain variable region; VH, heavy-chain variable region. *Present address: Sandoz Pharma, Preclinical Research, CH-4002 Basel, Switzerland.

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payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3576

Biochemistry: Gram et al. METHODS RNA Isolation and cDNA Synthesis. Three nonimmunized adult male (6 months old) BALB/cByJ mice (Scripps breeding colony) were used to prepare 5 x 107 bone marrow cells in 4% fetal calf serum in phosphate-buffered saline (PBS). To deplete for surface IgG-positive cells, the preparation was incubated with rat anti-mouse IgG2b (0.1 ml), goat antimouse IgG (0.1 ml), and rabbit anti-mouse IgG2b (0.1 ml) for 30 min at ambient temperature. The cells were pelleted, washed with PBS, and resuspended in PBS (9 ml). Rabbit complement was added (1 ml) and incubated at 370C for 30 min. The cells were pelleted and total RNA was isolated as described by Kang et al. (25). Total RNA was used as a template for cDNA synthesis for p and K chains with the following primers: A chain, 5'-ATTGGGACT AGI TTCTGC GAC AGC TGG ATT-3'; K chain, 5'-GCG CCG IC[ AiA ATT AAC ACT CAT TCC TGT TGAA-3', respectively, using a SuperScript kit (BRL). Briefly, 7 Ag of total RNA was mixed with 60 pmol of primer, heated to 70'C for 10 min, and immediately cooled on ice. Two microliters of RNase inhibitor, 10 pl of 5 x synthesis buffer, 8 A.l of dNTP mixture (to give a final concentration of 200 AM each NTP), 5 jul of 0.1 M dithiothreitol, and 1 Al of BRL SuperScript reverse transcriptase (200 units/,l) were added, and the reaction mixture was made up to 50 pl with diethylpyrocarbonatetreated water. The reaction was allowed to proceed at room temperature for 10 min and then at 42°C for 50 min. The reaction was terminated by incubating at 90°C for 5 min and then by placing on ice for 10 min followed by adding 1 pul of RNase H and incubating at 37°C for 20 min. PCR amplification was performed in a 100-pl reaction mixture as described, using VH1-9 and the ,i-chain primer for the H chains and VL3-7 and the K-chain primers for the L chains (25). Naive Immunoglobulin pl/K Library Construction. The PCR amplified ,u-chain and K-chain DNAs were cleaved as described (25). The resulting ,-chain Xho I/Spe I fragments were inserted into the pComb8 phagemid vector (15) to generate a ,-chain-gene VIII (gVIII) fusion library. Transformation into E. coli XL1 blue and phage production was carried out essentially as described (26). Subsequently, the K-chain Sac I/Xba I fragments were cloned into the H-chain Fd p-gVIII fusion library. Panning. The panning procedure used is a modification of that originally described by Parmley and Smith (27). Wells of a microtitration plate (Costar 3690) were coated at 4°C with 50 ,ul of progesterone-3-(O-carboxymethyl)oxime-bovine serum albumin (BSA) conjugate (100 ,ug/ml) (Sigma P4778) in PBS. The wells were washed twice with water and blocked by completely filling with 1% (wt/vol) BSA in PBS and incubating the plates at 37°C for 1 h. Blocking solution was flicked out and 50 pl of the phage library (typically 1011 colony-forming units) in PBS/BSA (0.1%) was added to each well and the plates were incubated for 2 h at 37°C. The washing steps, elution, and multiplication of the phage were done essentially as described (16). Colony Screening of Panned Libraries. The bacterial colonies were prepared essentially as described (16) and probed for progesterone binding with a progesterone-3-(O-

carboxymethyl)oxime-horseradish peroxidase conjugate (Sigma P3659). The filters were developed with 4-chloronaphthol (28). Sc Fv-gIII Fusion. The plasmid pComb3 (16) was digested with the restriction endonucleases Xba I and Nhe I and

religated, thus eliminating the L-chain cloning cassette. A synthetic DNA linker, which encodes a 15-amino acid linker sequence (29) consisting of two oligonucleotides (5'TCGAGAAAGTCTCTAGAGGTAAATCTTCTGGTTCTGGTTCCGAATCTAAATCTACTGAGCTCAAAGTCA-3' and 5'-CTAGTGACTTTGAGCTCAGTAGAT-

Proc. Natl. Acad. Sci. USA 89 (1992)

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TTAGATCGGAACCAGAACCAGAAGA TTT ACC TCTAGAGACTTTC-3'), was inserted into the Xho I/Spe I digested truncated pComb3 vector forming the phagemid ScpComb3. The internal recognition sequences for restriction endonucleases Xba I (TCTAGA) and Sac I (GAGCTC) are underlined. The VH and VL segments of the progesterone binders were amplified by PCR using the upstream primer described above and the oligonucleotides 5'-ATTTGGGAAGGACTGTCTAGATGMRGAGAC-3' (M is A or C and R is A or G) and 5'-GAGGACTAGTTACAGTTGGTGCAGCATCAG-3', respectively. The internal recognition sequences for Xba I (TCTAGA) and Spe I (ACTAGT) are underlined. The VH and VL Fv PCR fragments were digested with Xho I/Xba I and Sac I/Spe I, respectively, and subsequently inserted into ScpComb3. Expression and Detection of Soluble Fabs and Sc Fv Antibodies. For Fab production, the gVIII moiety in the phagemids encoding the progesterone binders PgA11, PgB6, and PgF1 was excised with restriction endonucleases Spe I and EcoRI and was subsequently replaced by a synthetic linker encoding a TAA stop codon (underlined). The linker was formed by the oligonucleotides 5'-CTAGTTAACTGAGTAAG-3' and 5'-AATTCTTACTCAGHIAA-3'. The production and detection of antibody Fab fragment was performed essentially as described (18, 26) except that the E. coli cells were disrupted by three freeze-thaw cycles. For producing soluble ScFv antibody fragments, the VH-linker-VL fusions were excised from the ScpComb3 phagemid with Xho I and Spe I and subcloned into the expression vector pTAC01 (to be described in detail elsewhere), a derivative of pFl260 (30). It has the inducible tac promoter and the pelB leader sequence for secretion, and it allows in-frame fusion of the inserted ScFv with a decapeptide sequence (YPYDVPDYAS) (31) as a tag for immunochemical detection. Expression and detection of the ScFv antibody fragments was as described above, except that an anti-decapeptide antibody conjugated to alkaline phosphatase was used for the ELISA. VH/VL Targeted Mutagenesis by PCR. Equal amounts of undigested PgF1, PgB6, and PgA11 ScpComb3 plasmids were mixed and serially diluted. Then 100, 10, 1, 0.1, and 0.01 ng of the mixture were subjected separately to 35 cycles (1 min at 94°C, 2 min at 50°C, 1 min at 72°C) of amplification under the following reaction conditions: 50 mM KCI/10 mM Tris'HCI, pH 9.0/6.5 mM MgCl2/0.5 mM MnCl2/0.01% gelatin/0.1% Triton X-100/1 mM each dCTP, dGTP, dTTP/ 0.2 mM dATP/0.1 mM dITP, using the M13 reverse sequencing primer 5'-AACAGCTATGACCATG-3' and a backward primer complementary to the gIII moiety 5'-GACAGGAG100 GTTGAGGCAGGT-3' at lM. The basic method of error-prone PCR was as originally described by Leung et al. (21). The PCRs of all template dilutions were pooled and treated with phenol before digestion with Xho I and Spe I. The gel-purified and digested PCR fragments were ligated back into the Xho I/Spe I-digested ScpComb3 plasmid. The ligation products were electroporated into E. coli XL1 blue giving rise to -106 transformants. Subsequent steps of phage production and panning were carried out as described above, except that the phage were panned in the absence of BSA. Affinity Determination. The binding constants of the soluble antibody fragments were determined by competitive ELISA (18, 32). Briefly, wells of a microtitration plate (Costar 3690) were coated at 4°C with 50 ,ul of progesterone3-(O-carboxymethyl)oxime-BSA conjugate (100 ,ug/ml) (Sigma P4778) in PBS. The wells were washed twice with water and blocked with 1% BSA in PBS at 37°C for 1 h. Fab or ScF, supernatants were mixed with progesterone-3-(O-carboxymethyl)oxime-BSA in PBS/BSA (0.1%) and incubated in the wells at 370C for 2 h. The plates were washed with PBS/ Tween (0.05%; vol/vol), and goat anti-mouse K-chain alkaline phosphatase conjugate (Southern Biotechnology Asso-

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Biochemistry: Gram et al.

Proc. Natl. Acad. Sci. USA 89 (1992)

ciates, B-' mingham, AL) or mouse anti-decapeptide monoclonal antibodies conjugated to alkaline phosphatase was added and incubated (370C; 1 h). The plates were washed as described, and substrate was added [0.1 ml of p-nitrophenyl phosphate at 1 mg/ml in 0.1 M Tris HCl (pH 9.4) containing 50 mM MgC1J. After incubation (250C; 60-180 min) the absorbance was read at 405 nm. Apparent affinities were determined as the reciprocal of the hapten concentration required to inhibit 50% maximal binding in a competitive ELISA. This is a close approximation to the affinity (32) and permitted the ranking of the binding activities. Nucleic Acid Sequencing. The complete nucleotide sequences of the V regions of the H and L chains were determined from double-stranded DNA by using Sequenase 2.0 (United States Biochemical).

RESULTS Progesterone Binding Fabs from a Naive Murine Repertoire. Bone marrow depleted for surface IgG-positive cells from unprimed mice was chosen as the source for the native antibody repertoire. Total RNA was extracted, and the cDNA fragments encoding the immunoglobulin ,. Fd and K chains were amplified by PCR. A combinatorial library of 5 x 106 members was established by subsequently cloning the g-immunoglobulin Fd and K-chain fragments into the vector pComb8, which allows fusion of the H-chain Fd fragment to gVIII. Since the Fab antibody fragments are displayed at a high copy number on the phage surface (15), this vector seemed most suitable for accessing low-affinity antibodies, which are expected to be found in an unselected and unprimed repertoire (2, 33). The recombinant phagemids were packaged into M13 phage particles and five rounds of panning on progesterone-3-(O-carboxymethyl)oxime-BSAcoated ELISA wells were performed. Phage eluted after the first and third round were analyzed for expression of antiprogesterone Fabs by bacterial colony lifts. Care was taken to exclude artifacts caused by phagemids expressing the immunoglobulin u-chain Ft-gVIII fusion without a corresponding L chain. These H-chain-only phages reacted nonspecifically to unrelated antigens (BSA, horseradish peroxidase, hen egg lysozyme), presumably due to the hydrophobic patch displayed on an unpaired H chain (9). Those colonies producing the strongest signal in the Western blot were further examined, and three clones-PgA11, PgB6, and PgF1-were isolated for subsequent analysis. The first two emerged from the first round of panning, and the

latter was isolated after the third round of selection. All three Fabs, produced in their soluble form, bound specifically to progesterone-3-(O-carboxymethyl)oxime-BSA and progesterone-lla-hemisuccinyl-BSA. In addition, all three Fabs displayed a significant crossreactivity against an epitope on cytochrome c (Fig. 1). Their apparent binding constants for progesterone-3-(O-carboxymethyl)oxime-BSA were determined as 4104 M-1 for PgA11 and =3 x 104 M'1 and =105 M1 for PgF1 and PgB6, respectively. These binding constants are much below that reported for anti-progesterone monoclonal antibodies with affinities of 2-5 x 108 M-1. These high-affinity antibodies show little diversity in V gene usage, with an exclusive restriction to the rarely found VH IX family and the widely used Vk1 family (23, 24). Since we established 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 results), we reasoned that the cited VH IX or Vk1 family or a proper combination of both was not represented in the library. Indeed, none of our progesterone binders utilized V genes of either the VH IX or Vk1 family. Our clones PgB6 and PgF1 utilize the same combination of closely related VH and VL genes. Both of their VH genes are identical to two closely related but distinct germ-line genes (34, 35) with no evidence of somatic mutation, as one would expect for a naive repertoire (Fig. 2). The true Aerm-line gene(s) for their closely related VL genes are not yet known. Since both the VH genes have joined to different diversity and joining region segments (see Fig. 2), the clones PgB6 and PgF1 cannot be of the same origin but must have been selected from two independent cloning events, pointing toward a possible importance of this particular combination for progesterone binding. The VL and VH genes used by PgAll are not closely related to the other two clones. Thus, this part of our study demonstrated that by using a multivalent display vector, Fabs cap be isolated from naive combinatorial libraries with affinities comparable to those observed for the primary immune response to haptens, such as phosphocholine (33) and pitrophenol (39). Furthermore, the combinatorial library approach can yield V genes or V gene combinations that would not have been selected in vivo. Affinity Maturation by PCR-Directed Mutagenesis. To mimic the process of somatic mutation, which leads to the selection of antibodies with a higher affinity, we set out to create random mutations in both the VL and VH regions and subsequently to select antibodies with an increased affinity to the hapten. To target the mutations' by error-prone PCR

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Proc. Natl. Acad. Sci. USA 89 (1992)

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In vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library.

We have used a combinatorial immunoglobulin library approach to obtain monoclonal antibodies from nonimmune adult mice, thereby establishing the princ...
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