Building antibodies from their genes.

Immunohgicat Reviews 1992, No. 130 Published by Munksgaard. Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)

Building Antibodies from their Genes HENNIE R . HOOGENBOOM*, JAMES D . MARKS*. ANDREW D . GRIFFITHS* & GREG WINTER*!

INTRODUCTION The immune system builds antibodies that bind to a vast range of antigens with high avidity and specificity, and trigger effector mechanisms. Antibodies have been used in industry, research and medicine, and as diagnostic and therapeutic agents, and their potential has been successively enhanced with the advent of new technologies. Hybridoma technology allowed isolation of cell lines secreting antibodies ofa single specificity (Kohler & Milstein 1975) and gene technology has allowed the construction ofa range of engineered antibodies from hybridomas (reviewed by Winter & Milstein 1991). The engineering of antibodies is facilitated by their domain structure. The antigen-binding site is fashioned by a heavy and a light chain variable domain, each domain consisting of two ^-sheets pinned together with a disulphide bridge. At the top of each domain, the ^-strands are linked by three loops which are hypervariable in length and sequence. These loops appear to fashion the antigenbinding site (and were termed complementarity-determining regions or CDRs) (Kabat & Wu 1971). The antigen-binding activity can be grafted from one antibody to another by transplanting the CDRs (Jones et al. 1986, Verhoeyen et al. 1988). showing that the ^-sheet is a structural framework for the CDRs, that the CDRs are responsible for binding to antigen, and that the same ^-sheet framework can provide a scaffold for mounting different antigen-binding loops. "CDR grafting" has been used to "humanize" rodent antibodies for human therapy, including antibodies against a human lymphocyte antigen (Riechmann et al. 1988a), the IL2 receptor (Queen et al. 1989), CD4 (Gorman et al. 1991), respiratory syncylial virus (Tempest et al. 1991), herpes simplex virus (Co et al. 1991), human immunodeficiency virus (Maeda et al. 1991), epidermal growth factor receptor (Kettleborough et al. 1991), carcinoembryonic antigen (Giissow & Seemann 1991), CD3 (Shalaby et al. 1992), and human epidermal growth factor receptor 2 (Carter et al. 1992a). • MRC Centre for Protein Engineering and tMRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.

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The binding sites for effector functions (serum complement and cell receptors) are provided by the constant domains. In IgG, the CH2 domain appears to include the binding site for Clq, and three adjacent residues (Glu318, Lys320 and Lys322) on a section of ^-strand within the CH2 domain were identified by mutagenesis as the "core" Clq-binding motif (Clackson & Winter 1989, Duncan & Winter 1988). The domains of antibodies in each chain are connected by short "link" peptides, allowing "elbow bending" between the variable domains and constant domains, and "hinge bending" between the Fab arms and Fc. In IgG, the hinge peptide also appears to include binding sites for Fc receptors, and Leu234 and 235 have been identified as critical determinants for binding of both FcyRI and FcyRII by mutagenesis (Duncan et al. 1988, Lund et al. 1991). The domain structure of the antibody allows the reshuffling of domains of complete antibodies, and the construction of antibody fragments. Simple chimaeric antibodies have been constructed, in which rodent variable domains were linked to human constant domains (reviewed in Morrison 1992). This has allowed the effector functions of different isotypes to be compared (Bruggemann et al. 1987), and prompted the choice of the human >'l isotype to kill human lymphocytes (Richmann et al. 1988a) for therapeutic application against lymphoma (Hale et al. 1988) and vasculitis (Mathieson et al. 1990). Likewise, antibody fragments, including Fab (Better et al. 1988, Neuberger et al. 1984), Fv (Riechmann et al. 1988b, Skerra & Pluckthun 1988) and single-chain Fv (scFv) fragments (Bird et al. 1988, Huston et al. 1988, Glockshuber et al. 1990), have been engineered for secretion from mammalian cells or bacteria, and also linked together chemically to make bispecific fragments that recruit effector functions (Carter et al. 1992b). Fusion of fragments with peptides can allow formation of homodimeric (Pack & Pluckthun 1992) or heterodimeric (Kostelny et al. 1992) antibody fragments to improve avidity of binding or make bispecific antibodies. Fusion with other proteins can endow immunoglobulins with novel properties; antigen-binding fragments (as either scFv, Fv, Fab or Fab' fragments) have been fused to enzymes, bacterial or plant toxins, T-cell receptor domains, growth factors, interleukins, a luminescent protein (reviewed in Morrison 1992), a cytokine (Hoogenboom et al. 1991b, 1991a) and human angiogenin (Ryback et al. 1992). The fusion of Fab and enzymes (Neuberger et al. 1984) can equip the antibody with novel effector mechanisms, with the potential to target tissue plasminogen activator to blood clots (Schnee et al. 1987), or enzymes that activate prodrugs to tumors (Bosslet et al. 1992). Antibody V-genes derived from hybridomas have provided the raw genetic material for this range of engineered antibodies and antibody fusions. We now describe technologies, based on the strategies of the immune system, to isolate the V-genes directly. Now the display of antibodies on filamentous phase (McCafferty et al. 1991) is allowing the construction of engineered antibody fragments directly from repertoires of antibody V-genes (Clackson et al. 1991a, Marks et

BUILDING ANTIBODIES FROM THEIR GENES

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al. 1991a). and the expression of the fragments in bacteria (Better et al. 1988, Skerra & Pluckthun 1988). MIMICKING IMMUNE SELECTION In the immune system, antibodies are created by selection with antigen. Briefly, the differentiation of hematopoietic stem cells into B lymphocytes involves extensive gene rearrangements (reviewed in Tonegawa 1983) in which the rearranged V-genes are created from a limited number of V-gene segments. Each lymphocyte displays only one species of immunoglobulin on its surface, and since the repertoire of rearranged antibody segments is large, each lymphocyte is likely lo display an immunoglobulin of unique structure and antigen-binding specificity. The newly-generated B lymphocyte migrates from the bone marrow (or fetal liver) to the periphery, where, after binding of antigen to its surface Ig receptor, it is triggered to proliferate and difTerentiate. Antigen that has been bound by the Ig receptor is endocytosed, processed and presented, in association with molecules of the major histoeompatibility complex, to helper T cells. These induce the proliferation and differentiation of the B lymphocyte to short-lived plasma cells, which secrete large amounts of the soluble Ig in the bloodstream, or to long-lived memory cells, which retain their surface Ig and are involved in future (secondary/tertiary) immune response to the same or cross-reactive antigen. The rearranged V-genes of memory cells are subjected to somatic mutation, allowing the creation of mutant antibodies with higher affinities. The immune strategy of selection suggests a process for building artificial antibodies (Milstein 1990, Winter & Milstein 1991). The process is summarized in Fig. 1.

Mimicking the B cell In the immune system, the B lymphocyte provides a "genetic display package" with antibody displayed on the outside of the cell to encounter and bind to antigen, and the genes encoding the antibody within. In principle, such genetic display packages could be created by display of antibodies on the surface of mammalian cells by fusion to a membrane anchor sequence, or on bacteria by fusion to outer membrane proteins or on virus by fusion to a coat protein. The package could then be selected by binding of the antibody to solid-phase antigen, and multiplied by growth, so mimicking the antigen-driven proliferation of B cells. Indeed, a similar strategy has been used to clone the genes encoding cell surface antigens. cDNA from human lymphocytes was cloned into vectors for transient expression on the surface of COS cells, and the protein antigens identified after rounds of panning of the cells with monoclonal antibodies (Aruffo & Seed 1987, Seed & Aruffo 1987). Proteins and peptides have been built into a variety of E. coli outer membrane

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Ill

.•0 Unrearranged V-genes Steps 1.2

Rearranged V genes I Step 3

Assembled V-genes in phage(mid) Step 3 I Antigen

I

Steps

Figure I. The strategy of the immune system in vivo (left) and using pliage (right). Step 1: rearrangement or assembly of germ line V-genes; step 2: surface display of antibody; step 3: antigen-driven or affinity selection; step 4: affinity maturation; step 5: production of soluble antibody (or antibody fragment).

proteins (for review see Hofnung 1991, Fuchs et al. 1991, Francisco et al. 1992). But more promising is the use of filamentous phage fd, a non-lytic bacteriophage, which has been used to display peptides (Greenwood et al. 1991, Il'ichev et al. 1990, Parmley & Smith 1988, Smith 1985). The single-stranded DNA genome of phage fd is coated with approximately 2700 copies of the major coat protein, pVIII, forming a long, thin virion, with three copies of pIII located at one tip of the phage (Glaser-Wuttke et al. 1989, Goldsmith & Konigsberg 1977). Libraries of peptides (up to 15 mers) have been presented on phage by cloning degenerate oligonucleotides as fusions with either gene III (Cwirla et al. 1990, Devlin et al. 1990, Scott & Smith 1990), or gene VIII (Felici et al. 1991). Panning with monoclonal antibody can enrich for phage-displaying epitopes (Parmley & Smith 1988, Stephen & Lane 1992) or mimotopes (peptides sequences that mimic the epitope) (Geysen et al. 1987). However, the estimated affinities of selected peptides are low, at best Kd^O.35 /iM (Cwirla et al. 1990). Peptide libraries have also

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been used to select peptides that mimic the binding of non-peptide ligands to such molecules as streptavidin (Devlin et al. 1990) and concanavalin A (Oldenburg et al. 1992, Scott et al. 1992). We chose the pIU protein of the filamentous phage in an attempt to display folded and functional antibody fragments. The pIII protein has two domains. The amino-terminal domain extends from the phage particle as a knob-like structure, and binds to the bacterial F-pilus to enable infection of male Eseherichia coli bacteria, while the carboxy-terminal domain anchors pIII into the phage coat. During phage assembly, pIII is anchored to the inner membrane of the bacterium by the C-terminal domain, with the N-terminal domain in the periplasm. As antibody fragments directed to the periplasm can fold and assemble heavy and light chains (Better et al. 1988, Skerra & Pluckthun 1988), we fused the antibody fragments to the N-terminus of pIII (McCafierty et al. 1990, Hoogenboom et al. 1991c). Antibody fragments have also been fused to the N- (Breitling et al. 1991) or C-tenninal domain of pIII (Barbas et al. 1991, Garrard et al. 1991), or to pVIII (Chang et al. 1991, Huse 1991, Kang et al. 1991a). First, we tried a single-chain antibody fragment (scFv) (Bird et al. 1988, Huston et al. 1988) (Fig. 2A), derived from a monoclonal antibody (Dl .3) specific for hen eggwhite lysozyme. The fusion protein was expressed and incorporated into the phage, and the phage shown to bind to hen egg-white lysozyme (but not turkey or human lysozyme, so mirroring the specificity of the parent D1.3 antibody) (McCafferty et al. 1990). We also succeeded in displaying heterodimeric Fab fragments specific for the hapten 2-phenyloxazol-5-one (phOx) on the phage, by linking the heavy or light chain to pIII, and secreting the other chain into the bacterial periplasm where the two chains associate (Hoogenboom et al. 1991c). After production of the pIII protein, the bacterium becomes resistant to further infection (Boeke et al. 1982), and therefore phage, like the B cell, should each carry only a single antibody species, whether Fab or scFv. We have also used phagemids for display of antibody fragments (Fig. 2B) (Hoogenboom et al. 1991c). Phagemids are plasmids that can be packaged into phage particles, but require the use ofa helper phage (Vieira & Messing 1987). Phage vectors should lead to three copies of the fragments on each phage (assuming no proteolysis), but with phagemids, the pIII coat protein from helper phage competes for incorporation into the phage. Affinity and kinetic selection Binding of the phage with antigen can be used to select and recover the phage encoding the displayed antibody (McCafierty et al. 1990). We have used several formats to select the phage, including direct binding of the phage to columns of antigen linked to a matrix (Clackson et al. 1991a, McCafferty et al. 1990); or to dishes or tubes to which the antigen is adsorbed (Marks et al. 1991a); or to

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phage vedorfd-lei-OOGI B. Phajtmld dltplay and t«cr«tlon

priagemid pHENt

Figure 2. Phage and phagemid vectors for display of antibody fragments(s) on the surface of filamentous phage. Panel A. Vector fd-tet-DOGI for display of three copies of the antibody fragment (scFv or Fab), Panel B. Phagemid vector pHENI fordisplay of antibody after rescue with helper phage from an E. coli suppressor strain. The amber codon allows production of (tagged) soluble antibody fragment in non-suppressor cells. TET = tetracycline resistance gene, AMP = ampicillin resistance gene, L = leader peptide sequence, tag = c-myc peptide sequence.

antigen on the surface of cells (unpublished data). Phages have also been allowed to bind to biotinylated antigen in solution, followed by capture on streptavidincoated paramagnetic beads (Hawkins et al. 1992b). Non-binders are removed by washing, and bound phage recovered by elution with antigen, acid or alkali (or with DTT where the antigen is biotinylated with a cleavable disulphide biotin), and give enrichment factors ranging from 20- (Marks et al. 1991a) to 1000-fold (McCafferty et al. 1990) for a single round of panning. By infecting bacteria with the eluted phage, more phage can be grown and subjected to another round of panning. An enrichment of 1000-fold in one round of panning can become a factor of one million-fold over two rounds (McCafferty et al. 1990) and, even when enrichments are low, several rounds of selection can lead to the isolation of very rare phage (1/10') (Marks et al. 1991a). The power of selection is limited mainly by the efficiency at each round of selection: it seems that only a small fraction of the phage ( < 1%) can be recovered by binding and elution from antigen (unpublished data). If a phage library contained less than a


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hundred phages with binding activities, it is unlikely that any of these phages would survive the first round of selection. To guarantee selection of at least one copy of a binding clone, it is necessary to have many copies present. Typically, we use 10'^ phages grown from a library with 10* clones, to provide more than 10'' copies of each phage. If the diversity of libraries is to be increased in order to recover higher aflinity antibodies (Perelson 1989, Perelson & Oster 1979), it will be necessary to increase the number of phages or the efilciency of selection. In the immune system, the selection of antibodies with higher affinities (in affinity maturation) appears to have both an affinity and a kinetic component (Foote & Milstein 1991). We have also selected phage by their afTinities of binding by a variation of the biotin capture method (Parmley & Smith 1988, Scott & Smith 1990) using monovalent biotinylated antigen in solution. Instead of using limiting amounts of antigen (such that competition occurs between phages), the antigen was used in excess over the phage, but with the concentration of antigen lower than the "target" equilibrium dissociation constant. This favors the binding of these phage binding more tightly than the target affinity. The phage in complex with antigen was then captured on streptavidin-coated paramagnetic beads. The technique is able to sort phages with small differences in affinity, albeit using many rounds of selection (Hawkins et al. 1992b). We have also selected phage by the kinetics of dissociation from antigen, for example by binding phage to biotinylated antigen in solution, diluting into a large excess of unlabelled antigen and capturing the phage in complex with biotinylated antigen on streptavidin-coated beads over time (Hawkins et al. 1992b). Alternatively, by binding the phage directly to solid-phase antigen, as in panning, and washing extensively, we can enrich for those phages with slower off-rates (Marks et al. 1992a). OfT-rate selection is very powerful in selecting for higher affinity phage as. due to the exponential nature of the decay, small differences in the ofT-rate are amplified over time. The rate of dissociation of phage from antigen on solid phase depends not only on the dissociation rate for each antibody head, but the number of heads engaged with antigen. The number of antibody heads attached to pIII protein on phage is dictated by the use of phage or phagemid vectors, proteolysis of the fusion protein, or self-association of antibody fragments on the phage to form scFv or Fab dimers, while the number of antigen molecules on the solid phase is dictated by the conditions of coating. Avidity effects can be powerful, allowing phages with low afTmities to be selected by binding to antigen (Clackson et al. 1991a), and improving enrichment factors, but they may also hinder the discrimination between phages with different affinities (Barbas et al. 1991). Mimicking the plasma cell Filamentous phage can therefore provide a genetic display package for antibody fragments, and can be selected with antigen, so mimicking the B lymphocyte.

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However, it is also possible to mimic the switch from surface display to secretion of soluble antibody from plasma cells, by introducing an amber stop codon between the antibody gene and gene III. When phages are grown in a supE suppressor strain of £. coli, the amber codon is read as glutamine and the antibody fusion is displayed on the surface of the phage. (Due to the incomplete suppression, some soluble antibody is also produced). In non-suppressor strains, the amber codon is read as a stop codon and soluble protein is secreted into the bacterial periplasm (Hoogenboom et al. 1991c, see also Lowman et al. 1991), where it folds and can be secreted into the culture from the bacteria (Better et al. 1988, Glockshuber et al. 1990, Skerra & Pluckthun 1988). In this way, soluble antibody fragments can be produced by infection of bacteria with the phage. Fragments in the culture medium can be screened for binding in ELISA (Marks et al. 1991a), or for slower dissociation rates using surface plasmon resonance with Biacore (Marks et al. 1992a). Fab fragments can be detected and purified from bacterial culture supernatants using antibodies or protein G (Carter et al. 1992b), and scFv fragments of the human VH3 family can be purified with protein A (Hoogenboom & Winter 1992). However, we have generally used peptide tags located at the C-terminus of the fragments (see Fig. 2B); for example, a peptide tag encoding a portion of the cmyc protein and recognised by the monoclonal antibody (9E10) (Munro & Pelham 1986). (The peptide provides a link to the pill protein when the fragment is displayed on phage). The peptide tag allows detection of antibody fragments in ELISA or Western blots, and the purification of large amounts of antibody fragments directly from bacteria! culture medium on columns of immobilized 9E10 antibody (Clarkson et al. 1991a, Marks et al. 1991a). Other C-terminal tags can also be used, for example six histidine residues (Hochuli et al. 1988, Skerra et al. 1991), or a peptide binding to streptavidin (Skerra, personal communication).

MAKING V-GENE REPERTOIRES In the immune system, recombination ofa limited number of V-gene segments (VH, D and JH; V^ and J^; V« and J J and pairing of different rearranged VH and V^ genes generates a large number of different antibody structures. In vivo., antibodies are selected from this primary library by antigen. To create a repertoire of structures for phage display, we can clone the rearranged V-genes provided by a population of lymphocytes. In principle, we could try to retain the pairings of heavy and light chain in each lymphocyte ofthe population, or take the repertoire of heavy chains and combine it with the repertoires of light chain, so scrambUng the original VH and VL combinations ofthe lymphocyte. The rearranged V-genes could be derived from immune or non-immune sources, mRNA or genomic DNA, from IgG or IgM heavy chains, K or A light chains, memory or plasma cells, and are likely to contain both unmutated and somatically mutated V-genes. As an alternative to using repertoires


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of rearranged V-genes made in vivo, we could create synthetic repertoires of V-genes rearranged in vitro. Here, we describe the technologies for making the heavy and light chain repertoires from rearranged V-genes, for linking the chains together and for making synthetic V-gene repertoires. Repertoires from rearranged V-genes Rearranged V-genes have been cloned from genomic DNA or cDNA of hybridomas, and expressed in mammalian cells (Neuberger 1983, Ochi et al. 1983, Oi et al. 1983) or bacteria (Better et al. 1988, Skerra & Pluckthun 1988). Likewise, rearranged V-genes ofa hybridoma have been amplified by the polymerase chain reaction (PCR, Saiki et al. 1985), using degenerate PCR primers based on an Nterminal sequence analysis ofthe hybridoma protein (Larrick et al. 1988). However, the key step towards making expression repertoires of rearranged V-genes came from the use of "universal" PCR primers, suitable for amplifying a range of heavy and light chain V-genes, and the cloning of the amplified DNA directly into expression vectors (Orlandi et al. 1989). To amplify a target sequence by PCR, the primers must anneal to sequences at each end of the target (Saiki et al. 1985). For Tg V-genes, the sequences of the J-segments are sufficiently conserved to allow the design of (forward) PCR primers for copying of the sense strand, as used for ftrst strand cDNA synthesis. It emerges that the nucleotide sequence at the 5' end of the exon encoding the Location of PCR primers in V^ genes unrearranged garm line segmsnts rearranged DNA B-fymphocy(B mflNA

Cloned germ line \ sagmeni

J_p "

D J

VH gene repertoires from B-iymphocytes

Synthetic V-gene repertoires from VH segments

Figttre 3. Location of PCR primers in VH-genes. Universal primers designed to anneal to the 5' end of the VH segment and in the J-segments, and useful for PCR amplification from both rearranged DNA and eDNA from B-lymphocytes are marked with thiek arrows. Other primers (thin arrows) anneal to the leader peptide sequence and immunoglobulin constant regions, and are useful for PCR from eDNA. Equivalent primer locations are used for amplifieation of VL-genes. For construetion of synthetie V-gene repertoires, the forward primer anneals to the 3' end ofthe unrearranged V-segment and earries the genetic information encoding CDR 3 and framework 4.

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V-genes is also sufTidently conserved to allow the design of degenerate "universal" (back) primers for copying of the antisense strand (Orlandi et al. 1989) (Fig. 3). These PCR primers were used to amplify the heavy and light chain V-genes from RNA of five mouse hybridomas, and contained restriction sites to allow the cloning ofthe amplified DNA directly into expression vectors. By this means, the VH and V^ cDNA of the MBrl hybridoma (directed against a human mammary carcinoma line) was amplified, cloned and expressed in mammalian cells (Orlandi et al. 1989). The primers were then used to produce a repertoire of VH genes. Rearranged VH genes from the genomic DNA of mouse splenoeytes were amplified and cloned into a plasmid vector to express a repertoire of VH domains in bacteria (Ward et al. 1989). The diversity of the repertoire was shown by sequencing 48 clones: most of the mouse VH gene families were present (Gussow et al. 1989, Ward et al. 1989). We also designed sets of PCR primers for amplification of human Vgenes based on the available sequences of each family of human VH genes, V^ genes and Vx genes. The "family-based" primers were used to amplify the VH genes from the mRNA of nine human hybridomas (Bye et aj. 1992) and to produce a repertoire of VH, V^, and V; genes from human peripheral blood lymphocytes. The diversity of the repertoire was demonstrated by sequencing 52 VH clones and 32 V^ clones in which all human VH families (apart from VH6), and all V^ families were represented (Marks et al. 1991a, 1991b). Other sets of PCR primers have emerged, either as degenerate primers or a mixture of unique primers, and with back primers based in the leader sequence or the V-gene exon, and forward primers in the J-segment or constant regions, for example, for amplification of the V-genes of mouse (Jones & Bendig 1991, LeBoeuf et al. 1989, Sastry et al. 1989) or human (Larrick et al. 1989a) hybridomas, or mouse (Huse et al. 1989) or human (MulHnax et al. 1990, Persson et al. 1991) lymphocyte populations. Primers based at 5' and 3' ends of the exon encoding the V-domains may be used for amplification of cDNA or genomic DNA (Orlandi et al. 1989, Ward et al. 1989), for direct cloning into vectors for expression in mammalian cells (Orlandi et al. 1989) or bacteria (Ward et al. 1989) of Fv fragments, scFv fragments, Fab fragments or complete antibodies. Other PCR primers are less general. For example forward primers based in the constant domains are not suitable for amplification of genomic DNA as the V and C genes are separated by a large intron; back primers based in the Ig signal peptides would require the expression of antibody fragments in bacteria using the Ig signal sequences (rather than a bacterial signal sequence) (Fig. 3). Linking the V-genes together PCR amplification of the V-genes from cloned hybridomas (Orlandi et al. 1989) or single hybridoma cells (Larrick et al. 1989b) allows us to retain and express


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the original pairings of heavy and light chains of the antibody. The original pairings of heavy and light chains are lost when repertoires of rearranged Vgenes are prepared from heterogeneous populations of lymphocytes. However, the separate heavy and light chain repertoires can be recombined to create combinatorial repertoires in which the pairings are scrambled. For example, by cloning mouse heavy and light chain gene repertoires separately into the left and right arms of a modified lambda zapll vector, and recombining the arms, a "random combinatorial" library of Fab fragments was created, and expressed in bacteria (Huse et al. 1989) (Fig. 4A). PCR "splicing by overlap extension" (Ho et al. 1989) offers a more general way of linking the heavy and light chains. We used the process to assemble (Clackson et al. 1991a) the V-genes of mouse (Clackson 1991b) and human (Marks 1992b) hybridomas for expression of soluble scFv fragments and Fab fragments in bacteria, and to create random combinatorial libraries of scFv fragments displayed on the surface of filamentous phage (Clackson et al. i991a, Marks et al. 1991a). The V-genes were amplified by PCR and the repertoires combined with 'linker' DNA, which has regions of sequence homology with the 3' end of the amplified VH gene and 5' end of the amplified V^ gene. The 'linker' provides the sequence between the genes; for example, for expression of scFv fragments it would encode the (Gly^Ser), linker peptide. A further PCR amplifi-

Linking the V-genes logether A. Parallel or sequential cloning

B. PCR assembly

B - ly mphocy I w

B- ry mphocy tes

Scrambled pairings

Scrambled pairings

C. In-cell PCR assembly B-lymphe«yies

Originai pairings

Figure 4. Linking the V-genes together. Panel A; parallel (Huse et al. 1989) or sequential (Barbas el al. 1991) cloning of the amplified V^ and V^ repertoires. Panel B: VH and VL repertoires are assembled using PCR and both chains cloned simultaneously (Clackson et al. 1991a). Panel C: VH and V^ genes are amplified and assembled within individuals cells (Embleton et al. 1992). V-genes (large boxes); restriction sites (small boxes).

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cation v^ith outer flanking PCR primers splices together the two repertoires and, by including restriction sites appended to the 5' ends of the flanking primers, the assembled genes are readily cloned for expression (Fig. 4B). Furthermore, the use of PCR assembly suggests a way of retaining the heavy and light chain pairings of each lymphocyte. We have amplified the V-genes within a single cell by PCR (Haase et al. 1990), and linked them together by PCR assembly (Embleton et al. 1992). As a model, cells of two hybridomas were mixed together, and fixed and permeabilized to stabilize the cells and allow the access of reagents and primers. The mRNA was reverse-transcribed within the cell to cDNA, and the cDNA amplified by PCR. To facilitate linking of the Vgenes within the same cell, the PCR primers for the 3' end of the VH gene and the 5' end of the VL gene were complementary, to allow splicing of the VH and VL genes together. The cells were washed to remove amplified (and linked) DNA from their surface or outer layers, then subjected to a second PCR using "nested" primers to amplify those V-genes assembled within individual cells (Fig. 4C). After cloning the assembled V-genes, the heavy and light chain pairings were shown to correspond to those ofthe parent hybridomas (Embleton et al. 1992). This approach may allow the construction of repertoires of V-genes in which the original pairings of heavy and light chains of each B lymphocyte in a heterogeneous population of lymphocytes are retained, or at least preferentially linked.

Synthetic V-gene repertoires To make entirely synthetic V-gene repertoires, we made a bank of human V-gene segments for assembly into "synthetic" rearranged V-genes. Each Vj, segment is flanked at the 3' end by two recombination signals consisting ofa highly conserved heptamer, a 23-base pair spacer and a less well conserved nonamer. We designed family-specific primers (one for each of the six human VH families) for the PCR amplification of VH segments, based on the heptamer at the 3' end of the VH exon (forward primers), and regions of the leader exon or intron at the 5' end (back primers). By cloning and sequencing the amplified VH segments from the genomic DNA of a single individual, we made a bank of 52 cloned VH segments with open reading frames (Tomlinson et al. 1992). Each human VH segment encodes a 5' signal sequence and 94 or 95 amino acid residues of the mature domain, including the first two CDRs. The third CDR is generated from the recombination of these segments with about thirty D and six J segments, and provides most of the sequence diversity of rearranged VH genes. We therefore created a hypervariable sequence for CDR3 using an oligonucleotide primer that includes random sequence. The repertoire of synthetic rearranged VH genes was assembled by PCR from the bank of rearranged VH segments using the primer (Hoogenboom & Winter 1992) (Fig. 3). Such VH repertoires can be linked with a light chain, or light chain repertoires as described above.


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BY-PASSING HYBRIDOMAS With hybridoma technology, the B-lymphocytes making an immune response to antigen are immortalized. By contrast, PCR can immortalize the rearranged Vgenes not only from immune lymphocytes, but also from plasma cells, which are rich in mRNA and do not fuse in hybridoma technology. Immunization leads to the cellular proliferation and production of mRNA in the plasma cells. As a result, V-gene repertoires prepared from mRNA (Huse et al. 1989) are enriched for sequences of heavy and light chains encoding part of an antigen-binding site. For example, a repertoire of VH genes was prepared from either the DNA or mRNA of the splenoeytes of mice immunized with the hapten NP and expressed along with a V^l light chain as Fv fragments. (The primary and early secondary response to NP is restricted, since almost all antibodies use the V^l light chain (Cumano & Rajewsky 1985).) As expected, the frequency of binding clones was much higher (> 50-fold) in the repertoire prepared from mRNA than from DNA (Hawkins & Winter 1992a). Alternatively, V-gene repertories can be derived from the DNA of memory B cells by preselecting splenoeytes with antigen before PCR amplification. When a library of Fv fragments was prepared in this manner from a mouse immunized with NP (as described above), the frequency of binding clones was similar to the frequency obtained with libraries prepared from RNA, but the sequences of the binders were more diverse and more typical of the later immune response to this hapten. This suggests that the DNA repertoire from the antigen-selected cells was derived in part from cells destined for the memory compartment. Given their long life, memory cells may prove a useful source of V-gene repertoires in the absence of recent immunization (Hawkins & Winter 1992a). V-gene repertoires can also be derived from lymphocytes from difTerent compartments, for example from the bone marrow or spleen (enriched in plasma cells), from peripheral blood lymphocytes, or from different antibody isotypes (IgM or IgG, K or X light chains) by choice of specific constant region primers for cDNA synthesis or PCR. For example, to take advantage of immunization, we derived the V-genes from the IgG mRNA of splenoeytes (Clackson et al. 1991a). Antihodies from immune repertoires Lambda phage was first used to express a random combinatorial library of Vgenes from hyperimmunized mice. The phage plaques (90000) were screened on filters with a transition state analogue to identify Fab fragments with binding activities (1/10000 plaques) (Huse et al. 1989). As shown later, the fragments had binding affinities in the micromolar range (apparent Ka=10^-10' M*') (Kang et al. 1991b). It is unlikely that the original heavy and light chain pairings

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of each B cell are present in small combinatorial libraries derived from a large number of heavy and light chain genes, especially from a hyper-immune response in which the sequences are extensively diversified. Most of the pairings with binding activities are likely to be artificial, and since the original B-cell pairings are selected by the immune system according to their afTinity of binding, many of the artificial pairs are also likely to have lower affinity (but some could have an improved affinity). However in a very large library (> 10^ members), some of the original pairings should be present (Winter & Milstein 1991). Phage selection should allow the recovery of original pairings from a large library, and many other artificial pairings, including those with improved affinities. In contrast to the filter screening of random combinatorial libraries, where only a small number of fragments was isolated (Caton & Koprowski 1990, Huse et al. 1989, Kang et al. 1991b, Mullinax et al. 1990, Persson et al. 1991), we were able to isolate many antibody fragments by using phage display and selection, even from a library of only 2 x 10^ clones (Clackson et al. 1991a). The repertoires of VH and V^ genes were amplified from the spleen mRNA of mice immunized with the hapten phOx, assembled by PCR, and cloned to create a diverse repertoire of scFv fragments expressed on the surface of fd phage. The repertoire was selected for antigen-binding phage by passing down a hapten affmity column. The sequences of 23 ofthe hapten-binding phage clones revealed 8 different VH sequences in combination with 7 difTerent V^ sequences (producing 14 different combinations): most of the VH and V^ domains were "promiscuous", and able to bind hapten with any of several partners. Indeed new binding combinations could be forced by taking one ofthe pairs, and recombining the VH gene or V^r gene with repertoires of partner chains: the VH gene elicited 16 new VK partners, and the WK gene elicited 14 new VH partners. The sequences of the VH and V^. genes in the original or "hierarchical" libraries were related to those seen in the phOx response in hybridomas, and the affmity of binding (Kd=1.0x 10"" M) ofa soluble scFv proved comparable to that of hybridomas from the secondary immune response to phOx. However, the VH and V^ pairings were different from those in hybridomas produced from the same immunized spleens (Clackson et al. I991a, Gherardi & Milstein 1992). Fab fragments have also been selected from immune repertoires displayed on phage (Burton etal. 1991, Zebedee etal. 1992). It has been suggested that random combinatorial libraries of V-genes can be used to "report on the current antibody response ofthe donor" (Burton et al. 1991) and to clone the "fossil record" (Lemer et al. 1991). Although such libraries should certainly be enriched for either heavy or light chains of the response, it seems unlikely that the original V-gene combinations of the immune response, if present, could be distinguished from artificial combinations that encode binding activities. Ideally the V-gene combinations ofthe immune lymphocytes would be retained, for example by assembly ofthe V-gene combinations within the eel! (Embleton et al. 1992). In conclusion, phage display of random combinatorial libraries from immun-


55

ized sources allows the ready isolation of numerous fragments with good affmities, but does not seem ideal for analysis of an immune response. BY-PASSING IMMUNIZATION In principle it should be possible to construct a phage display library to mimic the immune repertoire: phage clones would be selected by panning the same library with any desired epitope. This could by-pass immunization with foreign antigens, and allow the isolation, for therapy, of human antibodies against selfantigens, However the probability of isolating a clone is dependent on the size of the library, its diversity, and the threshold binding affinity (Perelson 1989, Perelson & Oster 1979): larger libraries should lead to the isolation of antibodies against more epitopes on individual antigens and also to antibodies of higher affmities (Perelson 1989, Perelson & Oster 1979). For immune systems, the repertoire size has been estimated as 10^-10' in tadpoles (Du Pasquier 1973, Du Pasquier & Haimovitch 1976), and between 10^ and 5 x 10' in mice (Holmberg et al. 1986, Klinman & Press 1975). Typically 1/ 10' B-cells recognise a particular epitope with an afiinity greal enough to stimulate B-ceil proliferation (Klinman & Press 1975). It is possible to make phage libraries of comparable size (10^-10* clones). At present the size ofthe phage library is limited by the transfection efficiency of bacteria, but in principle it could be increased by the use of two replicon libraries, where the heavy and light chain repertoires are encoded on two difTerent replicons, and are brought together in the same bacterium by infection (Hoogenboom et al. 1991c). After infection, the heavy and light chains could be combined onto the same replicon by in vivo recombination within the bacterium. The size of the primary antibody repertoire, the diversity and distribution of sizes and shapes of antigen-binding sites, and usage of difTerent germ-line V-gene segments have presumably evolved to match the likely universe of foreign antigens. We therefore made a large phage library (> 10' members) using repertoires of rearranged V-genes from "naive" lymphocytes to recreate the range and diversity of binding-site structures on natural antibodies. We also built a large phage library from defined V-gene segments and loops to create a "designer" universe of binding-site structures. Antibodies from "naive" repertoires We created a large "naive" repertoire (3 x 10' clones) by amplifying and cloning the V-genes derived from human cells (Marks et al. 1991a). To make a highly diverse library, we used human peripheral blood lymphocytes (so avoiding plasma cells and their mRNA), and made cDNA from the IgM heavy chains using a primer based in the /i constant regions. To maximize the diversity, PCR primers

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were designed for each of the families of human heavy and light chain {X and K) V-gene segments, and for the difTerent J-segments. The VH and VL gene repertoires were combined by PCR assembly to encode human scFv fragments on phage. The library was subjected to multiple rounds of affmity selection to ensure that even a single clone in the original library could be isolated. From the same library, antibody fragments against 12 different antigens were isolated. Binding activities were identified against "foreign antigens", including phOx, turkey egg-white lysozyme (TEL), bovine serum albumin (Marks et al. 1991a) and bovine thyroglobulin. Furthermore, scFv fragments against a variety of "self antigens" were isolated, including human thyroglobulin, a human monoclonal antibody (Fogl), and human lumor-necrosis factor-a (TNFa), the cellsurface markers carcinoembryonic antigen and mucin (selected with soluble antigen), and the human blood group B, D and E antigens (selected on antigenpositive cells) (unpublished data). For some antigens, many different scFv fragments were isolated, for example seven against TNFa and 12 against bovine thyroglobulin. Most of the scFv fragments isolated from the library were highly specific to the antigen used in panning (Fig. 5). For example, the scFv fragments isolated using TEL did not bind to a range of other protein antigens, including hen eggwhite lysozyme, which differs by only a few amino acids. This contrasts with the poor specificities of murine Fab fragments isolated from a naive library displayed on pVIII (Gram et al. 1992). scFv fragments were also raised against multiple epitopes on a single antigen: of three different antibodies isolated against Fogl (a human y\ IK antibody), one bound to the heavy chain constant region and the two others to the Fogl idiotype (Fig. 5; unpublished data). The sequences ofthe V-genes of the binders demonstrated the use of many families of V-genes, both germ line and mutated. The affinities of two of the soluble antibody fragments, specific for phOx and turkey egg-white lysozyme, were determined by fluorescence quench titration as K3 = 2x 10^ M " ' and 10' M"' respectively. The affinities are typical of monoclonal antibodies produced after primary immunization (Foote & Milstein 1991, Hughes-Jones et al. 1991). As expected, the affmities and numbers of scFv fragments binding to each antigen from the "naive" repertoires are lower than those from immunized sources. However, the naive repertoires already offer a way of making human antibody fragments with good specificities against self-antigens, and the affmities can be improved; see below and (Marks et al. 1992a). , I Antibodies from synthetic repertoires The ^-sheet framework structure of antibodies seems attractive for the de novo design of binding sites, as loops can be transplanted from one framework to another (Jones et al. 1986). Indeed, it might be possible to design repertoires of

BUILDING ANTIBODIES FROM THEIR GENES Anti-Human TNF

Anti-Bovlne Thyroglobulin

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Figure 5. Specificity of soluble single-chain Fvs (scFvs) isolated from a human "naive" repertoire. Panels A and B: specificity ofa scFv fragment for TNFa (Panel A) and bovine thyroglobulin (Panel B). (TTie panel of lest proteins is 1, plastic; 2, hen egg trypsin inhibitor; 3, chymotrypsinogen A; 4. hen egg ovalbumin; 5, keyhole limpet haemocyanin; 6, bovine thyroglobulin; 7, human TNFa; 8, turkey egg-white lysozyme; 9, horse heart cytochrome c; 10, bovine serum albumin.) Panels C and D show the binding of two scFvs, isolated by selection on a human monoclonal antibody Fogl (yl, K), to a panel of human antibodies of varying isotype (see Marks 1992b and Bye et al. 1992 for reference to these antibodies). The panel of test antibodies is 1, Fogl (IgGl, K); 2, the Fv fragment of Hulysll (Foote & Winter 1992); 3, Hulysll antibody, (IgGl, K); 4, RegA (IgGl, K); 5, FogC (IgG3, *c); 6, Pagl (IgGl. A); 7, myeloma agG2, /L); 8. Oak3, (lgG3,1); 9. myeloma (IgG4, A); 10. Foml (IgM, X); II, FomA (IgM, X). Clone H6 binds to the Fogl idiotope, whereas clone A4 binds to a common epitope found on the heavy chain constant regions of IgGl, 2 and 3. but not on IgG4 or IgM.

antibodies with binding sites broadly complementary to a defined universe of antigens, for example to haptens, peptides or carbohydrates. Such "designer" repertoires, enriched in complementary structures, might allow the creation of antibody fragments binding to defmed epitopes on an antigen, or lead to fragments with high binding affmities. Furthermore, diversity could be focussed to order, in the CDRs or in framework residues that may "fine tune" the conformation of CDRs (Chothia & Lesk 1987, Tramontano et al. 1990, Foote & Winter

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1992). However, design requires that the encoding V-gene repertoires are built from defined structural elements, such as ^-sheet and loops, or indeed from defined human V-gene segments. A range of structures is Implicit in the human VH gene segments. The diversity for antigen binding is fixed by about 50 groups of segments, each group encoding identical CDRs (1 and 2) (Tomlinson et al. 1992). However, the main chain conformation of the loop structures of CDRs 1 and 2 are more limited, with three folds for CDRl and five folds for CDR2, and together forming seven sets of loop structures (Chothia et al. 1992). V H - C D R 3 multiplies the structural diversity. It is fashioned by recombination of D- and J-segments, and with Nsegment addition, is highly variable in length (from 4 to 25 residues) and sequence (Sanz 1991) and structure (Chothia et al. 1992). Furthermore, it is located in the center ofthe antigen-binding site (Chothia & Lesk 1987). We created a repertoire of rearranged V^ genes from defmed VH gene segments and CDR3 loops (Hoogenboom & Winter 1992). Using each of 49 cloned VH gene segments (Tomlinson et al. 1992), we introduced a synthetic V H - C D R 3 of five or eight residues by PCR. The five-residue CDR3 (all five random) is close to the minimal size for this loop, and does not include the salt bridge from AsplOl ofthe loop to Arg 94 ofthe framework. In the eight-residue CDR3, the salt bridge was retained by including three residues from the human 1^4 segment (Phel00-AspI01-Tyrl02). The repertoire of "in vitro rearranged" VH genes was then combined with a single V; light chain. Although we exploited the entire structural repertoire of the VH segments, there was no light chain diversity and the lengths of CDR3 were restricted and short. The repertoire was displayed on phage, and a range of scFv fragments isolated from phage after rounds of panning with two haptens, phOx and 3-iodo-4hydroxy-5-nitrophenyl-acetate (NIP). Most were encoded by germ line segments ofthe human VH3 family, but there were a few examples of V^l and VH4 families. Together, the segments used three of the seven folds available to the first two CDR loops (Chothia et al. 1992). The 20 phOx binders used only eight-residue CDR3 and mainly the germ line DP-47 segment with related sequences in CDR3 (a branched aliphatic residue in the first position, and an aromatic residue in the fourth). The 10 hflP binders used both five- and eight-residue CDR3s and mainly the germ line DP-38 segment with a central glycine in the five-residue CDR3. One segment (DP-45) was seen in two clones binding to phOx and two clones binding to NIP, indicating that antigen-binding sites with different specificities can be created on the same antibody framework by substitution of CDR3 alone (see also Barbas et al. 1992). In all cases the binders were highly specific for the antigen, and for both phOx and NIP the best binders had affinities in the micromolar range and were comparable to affinities of antibodies for these haptens in a primary immune response (Cumano & Rajewsky 1985). However, it was more difficult to isolate


551

fragments with binding activities to protein antigens from this phage library. The use of highly related C D R 3 sequences in the hapten-binding fragments suggests that the repertoire may have been over-represented for hapten binding sites, at the expense of sites for other antigens. We suspect that synthetic libraries with short V H - C D R 3 and confined to a single light chain poorly recreate the diversity of shapes needed to recognise a wide range of antigens. To extend the range of antigens recognised by synthetic libraries, we could increase the length o f t h e VHC D R 3 , use a repertoire of light chains, and increase the library size. However for a repertoire suited to binding the entire universe of antigens, it may be simpler to use repertoires of V-gene rearranged in vivo.

Antibodies by affinity

maturation

The affinities of binding of antibody fragments derived from the primary phage repertoires (Marks et al. 1991a, Hoogenboom & Winter 1992) seem to lie in the micromolar range (Ka = IO^ M " ' - 1 0 ' M " ' ) and are characteristic of primary immune responses. In principle we could mimic the immune system to improve the affinities of binding of these antibodies, and subject the V-genes to random somatic mutation, and select the mutants with improved affinity. Somatic mutation at a few sites can lead to large increases (10- to 200-fold) in the affinity of binding (Allen et al. 1988, Sharon 1990), but for many antibodies the affinity appears to increase in small stepwise increments during sequential rounds of mutation and selection of B-cell clones (Kocks & Rajewsky 1988). As a model, the V-genes of an anti-hapten scFv derived from the primary response hybridoma BI.8 were mutated (1.7 mutations per VH gene) by using reaction conditions which decrease the fidelity of Taq polymerase (Leung et al. 1989). The mutant scFv repertoires were cloned into vectors for display on the surface of phage and a mutant with a 4-fold improved affinity for hapten isolated after many rounds of selection (Hawkins et al. 1992b). The increase in affinity is comparable to that seen for the BI-8 antibody during the secondary immune response, and suggests that it may be difficult to improve the affinity of this antibody by single point mutations. Single point mutations could also be introduced inlo the selected V-genes by growth of the phage in an E. coli mutD strain in which the spontaneous mutation frequency is 10^-10^ times higher than in wild-type strains (Schaaper 1988, Yamagishi et al. 1990). Multiple point mutations would be more readily introduced by spiked oligonucleotides (Hermes et al. 1989), and could even be focussed into "hot-spots", such as key residues of the C D R s or framework residues that "fine tune" the conformation of the CDRs (Foote & Winter 1992). However, it is also possible to harness the somatic mutation of a natural immune system, by shuffling the loops or entire heavy or light chains with a repertoire of in vivo somatically mutated V-genes (Marks et al. 1992a). The

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structure of a scFv fragment (Q:phOx-15) derived from the naive phage library (and with moderate affinity for phOx, Kd = 3 x 10"' M), was diversified by first shuffling light chains, then heavy chains. However, to avoid disrupting key features of antigen binding, the V H - C D R 3 of the heavy chain was retained. We isolated a mutant scFv fragment (aphOx-B2) with a 20-foId improved affinity on shuffling the light chain of aphOx-15 and, on shuffiing the heavy chain of aphOxB2, improved its afTmity 16-fold, leading to a 320-fold improvement in the affinity compared to the original scFv fragment. The affinity of binding (Kj = 1.1 x 10"' M) is comparable to the affinities of mouse hybridomas from the tertiary immune response to the same hapten (Foote & Milstein 1991). In the mutant scFv fragments, both the light and heavy chains were derived from the same gennline genes as the parent ctphOx-15 and differed only by multiple point mutations. Nevertheless, the repertoire of chain-shufTled mutants differs from those generated by random mutation in two respects. Firstly, the Vgenes encoding the shuffled chains are derived from the mRNA of B lymphocytes and are likely to be functional. In contrast, in vitro random mutagenesis is likely to result in many mutants that would compromise chain folding, particularly if multiple mutations are introduced into the same gene. Secondly, with in vitro mutagenesis, mutations are introduced directly into the DNA of the clone to be affinity matured whereas, with chain swapping, mutations are introduced into the corresponding germline genes. This could allow any deleterious mutations in the original clone to be replaced more readily. Chain shuffling also appears to have wider applications. For example, by shuffiing the heavy and light chains of a mouse monoclonal antibody directed against TNFa with repertoires of human chains, we created an entirely human antibody with a similar specificity (unpublished data). If general, this may offer an alternative to CDR grafting as a means of humanizing rodent antibodies. , CONCLUSIONS The V-genes encoding antigen-binding sites can be used to build complete antibodies, antibody fragments and fusions with other proteins with potential in medicine and industry. The V-genes can be derived from hybridomas or, as described in the review, by mimicking the strategies of the immune system. Vgene repertoires are cloned for expression as a fusion to the pIII coat protein of filamentous phage. The display of antibody fragments on the surface ofthe phage allows antibodies (and their encoding V-genes) to be selected by afiinity or kinetics of binding to antigen; and the fragments are produced in a soluble form by infecting bacteria with the phage. Repertoires of V-genes have been made: (1) from the rearranged V-genes of populations of immune lymphocytes (mouse splenoeytes). Many difTerent antibody fragments were isolated with good specificities and affinities to the immunizing antigen. However, the pairings


61

of heavy and light chains did not seem to correspond to those ofthe immune lymphocyte. The assembly of V-genes within the same cell by PCR may offer a means of retaining the combinations and help in analysis of the V-gene pairings of an immune response. (2) as above but from human peripheral blood lymphocytes of an unimmunized donor. The antibody fragments isolated had good specificities to the selecting antigen, but only moderate affinities. In most cases at least one clone could be isolated with each selecting antigen, and fragments directed to 12 antigens, including several human self antigens and human cell surface markers, were isolated from the same library. To make higher affinity fragments or increase the numbers of fragments binding to each antigen, it will be necessary to increase the size of the libraries and their diversity. (3) from a synthetic V-gene repertoire derived from germ line V-gene segments. Many different human antibody fragments, and often utilizing different germ line segments, were isolated against two haptens. Again, these fragments had good specificities but only moderate affinities. For affinity maturation of the fragments on phage, it was possible to harness the somatic mutation in rearranged V-genes from lymphocytes by chain shuffiing. In this way, the affinity of a human anti-hapten fragment derived from the naive library was increased 300-rold, from K^ = 3x 10* M"' to 1.1 x 10' M"'. This showed that antibody fragments with high affinities, similar to those of hybridomas, can be made without immunization. We conclude that "repertoire selection" technologies, by mimicking the strategies of the immune system, can allow the creation of antibodies with high specificities and affinities of binding, without use of hybridomas or immunization. This will be particularly useful for making human antibodies for therapy, especially for those directed against self-antigens. The technologies seem to be leading towards a huge and diverse phage library - a Holy Grail - that provides an everlasting supply of useful antibody fragments on panning with antigen.

ACKNOWLEDGMENTS H.R.H. was supported by the D. Collen Research Foundation, Leuven, and the European Molecular Biology Organisation, A.D.G. by the Cancer Research Campaign, and J.D.M. by the MRC and the MRC AIDS Directed Programme.

REFERENCES Allen, D.. Simon, T., Sablitzky, F.. Rajewsky. K. & Cumano, A. (1988) Antibody engineering for the analysis of afTinity maturation of an anti-hapten response. EMBO J. 7. 1995. ArufTo, A. & Seed. B. (1987) Molecular cloning of two CD7 (T-cell leukemia antigen) cDNAs by COS cell expression system. EMBO J. 6, 3313.

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Barbas, C. F., Bain, J. D., Hoekstra, D. M. & Lerner, R. A. (1992) Semisynthetic combinatorial antibody libraries: A chemical solution to the diversity problem. Proc. Natl. Acad. Sci. USA 89, 4457. Barbas, C. F , Kang, A. S., Lerner, R. A. & Benkovic, S. J. (1991) Assembly of combinatorial antibody Hbraries on phage surfaces: The gene III site. Proc. Natl. Acad. Sci. USA 88, 7978. Better, M., Chang, C. P., Robinson, R. R. & Horwitz, A. H. (1988) Escherichia coli secretion of an active chimeric antibody fragment. Science 240, 1041. Bird, R. E., Hardman, K. D., Jacobson, J. W., Johnson, S., Kaufman, B. M., Lee, S. M., Lee T, Pope, S. H., Riordan, G. S. & Whitlow, M. (1988) Single-chain antigen-binding proteins. Science 242, 423. Boeke, J. D., Model, R & Zinder, N. D. (1982) Effects of bacteriophage fl gene III protein on the host cell membrane. Mol. Gen. Genet. 186, 185. Bossiet, K., Czech, J., Lorenz, P., Sedlacek, H. H., Schuermann, M. & Seemann, G. (1992) Molecular and functional characterisation ofa fusion protein suited for tumour specific prodrug activation. Br. J. Cancer 65, 234. Boulianne, G. L., Hozumi, N. & Shuiman, M. J. (1984) Production of functional chimaeric mouse/human antibody. Nature 312, 643. Breitling, S. D., Seehaus, T, Klewinghaus, L & Little, M. (1991) A surface expression vector for antibody screening. Gene 104, 147. Bruggemann, M., Williams, G. T, Bindon, C. I., Qark, M. R., Walker, M. R., Jefferis, R.. Waldmann, H. & Neuberger, M. S. (1987) Comparison of the effector functions of human immunoglobulins using a matched set of chimeric antibodies. J. Exp. Med 166, 1351. Burton, D. R., Barbas, C. F., Persson, M. A. A., Koenig, S., Chanock, R. M. & Lerner, R. A. (1991) A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial of asymptomatic seropositive individuals. Proc. Natt. Acad Sci. USA 88, 10134. Bye, J. M., Carter, C , Cui, Y., Gorick, B. D., Songsivilai, S., Winter, G., Hughes-Jones, N. C. & Marks, J. D. (1992) Germline variable region gene derivation of human monoclonal anti-Rh(D) antibodies: evidence for affinity maturation by somatic hypermutation and repertoire shift. J. Ctin. Invest, (in press). Carter, P., Kelley, R. F., Rodrigues, M. L,, Snedocor, B., Covarrubias, M.. Velligan, M. D., Wong, W. L. T, Rowland, A. M., Kotts, C , M. E., C , Yang, M., Bourell, J. H.. Shepard, H. M. & Henner. D. (1992h) High level Escherichia coli expression and production ofa bivalent humanized antibody fragment. Bio/Technology 10, 163. Carter, P., Prestal, L., Gorman, C. M., Ridgway, J. B., Henner, D., Wong, W. L. T., Rowland, A. M., Kotts, C , M. E., C. & Shepard, H. M. (1992a) Humanization of an anti-pl85"^'''antibody for human cancer therapy. Proc. Natt. Aead. Sci. USA 89,4285. Caton, A. J. & Koprowski, H. (1990) Influenza virus hemagglutinin-specific antibodies isolated from a combinatorial expression library are closely related to the immune response of the donor. Proc. Natt. Acad. Sci. USA 87, 6450. Chang, C. N., Landolfi, N. F. & Queen, C. (1991) Expression of antibody Fab domains on bacteriophage surfaces. J. Immunology 147, 3610. Chothia, C. & Lesk, A. M. (1987) Canonical structures for the hypervariable regions of immunoglobulins. J. Mot. Biot. 196, 901. Chothia, C , Lesk, A. M., Gherardi E., Tomlinson, I. M., Waiter, G., Marks, J. D., Llewelyn, M. B. & Winter, G. (1992) The structural repertoire ofthe human VH segments. J. Mol. Biot. 227, 799. Clackson, T. P. (1991b) Antibody engineering using the polymerase chain reaction. PhD thesis, Cambridge University.


&

Clackson, T. & Winter, G. (1989) 'Sticky feet'-directed mutagenesis and its application to swapping antibody domains. Nucleic Acids Res. 17, 10163. Clackson, T., Hoogenboom, H. R.. Griffiths, A. D. & Winter, G. (1991a) Making antibody fragments using phage display libraries. Nature 352, 624. Co, M. S., Deschamps, M., Whitley, R. J. & Queen, C. (1991) Humanized antibodies for antiviral therapy. Proc. Natt. Acad Sci. USA 88, 2869. Cumano, A. & Rajewsky, K. (1985) Structure of primary anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antihodies in normal and idiotypically supressed C57BL/6 mice. Eur. J. Immunot. 15, 512. Cwirla, S. E., Peters, E. A., Barrett, R. W. & Dower, W. J. (1990) Peptides on phage: a vast library of peptides for identifying ligands. Proc. Natl. Acad. Sci. USA 87, 6378. Devlin, J. J., Panganiban, L. C. & Devlin, P. E. (1990) Random peptide libraries: a source of specific protein binding molecules. Science 249, 404. Du Pasquier, L. (1973) Ontogeny ofthe immune response in cold blooded vertebrates. Curr. Top. Microbiol. Immunot. 61, 37. Du Pasquier, L. & Haimovitch, J. (1976) The antibody response during amphibian ontogeny. Immunogenetics 3, 381. Duncan, A. R. & Winter, G. (1988) The binding site for Clq on IgG. Nature 332, 738. Duncan, A. R., Woof, J. M., Partridge, L. J., Burton, D. R. & Winter, G. (1988) Localization of the binding site for the human high-afiinity Fc receptor on IgG. Nature 332, 563. Embleton, M. J., Gorochov, G., Jones, P. T & Winter, G. (1992) Incell PCR from mRNA: amplifying and linking the rearranged immunoglobulin heavy and light chain V-genes within single cells. Nuct. Acids Res. 20, 3831. Felici, K, Castagnoli, L., Musacchio, A., Jappelli, R. & Cesareni, G. (1991) Selection of antibody Hgands from a large library of oligopeptides expressed on a multivalent exposition vector. / Mot. Biot. 222, 301. Foote, J. & Milstein, C. (1991) Kinetic maturation of an immune response. Nature 352, 530. Foote, J. & Winter, G. (1992) Antibody framework residues affecting the conformation of the hypervariable loops. J. Mol. Biot. 224, 487. Francisco, J. A., Earhart. C. F. & Georgiou, G. (1992) Transport and anchoring of ^lactamase to the external surface of Escherichia coti. Proc. Natl. Acad. Sci. 89, 2713. Fuchs, P., Breitling, F., Diibel, S., Seehaus, T & Little, M. (1991) Targeting recombinant antibodies to the surface of Escherichia coli: fusion to a peptidoglycan associated lipoprotein. Bio/technologv 9, 1369. Garrard, L. J., Yang. M., O'Connell, M. P., Kelley, R. F. & Henner, D. J. (1991) Fab assembly and enrichment in a monovalent phage display system. Bio/technology 9, 1373. Geysen, H. M., Rodda, S. J., Mason, T. J., Tribbick, G. & Schoofs, P G. (1987) Strategies for epitope analysis using peptide synthesis. J. Immunol. Methods 102, 259. Gherardi, E. & Milstein, C. (1992) Original and artificial antibodies. Nature 357. 201. Glaser-Wuttke, G., Keppner, J. & Rasched, I. (1989) Pore-forming properties of the adsorption protein of filamentous phage fd. Biochim. Biophys. Acta 985, 239. Glockshuber, R., Malia, M., Pfitzinger, 1. & PliJckthun, A. (1990) A comparison of strategies to stabilize immunoglobulin Fv-fragments. Biochemistry 29, 1362. Goldsmith, M. E. & Konigsberg, W. H. (1977) Adsorption protein of the bacteriophage fd: isolation, molecular properties, and location in the virus. Biochemistry 16, 2686. Gorman, S. D , Clark, M. R., Routiedge, E. G., Cobbold, S. P & Waldmann, H, (1991) Reshaping a therapeutic CD4 antibody. Proc. Natl. Acad. Sci. USA 88, 4181. Gram, H.. Marconi, L.-A., Barbas, C. F., Collet, T. A., Lerner, R. A. & Kang, A. S. (1992) In vitro selection and alTinity maturation of antibodies from a naive combinatorial immunoglobulin library. Proc. Natl. Acad. ScL USA 89, 3576.

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HOOGENBOOM ET AL.

Greenwood, J., Willis, A. E. & Perham, R. N. (1991) Multiple display of foreign peptides on a filamentous bacteriophage: Peptides from Plasmodium falciparum circumsporozoite protein as antigen. J. Mol. Biot. 220, 821. Gussow, D. & Seemann, G. (1991) Humanization of monoclonal antibodies. Methods in Enzymology 203, 99. Gussow, D , Ward, E. S., Griffiths, A. D., Jones, P T. & Winter, G. (1989) Generating binding activities from Escherichia coli by expression of a repertoire of immunoglobulin variable domains. Cold Spring Harb. Symp. Quant. Biol. 1, 265. Haase, A. T, Retzel, E. E. & Staskus, K. A. (1990) Amplification and detection of lentiviral DNA inside cells. Proc. Natl Acad. Sci. USA 87, 4971. Hale, G., Dyer, M. J., Clark, M. R., Phillips, J. M., Marcus, R., Riechmann, L., Winter. G. & Waldmann, H. (1988) Remission induction in non-Hodgkin lymphoma with reshaped human monoclonal antibody CAMPATH-IH. Lancet 2, 1394. Hawkins, R. E., Russell, S. J. & Winter, G. (1992b) Selection of phage antibodies by binding affinity. Mimicking affinity maturation. J. Mot. Biot. lid, 889. Hawkins, R. E. & Winter, G. (1992a) Cell selection strategies for making antibodies from variable gene libraries: trapping the memory pool. Eur. J. Immunol 22, 867. Hermes, J. D , Parekh, S. M., Blacklow, S. C , Koster, H. & Knowles, J. R. (1989) A reliable method for random mutagenesis: the generation of mutant libraries using spiked oligodeoxyribonucleotide primers. Gene 84, 143. Ho, S. N., Hunt, H. D , Horton, R. M., Pullen, J. K. & Pease, L. R. (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51. Hochuli, E., Bannwarth, W, Dobeli, H., Gentz, R. & Stuber, D. (1988) Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Bio/Technology (,, 1321. Hofnung, M. (1991) Expression of foreign polypeptides at the Escherichia coli cell surface. Methods of Enzymology 34, 77. Holmberg, D , Freitas, A. A., Portnoi, D , Jacquemart, F., Avrameas, S. & Coutinho, A. (1986) Antibody repertoires of normal BALB/c mice: B lymphocytes populations defined by state of activation. Immunot. Rev. 93, 147. Hoogenboom, H. R., Griffiths, A. D., Johnson, K. S., Chisweil, D. J., Hudson, P. & Winter, G. (1991c) Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucteic Acids Res. 19, 4133. Hoogenboom, H. R., Raus, J. C. M. & Volckaert, G. (1991b) Targeting of tumour necrosis factor to tumor cells: secretion by myeloma cells of a genetically engineered antibodytumor necrosis factor hybrid molecule. Biochim. Biophys. Acta 1096, 345. Hoogenboom, H. R., Volckaert, G. & Raus, J. C. M. (1991a) Construction and expression of antibody-tumor necrosis factor fusion proteins. Mot. Immunot. 28, 1027. Hoogenboom, H. R. & Winter, G. (1992) Bypassing immunisation: human antibodies from synthetic repertoires of germ line VH-gene segments rearranged in vitro. J. Mot. Biot 227,381. Hughes-Jones, N. C , Gorick, B. D. & Beagle, D. (199!) Multiple epitopes recognized by human monoclonal IgM anti-D antibodies. Vox Sanguinis 59, 112. Huse, W. D. (1991) Combinatorial antibody expression libraries in filamentous phage. In: Antibody engineering. A practical approach, ed. Borrebaeck, C. A. K. W H. Freeman & Co, New York, p. 103. Huse, W D., Sastry, L., Iverson, S. A., Kang, A. S., Alting, M. M., Burton, D. R., Benkovic, S. J. & Lemer, R. A. (1989) Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science 246, 1275. Huston, J. S., Levinson, D , Mudgett, H. M., Tai, M. S., Novotny, J., Margolies, M. N., Ridge, R. J., Bruccoleri, R. E., Haber, E., Crea, R. & Opperman, H. (1988) Protein


65

engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc. Natl. Acad. Sci. USA 85, 5879. irichev, A. A., Minenkova, O. O., Tat'kov, S. I., Karpyshev, N. N., Eroshkin, A. M., Ofilserov, V. I., Akimenko. Z. A., Petrenko, V A. & Sandakhchiev, L. S. (1990) The use of filamentous phage MI3 in protein engineering. Mot. Biot. Mosk. 24, 530. Jones, P T, Dear, P H., Foote. J., Neuberger, M. S. & Winter, G. (1986) Replacing the complementarity-determining regions in a human antibody with those from a mouse.

Nature yi\,S22. Jones, S. T.& Bendig, M. (1991) Rapid PCR-cloning of full-length mouse immunoglobulin variable regions. Bio/Technotogy 9, 88. Kabat, E. A. & Wu, T. T. (1971) Attempts to locate complementarity-determining residues in the variable positions of light and heavy chains. Ann. N. Y. Acad. Sci. 190, 382. Kang, A. S., Barbas, C. F., Janda. K. D , Benkovic, S. J. & Lemer, R. A. (1991a) Linkage of recognition and replication functions by assembling combinatorial antibody Fab libraries along phage surfaces. Proc. Natl Acad. Sci. USA 88, 4363. Kang, A. S., Jones, T. M. & Burton, D. R. (1991b) Antibody redesign by chain shuffling from random combinatorial immunoglobulin libraries. Proc. Natl Acad. Sci. USA 88, 11120. Kettleborough, C. A., Saldanha, J., Heath, V. J., Morrison, C. J. & Bendig, M. M. (1991) Humanization of a mouse monoclonal antibody by CDR-grafting: the importance of framework residues on loop conformation. Protein Eng. 4, 773. Klinman, N. R. & Press, J. L. (1975) The B-cell specificity repertoire: its relationship to definable subpopulations. Transptant. Rev. 24, 41. Kocks, C. & Rajewsky, K. (1988) Stepwise intraclona! maturation of antibody affinity through somatic hypermutation. Proc. Natt. Acad. Sci. USA 85, 8206. Kohler, G. & Milstelin, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495. Kostelny, S. A., Cole, M. S. & Tso, J. Y. (1992) Formation ofa bispecific antibody by the use ofleucine zippers. J. Immunol. 148, 1547. Larrick, J. W, Chiang, Y L., Sheng-Dong, R., Senck, G. & Casali, P. (1988) Generation of specific human monoclonal antibodies by in vitro expansion of human B cells: a novel recombinant DNA approach. In: In vitro immunisation in hybridoma technology, eds. Borrebaeck, C. A. K. Elsevier Science Publishers B.V, Amsterdam, p. 231. Larrick, J. W., Danielsson, L., Brenner, C. A., Abrahamson, M., Fry, K. E. & Borrebaeck. C. A. K. (1989a) Rapid cloning of rearranged immunoglobulin genes from human bybridoma cells using mixed primers and the polymerase chain reaction. Biochem. Biophys. Res. Commun. 160, 1250. Larrick, J. W, Danielsson, L., Brenner, C. A.. Wallace, E. F., Abrahamson, M., Fry, K. E. & Borrebaeck, C. A. K. (1989b) Polymerase chain reaction using mixed primers: Cloning of human monoclonal antibody variable region genes from single hybridoma cells. Bio/Technology 7, 934. LeBoeuf, R. D, Galin, F S.. Hollinger, S. K., Peiper. S. C. & Blalock, J. E. (1989) Cloning and sequencing of immunoglobulin variable-region genes using degenerate oligodeoxyribonucleotides and polymerase chain reaction. Gene 82, 371. Lerner. R. A.. Barbas III, C F., Kang, A. S. & Burton, D. R. (1991) On the use of combinatorial antibody libraries to clone the "fossil record" of an individual's immune response. Proc. Natl Acad Sci. USA 88, 9705. Leung. D. W.. Chen, E. & Goeddel, D. V. (1989) A method for random mutagenesis ofa defined DNA segment using a modified polymerase chain reaction. Technique 1, II. Lowman, H. B., Bass, S. H., Simpson, N. & Wells, J. A. (1991) Selecting high-affinity binding proteins by monovalent phage display. Biochemistry 30, 10832.

66

HOOGENBOOM ET AL.

Lund, J., Winter, G., Jones, P. T , Pound, J. D.. Tanuka, T., Walker, M. R., Artymiuk, P. J., Arata, Y., Burton, D. R., Jefferis, R. & Woof, J. (1991) Human FcgRI and FcgRII interact with distinct but overlapping sites on human IgG. J. Immunot. 147, 2657. Maeda, H., Matsushita, S., Eda, Y., Kimachi, K., Tokiyoshi, S. & Bendig, M. M. (1991) Construction of reshaped human antibodies with HIV-neutralizing activity. Human Antibodies and Hybridomas 2, 124. Marks, J. D. (1992b) Making human antibodies in bacteria and phage. PhD thesis, C.N.A.A., MRC, Centre for Protein Engineering. Marks, J. D , Griffiths, A. D., Malmqvist, M., Clackson, T, Bye, J. M. & Winter, G. (1992a) By-passing immunization: building high affinity human antibodies by chain shuffiing. Bio/Technotogy 10, 779. Marks, J. D , Hoogenboom, H. R., Bonnert, T. P., McCafferty, J., Griffiths, A. D. & Winter, G. (1991a) By-passing immunization: Human antibodies from V-gene libraries displayed on phage. /. Mot. Biol. 222, 581. Marks, J. D., Tristrem, M., Karpas, A. & Winter, G. (1991b) Oligonucleotide primers for polymerase chain reaction amplification of human immunoglobulin variable genes and design of family-specific oligonucleotide probes. Eur. J. Immunot. 21, 985. Mathieson, P W., Kobbold, S. P, Hale, G., Qark, M. R., Oliveira, D. B. J., Lockwood, C. M. & Waldmann, H. (1990) Monoclonal antibody therapy in systemic vasculitis. New. Engl J. Med 323, 250. McCafferty, J., Griffiths, A. D., Winter, G. & Chisweil, D. J. (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348, 552. Milstein, C. (1990) Tlie Croonian lecture, 1989. Antibodies: a paradigm for the biology of molecular recognition. Proc. R. Soc. Lond. Biol. 239, I. Morrison, S. L. (1992) In vitro antibodies: strategies for production and application. Ann. Rev. Immunol. 10, 239. Mullinax, R. L., Gross, E. A., Amberg, J. R., Hay, B. N., Hogrefe, H. H., Kubitz, M. M., Greener, A., Alting, M. M., Ardourel, D. & Short, J. M. (1990) Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage lambda immunoexpresslon library. Proc. Natt. Acad. Sci. USA 87, 8095. Munro, S. & Pelham, H. R. B. (1986) An Hsp-like protein in the ER: Identity with the 78 kd glucose regulated protein and immunoglobulin heavy chain binding protein. Cell 46, 291. Neuberger, M. (1983) Expression and regulation of immunogiobulin heavy chain gene transfected into lymphoid cells. EMBO J. 2, 1373. Neuberger, M. S., Williams, G. T. & Fox, R. O. (1984) Recombinant antibodies possessing novel effector functions. Nature 312, 604. Ochi, A., Hawley, R. G., Hawley, T., Shulman, M. J., Traunecker, A., Kohler, G. & Hozumi, N. (1983) Functional immunoglobulin M production after transfection of cloned immunoglobulin heavy and light chain genes into lymphoid cells. Proc. Natt. Acad. Sci. t/5,4 80, 6351. Oi, V. T., Morrison, S. L., Herzenberg, L. A. & Berg, P. (1983) Immunoglobulin gene expression in transformed lymphoid cells. Proc. Natl. Acad. Sci. USA 80, 825. Oldenburg, K. R., Loganathan, D., Goldstein, I. J., Schultz, P G. & Gallop, M. A. (1992) Peptide ligands for a sugar-binding protein isolated from a random peptide library. Proc. Natt. Acad Sci. USA 89, 5393. Orlandi, R., Gussow, D. H., Jones, P. T. & Winter, G. (1989) Qoning immunoglobulin variable domains for expression by the polymerase chain reaction. Proc. Natl Acad. Sci. USA 86, 3833. Pack, P. & Pluckthun, A. (1992) Miniantibodies: use of amphipathic helices to produce functional, flexibly linked dimeric FV fragments with high avidity in Escherichia coli. Biochemistry 31, 1579.


67

Pannley, S. F. & Smith, G. P. (1988) Antibody-selectable filamentous fd phage vectors: affinity purification of target genes. Gene 73, 305. Perelson, A. S. (1989) Immune network theory. Invnunot. Rev. 110, 5. Perelson, A. S. & Oster, G. F. (1979) Theoretical studies of cional selection: Minimal antibody repertoire size and reliability of self non-self discrimination. J. Theor. Biot. 81, 645. Persson. M. A., A., Caothien, R. H. & Burton, D. R. (1991) Generation of diverse highaffinity human monoclonal antibodies by repertoire cloning. Proc. Natl. Acad. Sci. USA 88, 2432. Queen, C , Schneider, W. P., Selick, H. E., Payne, P. W., Landolfi, N. R, Duncan, J. F., Avdalovic, N. M., Levitt, M., Junghans, R. P. & Waldmann, T. A. (1989) A humanized antibody that binds to the interleukin 2 receptor. Proc. Natl Acad. Sci. USA 86, 10029. Riechmann, L., Clark, M., Waldmann, H. & Winter, G. (1988a) Reshaping human antibodies for therapy. Nature 332, 323. Riechmann, L., Foote, J. & Winter, G. (1988b) Expression of an antibody Fv fragment in myeloma cells. J. Mol. Biot. 203, 825. Rybak, S. M., Hoogenboom, H. R., Meade, H. M., Raus, J. C. M., Schwatz, D. & Youle, R. J. (1992) Humanisation of immunotoxins. Proc. Natt. Acad. Sci. USA 89, 3165. Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Hom, G. T., Erlich, H. A. & Amheim, N. (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350. Sanz, I. (1991) Multiple mechanisms participate in the generation of diversity of human H chain CDR3 regions. / Immunot. 147, 1720. Sasso, E. H., Silverman, G. J. & Mannik, M. (1991) Human IgA and IgG F(ab')2 that bind to staphylococcal protein A belong to the VHIII subgroup. J. Immunol 147, 1877. Sastry, L., Alting, M. M., Huse, W. D., Short, J. M., Sorge, J. A., Hay, B. N., Janda, K. D., Benkovic, S. J. & Lerner, R. A. (1989) Cloning ofthe immunological repertoire in Escherichia coti for generation of monoclonal catalytic antibodies: construction of a heavy chain variable region-specific cDNA library. Proc. Natl. Acad. Sci. USA 86, 5728. Schaaper, R. M. (1988) Mechanisms of mutagenesis in the Escherichia coli mutator mutD5: role of DNA mismatch repair. Proc. Natl Acad Sci. USA 85, 8126. Schnee, J. M., Runge, M. S., Matsueda, G. R., Hudson, N. W, Seidman, J. G., Haber, E. & Quertermous, T. (1987) Construction and expression ofa recombinant antibodytargeted plasminogen activator. Proc. Natt. Acad. Sci. USA 84, 6904. Scott, J. K., Loganathan, D , Easley, R. B., Gong, X. & Goldstein, I. J. (1992) A family of concanavalin A-binding peptides from a hexapeptide epitope library. Proc. Natl. Acad Sci. USA 89, 5398. Scott, J. K. & Smith, G. P. (1990) Searching for peptide ligands with an epitope library. Science 249, 386. Seed, B. & ArufTo, A. (1987) Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapid inununoselection procedure. Proc. Natt. Acad. Sci. USA 84, 3365. Shalaby, M. R.. Shepard, H. M., Presta, L., Rodrigues, M. L., Beverley, P C , Feldmann, M. & Carter, P. (1992) Development of humanized bispecific antibodies reactive with cytotoxic lymphocytes and tumor cells overexpressing the HER2 protooncogene. J. Exp. Med 175,217. Sharon, J. (1990) Structural correlates of high antibody affinity: Three engineered amino acid substitutions can increase ihe affinity of an anti-p-azophenylarsonate antibody 200-fold. Proc. Natl Acad Sci. USA 87, 4814. Skerra, A., Pfitzinger, I. & Pluckthun, A. (1991) The functional expression of antibody Fv fragments in Escherichia coli: improved vectors and a generally applicable purification technique. Bio/Technology 9, 273.

68

HOOGENBOOM ET AL.

SkerTa, A. & Pluckthun, A. (1988) Assembly of a functional immunoglobuiin Fv fragment in Escherichia coti. Science 240, 1038. Smith, G. P. (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315. Stephen, C. W. & Lane, D. P. (1992) Mutant conformations of p53. Precise epitope mapping using a filamentous phage epitope library. J. Mot. Biol. 225, 577. Tempest, P. R., Bremner, P., Lambert, M., Taylor, G., Furze, J. M., Carr, F. J. & Harris, W. J. (1991) Reshaping a monoclonal antibody to inhibit human respiratory syncytial virus infection in vivo. Bio/Technology 9, 266. Tomlinson, I. M., Walter, G., Marks, J. D . Llewelyn, M. B. & Winter, G. (1992) The repertoire of human germline VH sequences reveals about fifty groups of VH segments with different hypervariable loops. / Mol. Biol. Ill, 116. Tonegawa, S. (1983) Somatic generation of antibody diversity. Nature 302, 575. Tramontano, A., Chothia, C. & Lesk, A. M. (1990) Framework residue 71 is a major determinant of the position and conformation of the second hypervariable region in the VH domain of immunoglobulins. J. Mot. Biol. 215, 175. Verhoeyen, M., Milstein, C. & Winter, G. (1988) Reshaping human antibodies: grafting an antilysozyme activity. Science 239, 1534. Vieira, J. & Messing, J. (1987) Production of single-slranded plasmid DNA. Methods Enzymol. 153. 3. Ward, E. S., Gussow, D , Griffiths, A. D., Jones, P. T & Winter, G. (1989) Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coti. Nature 341, 544. Winter, G. & Milstein, C. (1991) Man-made antibodies. Nature 349, 293. Yamagishi, J., Kawashima, H., Matsuo, N., Ohue. M., Yamayoshi, M., Fukui, T , Kotani, H., Furuta, R., Nakano, K. & Yamada, M. (1990) Mutational analysis of structureactivity relationships in human tumor necrosis factor-alpha. Protein Eng. 3, 713, Zebedee, S. L., Barbas, C. R, Hom, Y.-L., Caothien. R. H.. Graff, R. Degraw, J,, Pyati, J., LaPoIla, R., Burton, D. R., Lemer, R. A. & Thomthon, G. A. (1992) Human combinatorial antibody libraries to hepatitis B surface antigen. Proc. Natl. Acad. Sci. 89, 3175.

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