Constructs And Libraries Comprising Antibody Surrogate Light Chain Sequences

Abstract

The invention concerns constructs and libraries comprising antibody surrogate light chain sequences. In particular, the invention concerns constructs comprising VpreB sequences, optionally partnered with another polypeptide, such as, for example, antibody heavy chain variable domain sequences, and libraries containing the same.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Ser. No. 60/920,568, filed Mar. 27, 2007, the entire disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention concerns constructs and libraries comprising antibody surrogate light chain sequences. In particular, the invention concerns constructs comprising VpreB sequences, optionally partnered with another polypeptide, such as, for example, antibody heavy chain variable domain sequences, and libraries containing the same.

BACKGROUND OF THE INVENTION

[0003] Antibody (Ig) molecules produced by B-lymphocytes are built of heavy (H) and light (L) chains. The amino acid sequences of the amino terminal domains of the H and L chains are variable (V.sub.H and V.sub.L), especially at the three hypervariable regions (CDR1, CDR2, CDR3) that form the antigen combining site. The assembly of the H and L chains is stabilized by a disulfide bond between the constant region of the L chain (C.sub.L) and the first constant region of the heavy chain (C.sub.H1) and by non-covalent interactions between the V.sub.H and V.sub.L domains.

[0004] In humans and many animals, such as mice, the genes encoding the antibody H and L chains are assembled by stepwise somatic rearrangements of gene fragments encoding parts of the V regions. Various stages of B lymphocyte development are characterized by the rearrangement status of the Ig gene loci (see, e.g. Melchers, F. & Rolink, A., B-Lymphocyte Development and Biology, Paul, W. E., ed., 1999, Lippincott, Philadephia).

[0005] Precursors of B cells (pre-B cells) have been identified in the bone marrow as lymphocytes that produce .mu. heavy chains but instead of the fully developed light chains express a set of B lineage-specific genes called VpreB(1-3) and .lamda.5, respectively.

[0006] The main isoform of human VpreB1 (CAG30495) is a 145 aa-long polypeptide (SEQ ID NO: 1). It has an Ig V domain-like structure, but lacks the last .beta.-strand (.beta.7) of a typical V domain, and has a carboxyl terminal end that shows no sequence homologies to any other proteins. VpreB2 has several isoforms, including a 142-amino acid mouse VpreB2 polypeptide (P13373; SEQ ID NO: 2), and a 171-amino acid long splice variant of the mouse VpreB2 sequence (CAA019641SEQ ID NO: 3). VpreB1 and VpreB2 sequences have been disclosed in EP 0 269 127 and U.S. Pat. No. 5,182,205; Collins et al., Genome Biol. 5(10):R84 (2004); and Hollins et al., Proc. Natl. Acad. Sci. USA 86(14):5552-5556 (1989). The main isoform of human VpreB3 (SEQ ID NO: 4) is a 123 amino acid long protein (CAG30496), disclosed in Collins et al., Genome Biol. 5(10):R84 (2004).

[0007] VpreB(1-3) are non-covalently associated with another protein, .lamda.5. The human .lamda.5 is a 209-amino acid polypeptide (CAA01962; SEQ ID NO: 5), that carries an Ig C domain-like structure with strong homologies to antibody light chains and, towards its amino terminal end, two functionally distinct regions, one of which shows strong homology to the .beta.7 strand of the V.lamda. domains. A human .lamda.5-like protein has 213 amino acids (NP.sub.--064455; SEQ ID NO: 6) and shows about 84% sequence identity to the antibody .lamda. light chain constant region.

[0008] For further details, see the following review papers: Karasuyama et al., Adv. Immunol. 63:1-41 (1996); Melchers et al., Immunology Today 14:60-68 (1993); and Melchers, Proc. Natl. Acad. Sci. USA 96:2571-2573 (1999).

[0009] The VpreB and .lamda.5 polypeptides together form a non-covalently associated, Ig light chain-like structure, which is called the surrogate light chain or pseudo light chain. On the surface of early preB cells, the surrogate light chain is disulfide-linked to membrane-bound Ig p heavy chain in association with a signal transducer CD79a/CD79b heterodimer to form a B cell receptor-like structure, the so-called preB cell receptor (preBCR).

SUMMARY OF THE INVENTION

[0010] In one aspect, the invention concerns polypeptides comprising a VpreB sequence or a .lamda.5 sequence conjugated to a heterogeneous amino acid sequence, wherein the polypeptides are capable of binding to a target.

[0011] In a preferred embodiment, the polypeptide comprises a VpreB sequence, where VpreB may be any native VpreB, including human VpreB1 (SEQ ID NO: 1), mouse VpreB2 (SEQ ID NO: 2 and 3) and human VpreB3 (SEQ ID NO: 4), or a homologue thereof in another mammalian species, or a fragment or variant thereof, provided that the polypeptide retains the ability to bind to a target.

[0012] In a preferred embodiment, the heterogeneous amino acid sequence is a .lamda.5 sequence, which may be any native .lamda.5 sequence, or any fragment or variant thereof, including the native human .lamda.5 sequence of SEQ ID NO: 5, the human .lamda.5-like sequence of SEQ ID NO: 6, and fragments and variants thereof.

[0013] The VpreB sequence and the heterogeneous amino acid sequence, e.g. the .lamda.5 sequence, may be directly fused to each other, or may be non-covalently associated. In the former case, the fusion preferably takes place at or around the CDR3 analogous regions of VpreB and .lamda.5, respectively.

[0014] In another embodiment, the heterogeneous amino acid sequence is or comprises an antibody light chain variable region sequence. In a particular embodiment, the antibody light chain variable region sequence is fused to the VpreB sequence at a site analogous to an antibody light chain CDR3 region. In another embodiment, the fusion is between the CDR3 region of an antibody light chain and the CDR3 analogous region of a VpreB. In all embodiments, the antibody light chain can be a .lamda. chain or a .kappa. chain.

[0015] In particular embodiments, the polypeptides herein, including, without limitation, VpreB-.lamda.5 conjugates (including fusions, other covalent linkage, and non-covalent associations), and VpreB-antibody light chain conjugates, may be further associated with a sequence comprising an antibody heavy chain variable region sequence, such as an antibody heavy chain variable region, or a complete antibody heavy chain, including a variable region.

[0016] When the polypeptide comprises a .lamda.5 sequence, .lamda.5 may be any native .lamda.5, including human .lamda.5 of SEQ ID NO: 5 and human .lamda.5-like protein of SEQ ID NO: 6, or a homologue in another mammalian species, or any fragment or variant thereof, provided that the polypeptide retains the ability to bind to a target. In a particular embodiment, the heterogeneous amino acid sequence conjugated to the .lamda.5 sequence is a VpreB sequence.

[0017] In the polypeptide constructs of the present invention, the VpreB and .lamda.5 sequences, if both present, may be conjugates by any means, including direct fusion, covalent linkage by a linker sequence (e.g. a peptide linker), and non-covalent association.

[0018] In a particular embodiment, a fusion of a VpreB sequence and a .lamda.5 sequence is conjugated town antibody heavy chain sequence by non-covalent association, to form a dimeric complex.

[0019] In another embodiment, a trimeric complex is formed by non-covalent association of a VpreB sequence, a .lamda.5 sequence and an antibody heavy chain sequence. In certain embodiments, in these structures, which are also referred to as variant surrogate light chain structures of "SURROBODY.TM. variants," the characteristic tails (appendages) of one or both of the VpreB and .lamda.5 portions may be (but do not have to be) retained. It is possible to attach additional functionalities to such appendages. In addition, in various embodiments, beneficial appendage fusions can be designed and made in order to improve various properties of the constructs, such as PK and/or potency.

[0020] In all embodiments, when an antibody heavy chain comprising variable region sequences is present, the polypeptide of the present invention and the antibody heavy chain variable region sequences may bind to the same or to different targets.

[0021] In another aspect, the invention concerns a library of such polypeptides.

[0022] In yet another aspect, the invention concerns a library of such polypeptides associated with antibody heavy chains or fragments thereof comprising variable region sequences.

[0023] In a further aspect, the invention concerns a library comprising a collection of surrogate light chain sequences optionally associated with antibody heavy chain variable region sequences.

[0024] In all aspects, the library may be in the form of a display, such as, for example, a phage display, bacterial display, yeast display, ribosome display, mRNA display, DNA display, display on mammalian cells, spore display, viral display, display based on protein-DNA linkage, or microbead display.

[0025] The invention further concerns various uses of such polypeptides and libraries containing such polypeptides, for example, to design or select antibody-like molecules with desired binding specificities and/or binding affinities, which have important therapeutic applications.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 shows the alignment of human VpreB1 (SEQ ID NO: 1) and human .lamda.5 with antibody .lamda. chain variable and constant regions. VpreB1 shares some sequence similarity to antibody .lamda. chain variable regions, while .lamda.5 shares some similarly to antibody .lamda. chain constant regions and framework region 4. The boxed regions identify VpreB1 and .lamda.5 sequences that are similar to antibody .lamda. chain CDR1, CDR2 and CDR3 regions, respectively.

[0027] FIG. 2 is a schematic illustration of a surrogate light chain formed by VpreB and .lamda.5 sequences, illustrative fusion polypeptides comprising surrogate light chain sequences, and an antibody light chain structure derived from V-J joining.

[0028] FIG. 3 is a schematic illustration of various surrogate light chain deletion and single chain constructs.

[0029] FIG. 4 schematically illustrates the incorporation of combinatorial functional diversity into surrogate light chain constructs.

[0030] FIG. 5 shows the gene and protein structures of various illustrative surrogate light chain constructs.

[0031] FIG. 6 is the alignment of VpreB1 sequences with antibody .lamda.5 light chain variable region sequences. Regions with the highest degree of sequence similarity are boxed. As shown in the figure, VpreB1 shows only 56%-62% (amino acids 2 to 97) sequence identity to the .lamda.5 light chain variable region germline sequences.

[0032] FIG. 7 is the alignment of VpreB1 sequences with antibody .lamda.5 light chain constant region sequences. As shown in the figure, the aligned VpreB1 sequences show only 62% (amino acids 97 to 209) sequence identity to the corresponding antibody .lamda.5 light chain constant region sequences.

[0033] FIG. 8 is the alignment of VpreB1 sequences with antibody .kappa. light chain constant region sequences. As shown in the figure, the aligned VpreB1 sequences show only 35% (amino acids 105 to 209) sequence identity to the corresponding antibody .kappa. light chain constant region sequences.

[0034] FIG. 9 illustrates various representative ways of adding functionality to surrogate light chain (SLC) components.

[0035] FIG. 10 shows the human VpreB1 sequence of SEQ ID NO: 1. the mouse VpreB2 sequences of SEQ ID NOS: 2 and 3; the human VpreB3 sequence of SEQ ID NO: 4, the human .lamda.5 sequence of SEQ D NO: 5 and the human .lamda.5-like protein sequence of SEQ ID NO: 6, and sequences of various constructs used in the examples.

[0036] FIG. 11 illustrates various trimeric and dimeric surrogate light chain constructs of the invention.

[0037] FIG. 12: Detection of surrogate light chains and conjugated heavy chains. Lane 1: Full Length; Lane 2: Lambda 5 dT; Lane 3: VpreB dt; Lane 4; Short; Lane 5: SCL fusion I; Lane 6: SLC fusion 2; Lane 7: Antibody.

[0038] FIG. 13: SLC fusion proteins express and secrete well into the periplasm of E. coli and are best partnered with heavy chain CH1 from IgG1 rather than IgM. Panel A: SCL fusion protein expression in E. coli. Panel B: IgG1 gamma chain partners and purifies better than IgM .mu. chain with an SLC fusion.

[0039] FIG. 14: Phage surrogate light chain construct capture ELISA via anti-phage detection.

[0040] FIG. 15: Purified surrogate light chain constructs expressed in mammalian cells bind viral target.

[0041] FIG. 16: Purified surrogate light chain constructs expressed in mammalian cells contain stable complexes that bind viral antigen.

[0042] FIG. 17: Antigen binding with E. coli periplasmic lysates.

[0043] FIG. 18: Surrogate light chain fusion construct phage paired with neutralizing heavy chain readily binds H5 HA antigen.

[0044] FIG. 19: Surrogate light chain construct phage paired with neutralizing heavy chain binds antigen.

[0045] FIG. 20: Table summarizing the results of phage display experiments.

[0046] FIGS. 21 and 22: Results of clonal analysis of rounds 1 and 2 of surrogate light chain fusions 1 and 2.

DETAILED DESCRIPTION OF THE INVENTION

[0047] A. Definitions

[0048] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), provides one skilled in the art with a general guide to many of the terms used in the present application.

[0049] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0050] The term "surrogate light chain," as used herein, refers to a dimer formed by the non-covalent association of a VpreB and a .lamda.5 protein.

[0051] The term "VpreB" is used herein in the broadest sense and refers to any native sequence or variant VpreB polypeptide, specifically including, without limitation, human VpreB1 of SEQ ID NO: 1, mouse VpreB2 of SEQ ID NOS: 2 and 3, human VpreB3 of SEQ ID NO: 4 and isoforms, including splice variants and variants formed by posttranslational modifications, other mammalian homologues thereof, as well as variants of such native sequence polypeptides.

[0052] The term ".lamda.5" is used herein in the broadest sense and refers to any native sequence or variant .lamda.5 polypeptide, specifically including, without limitation, human .lamda.5 of SEQ ID NO: 5, human .lamda.5-like protein of SEQ ID NO: 6, and their isoforms, including splice variants and variants formed by posttranslational modifications, other mammalian homologous thereof, as well a variants of such native sequence polypeptides.

[0053] The terms "variant VpreB polypeptide" and "a variant of a VpreB polypeptide" are used interchangeably, and are defined herein as a polypeptide differing from a native sequence VpreB polypeptide at one or more amino acid positions as a result of an amino acid modification. The "variant VpreB polypeptide," as defined herein, will be different from a native antibody .lamda. or .kappa. light chain sequence, or a fragment thereof. The "variant VpreB polypeptide" will preferably retain at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98% sequence identity with a native sequence VpreB polypeptide. In another preferred embodiment, the "variant VpreB polypeptide" will be less then 95%, or less than 90%, or less then 85%, ore less than 80%, or less than 75%, or less then 70%, or less than 65%, or less than 60% identical in its amino acid sequence to a native antibody .lamda. or .kappa. light chain sequence. Variant VpreB polypeptides specifically include, without limitation, VpreB polypeptides in which the non-Ig-like unique tail at the C-terminus of the VpreB sequence is partially or completely removed.

[0054] The terms "variant .lamda.5 polypeptide" and "a variant of a .lamda.5 polypeptide" are used interchangeably, and are defined herein as a polypeptide differing from a native sequence .lamda.5 polypeptide at one or more amino acid positions as a result of an amino acid modification. The "variant .lamda.5 polypeptide," as defined herein, will be different from a native antibody .lamda. or .kappa. light chain sequence, or a fragment thereof. The "variant .lamda.5 polypeptide" will preferably retain at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98% sequence identity with a native sequence .lamda.5 polypeptide. In another preferred embodiment, the "variant .lamda.5 polypeptide" will be less then 95%, or less than 90%, or less then 85%, ore less than 80%, or less than 75%, or less then 70%, or less than 65%, or less than 60% identical in its amino acid sequence to a native antibody .lamda. or .kappa. light chain sequence. Variant .lamda.5 polypeptides specifically include, without limitation, .lamda.5 polypeptides in which the unique tail at the N-terminus of the .lamda.5 sequence is partially or completely removed.

[0055] Percent amino acid sequence identity may be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov or otherwise obtained from the National Institute of Health, Bethesda, Md. NCBI-BLAST2 uses several search parameters, wherein all of those search parameters arc set to default values including, for example, unmask=yes, strand=all, expected occurrences=10, minimum low complexity length=15/5, multi-pass e-value=0.01, constant for multi-pass=25, dropoff for final gapped alignment=25 and scoring matrix=BLOSUM62.

[0056] The term "VpreB sequence" is used herein to refer to the sequence of "VpreB," as hereinabove defined, or a fragment thereof.

[0057] The term ".lamda.5 sequence" is used herein to refers to the sequence of ".lamda.5," as hereinabove defined, or a fragment thereof.

[0058] The term "surrogate light chain sequence," as defined herein, means any polypeptide sequence that comprises a "VpreB sequence" and/or a ".lamda.5 sequence," as hereinabove defined. The "surrogate light chain sequence," as defined herein, specifically includes, without limitation, the human VpreB1 sequence of SEQ ID NO 1, the mouse VpreB2 sequences of SEQ ID NOS: 2 and 3, and the human VpreB3 sequence of SEQ ID NO: 4, and their various isoforms, including splice variants and variants formed by posttranslational modifications, homologues thereof in other mammalian species, as well as fragments and variants thereof. The term "surrogate light chain sequence" additionally includes, without limitation, the human .lamda.5 sequence of SEQ ID NO: 5, the human .lamda.5-like sequence of SEQ ID NO: 6, and their isoforms, including splice variants and variants formed by posttranslational modifications, homologues thereof in other mammalian species, as well as fragments and variants thereof. The term "surrogate light chain sequence" additionally includes a sequence comprising both VpreB and .lamda.5 sequences as hereinabove defined.

[0059] For the three-dimensional structure of the pre-B-cell receptor (pre-BCR), including the structure of the surrogate light chain (SCL) and its components see, e.g. Lanig et al., Mol. Immunol. 40(17):1263-72 (2004).

[0060] The "surrogate light chain sequence" may be optionally conjugated to a heterogeneous amino acid sequence, or any other heterogeneous component, to form a "surrogate light chain construct" herein. Thus, the term, "surrogate light chain construct" is used in the broadest sense and includes any and all additional heterogeneous components, including a heterogeneous amino acid sequence, nucleic acid, and other molecules conjugated to a surrogate light chain sequence, wherein "conjugation" is defined below. A "surrogate light chain construct" is also referred herein as a `SURROBODY.TM.," and the two terms are used interchangeably.

[0061] In the context of the polypeptides of the present invention, the term "heterogeneous amino acid sequence," relative to a first amino acid sequence, is used to refer to an amino acid sequence not naturally associated with the first amino acid sequence, at least not in the form it is present in the surrogate light chain constructs herein. Thus, a "heterogenous amino acid sequence" relative to a VpreB is any amino acid sequence not associated with native VpreB in its native environment, including, without limitation, .lamda.5 sequences that are different from those .lamda.5 sequences that, together with VpreB, form the surrogate light chain on developing B cells, such as amino acid sequence variants, e.g. truncated and/or derivatized .lamda.5 sequences. A "heterogeneous amino acid sequence" relative to a VpreB also includes .lamda.5 sequences covalently associated with, e.g. fused to, VpreB, including native sequence .lamda.5, since in their native environment, the VpreB and .lamda.5 sequences are not covalently associated, e.g. fused, to each other. Heterogeneous amino acid sequences also include, without limitation, antibody sequences, including antibody and heavy chain sequences and fragments thereof, such as, for example, antibody light and heavy chain variable region sequences, and antibody light and heavy chain constant region sequences.

[0062] The terms "conjugate," "conjugated," and "conjugation" refer to any and all forms of covalent or non-covalent linkage, and include, without limitation, direct genetic or chemical fusion, coupling through a linker or a cross-linking agent, and non-covalent association, for example through Van der Waals forces, or by using a leucine zipper.

[0063] The term "fusion" is used herein to refer to the combination of amino acid sequences of different origin in one polypeptide chain by in-frame combination of their coding nucleotide sequences. The term "fusion" explicitly encompasses internal fusions, i.e., insertion of sequences of different origin within a polypeptide chain, in addition to fusion to one of its termini.

[0064] As used herein, the term "target" is a substance that interacts with a polypeptide herein. Targets, as defined herein, specifically include antigens with which the VpreB-containing constructs of the present invention interact. Preferably, interaction takes place by direct binding.

[0065] As used herein, the terms "peptide," "polypeptide" and "protein" all refer to a primary sequence of amino acids that are joined by covalent "peptide linkages." In general, a peptide consists of a few amino acids, typically from about 2 to about 50 amino acids, and is shorter than a protein. The term "polypeptide," as defined herein, encompasses peptides and proteins.

[0066] The term "amino acid" or "amino acid residue" typically refers to an amino acid having its art recognized definition such as an amino acid selected from the group consisting of alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys); glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (Ile): leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val) although modified, synthetic, or rare amino acids may be used as desired. Thus, modified and unusual amino acids listed in 37 CFR 1.822(b)(4) are specifically included within this definition and expressly incorporated herein by reference. Amino acids can be subdivided into various sub-groups. Thus, amino acids can be grouped as having a nonpolar side chain (e.g., Ala, Cys, Ile, Leu, Met, Phe, Pro, Val); a negatively charged side chain (e.g., Asp, Glu); a positively charged side chain (e.g., Arg, His, Lys); or an uncharged polar side chain (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr). Amino acids can also be grouped as small amino acids (Gly, Ala), nucleophilic amino acids (Ser, His, Thr, Cys), hydrophobic amino acids (Val, Leu, Ile, Met, Pro), aromatic amino acids (Phe, Tyr, Trp, Asp, Glu), amides (Asp, Glu), and basic amino acids (Lys, Arg).

[0067] The term "polynucleotide(s)" refers to nucleic acids such as DNA molecules and RNA molecules and analogs thereof (e.g., DNA or RNA generated using nucleotide analogs or using nucleic acid chemistry). As desired, the polynucicotides may be made synthetically, e.g., using art-recognized nucleic acid chemistry or enzymatically using, e.g., a polymerase, and, if desired, be modified. Typical modifications include methylation, biotinylation, and other art-known modifications. In addition, the nucleic acid molecule can be single-stranded or double-stranded and, where desired, linked to a detectable moiety.

[0068] The term "variant" with respect to a reference polypeptide refers to a polypeptide that possesses at least one amino acid mutation or modification (i.e., alteration) as compared to a native polypeptide. Variants generated by "amino acid modifications" can be produced, for example, by substituting, deleting, inserting and/or chemically modifying at least one amino acid in the native amino acid sequence.

[0069] An "amino acid modification" refers to a change in the amino acid sequence of a predetermined amino acid sequence. Exemplary modifications include an amino acid substitution, insertion and/or deletion.

[0070] An "amino acid modification at" a specified position, refers to the substitution or deletion of the specified residue, or the insertion of at least one amino acid residue adjacent the specified residue. By insertion "adjacent" a specified residue is meant insertion within one to two residues thereof. The insertion may be N-terminal or C-terminal to the specified residue.

[0071] An "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence with another different "replacement" amino acid residue. The replacement residue or residues may be "naturally occurring amino acid residues" (i.e. encoded by the genetic code) and selected from the group consisting of: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys); glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (Ile): leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val). Substitution with one or more non-naturally occurring amino acid residues is also encompassed by the definition of an amino acid substitution herein.

[0072] A "non-naturally occurring amino acid residue" refers to a residue, other than those naturally occurring amino acid residues listed above, which is able to covalently bind adjacent amino acid residues(s) in a polypeptide chain. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine and other amino acid residue analogues such as those described in Ellman et al. Meth. Enzym. 202:301 336 (1991). To generate such non-naturally occurring amino acid residues, the procedures of Noren et al. Science 244:182 (1989) and Ellman et al., supra, can be used. Briefly, these procedures involve chemically activating a suppressor tRNA with a non-naturally occurring amino acid residue followed by in vitro transcription and translation of the RNA.

[0073] An "amino acid insertion" refers to the incorporation of at least one amino acid into a predetermined amino acid sequence. While the insertion will usually consist of the insertion of one or two amino acid residues, the present application contemplates larger "peptide insertions", e.g. insertion of about three to about five or even up to about ten amino acid residues. The inserted residue(s) may be naturally occurring or non-naturally occurring as disclosed above.

[0074] An "amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

[0075] The term "mutagenesis" refers to, unless otherwise specified, any art recognized technique for altering a polynucleotide or polypeptide sequence. Preferred types of mutagenesis include error prone PCR mutagenesis, saturation mutagenesis, or other site directed mutagenesis.

[0076] "Site-directed mutagenesis" is a technique standard in the art, and is conducted using a synthetic oligonucleotide primer complementary to a single-stranded phage DNA to be mutagenized except for limited mismatching, representing the desired mutation. Briefly, the synthetic oligonucleotide is used as a primer to direct synthesis of a strand complementary to the single-stranded phage DNA, and the resulting double-stranded DNA is transformed into a phage-supporting host bacterium. Cultures of the transformed bacteria are plated in top agar, permitting plaque formation from single cells that harbor the phage. Theoretically, 50% of the new plaques will contain the phage having, as a single strand, the mutated form; 50% will have the original sequence. Plaques of interest are selected by hybridizing with kinased synthetic primer at a temperature that permits hybridization of an exact match, but at which the mismatches with the original strand are sufficient to prevent hybridization. Plaques that hybridize with the probe are then selected, sequenced and cultured, and the DNA is recovered.

[0077] In the context of the present invention, the term "antibody" (Ab) is used to refer to a native antibody from a classically recombined heavy chain derived from V(D)J gene recombination and a classically recombined light chain also derived from VJ gene recombination, or a fragment thereof.

[0078] A "native antibody" is heterotetrameric glycoprotein of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by covalent disulfide bond(s), while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has, at one end, a variable domain (V.sub.H) followed by a number of constant domains. Each light chain has a variable domain at one end (V.sub.L) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains, Chothia et al., J. Mol. Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A. 82:4592 (1985).

[0079] The term "variable" with reference to antibody chains is used to refer to portions of the antibody chains which differ extensively in sequence among antibodies and participate in the binding and specificity of each particular antibody for its particular antigen. Such variability is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework region (FR). The variable domains of native heavy and light chains each comprise four FRs (FR1, FR2, FR3 and FR4, respectively), largely adopting a .beta.-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the .beta.-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), pages 647-669). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0080] The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" (i.e., residues 30-36 (L1), 46-55 (L2) and 86-96 (L3) in the light chain variable domain and 30-35 (H1), 47-58 (H2) and 93-101 (H3) in the heavy chain variable domain; MacCallum et al., J Mol Biol. 262(5):732-45 (1996).

[0081] The term "framework region" refers to the art recognized portions of an antibody variable region that exist between the more divergent CDR regions. Such framework regions are typically referred to as frameworks 1 through 4 (FR1, FR2, FR3, and FR4) and provide a scaffold for holding, in three-dimensional space, the three CDRs found in a heavy or light chain antibody variable region, such that the CDRs can form an antigen-binding surface.

[0082] Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of antibodies IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

[0083] The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called .alpha., .delta., .epsilon., .gamma., and .mu., respectively.

[0084] The "light chains" of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (.kappa.) and lambda (.lamda.), based on the amino acid sequences of their constant domains. Any reference to an antibody light chain herein includes both .kappa. and .lamda. light chains.

[0085] "Antibody fragments" comprise a portion of a full length antibody, generally the antigen binding or a variable domain thereof. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab').sub.2, scFv, and (scFv).sub.2 fragments.

[0086] As used herein the term "antibody binding region" refers to one or more portions of an immunoglobulin or antibody variable region capable of binding an antigen(s). Typically, the antibody binding region is, for example, an antibody light chain (VL) (or variable region thereof), an antibody heavy chain (VH) (or variable region thereof), a heavy chain Fd region, a combined antibody light and heavy chain (or variable region thereof) such as a Fab, F(ab').sub.2, single domain, or single chain antibody (scFv), or a full length antibody, for example, an IgG (e.g., an IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

[0087] The term "epitope" as used herein, refers to a sequence of at least about 3 to 5, preferably at least about 5 to 10, or at least about 5 to 15 amino acids, and typically not more than about 500, or about 1,000 amino acids, which define a sequence that by itself, or as part of a larger sequence, binds to an antibody generated in response to such sequence. An epitope is not limited to a polypeptide having a sequence identical to the portion of the parent protein from which it is derived. Indeed, viral genomes are in a state of constant change and exhibit relatively high degrees of variability between isolates. Thus the term "epitope" encompasses sequences identical to the native sequence, as well as modifications, such as deletions, substitutions and/or insertions to the native sequence. Generally, such modifications are conservative in nature but non-conservative modifications are also contemplated. The term specifically includes "mimotopes," i.e. sequences that do not identify a continuous linear native sequence or do not necessarily occur in a native protein, but functionally mimic an epitope on a native protein. The term "epitope" specifically includes linear and conformational epitopes.

[0088] The term "vector" is used to refer to a rDNA molecule capable of autonomous replication in a cell and to which a DNA segment, e.g., gene or polynucleotide, can be operatively linked so as to bring about replication of the attached segment. Vectors capable of directing the expression of genes encoding for one or more polypeptides are referred to herein as "expression vectors. "The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0089] Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0090] A "phage display library" is a protein expression library that expresses a collection of cloned protein sequences as fusions with a phage coat protein. Thus, the phrase "phage display library" refers herein to a collection of phage (e.g., filamentous phage) wherein the phage express an external (typically heterologous) protein. The external protein is free to interact with (bind to) other moieties with which the phage are contacted. Each phage displaying an external protein is a "member" of the phage display library.

[0091] The term "filamentous phage" refers to a viral particle capable of displaying a heterogenous polypeptide on its surface, and includes, without limitation, f1, fd, Pf1, and M13. The filamentous phage may contain a selectable marker such as tetracycline (e.g., "fd-tet"). Various filamentous phage display systems are well known to those of skill in the art (see, e.g., "Lacher et al. Gene 9: 127-140 (1980), Smith et al. Science 228: 1315-1317 (1985); and Parmley and Smith Gene 73: 305-318 (1988)).

[0092] The term "panning" is used to refer to the multiple rounds of screening process in identification and isolation of phages carrying compounds, such as antibodies, with high affinity and specificity to a target.

[0093] B. Detailed Description

[0094] Techniques for performing the methods of the present invention are well known in the art and described in standard laboratory textbooks, including, for example, Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997); Molecular Cloning: A Laboratory Manual, Third Edition, J. Sambrook and D. W. Russell, eds., Cold Spring Harbor, N.Y., USA, Cold Spring Harbor Laboratory Press, 2001; O'Brian et al., Analytical Chemistry of Bacillus Thuringiensis, Hickle and Pitch, eds., Am. Chem. Soc., 1990; Bacillus thuringiensis: biology. ecology and safety, T. R. Glare and M. O'Callaghan, eds., John Wiley, 2000; Antibody Phage Display, Methods and Protocols, Humana Press, 2001; and Antibodies, G. Subramanian, ed., Kluwer Academic, 2004. Mutagenesis can, for example, be performed using site-directed mutagenesis (Kunkel et al., Proc. Natl. Acad. Sci USA 82:488-492 (1985)). PCR amplification methods are described in U.S. Pat. Nos. 4,683,192, 4,683,202, 4,800,159, and 4,965,188, and in several textbooks including "PCR Technology: Principles and Applications for DNA Amplification", H. Erlich, ed., Stockton Press, New York (1989); and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., Academic Press, San Diego, Calif. (1990).

[0095] The present invention concerns constructs and libraries comprising antibody surrogate light chain sequences.

[0096] Surrogate Light Chain Constructs

[0097] As discussed above, pre-B cells have been identified in the bone marrow as lymphocytes that produce .mu. heavy chains but instead of the fully developed light chains express a set of B lineage-specific genes called VpreB(1-3) and .lamda.5, respectively. The VpreB and .lamda.5 polypeptides together form a non-covalently associated, Ig light chain-like structure, which is called the surrogate light chain. The surrogate light chain, although not an antibody chain, naturally associates with all antibody heavy chains, and surrogate light chain-antibody heavy chain complexes have been shown to bind self-antigens.

[0098] In one aspect, the present invention provides polypeptides comprising VpreB and/or .lamda.5 sequences and having the ability to bind a target. The target can be any peptide or polypeptide that is a binding partner for the VpreB and/or .lamda.5 sequence-containing polypeptides of the present invention. Targets specifically include all types of targets generally referred to as "antigens" in the context of antibody binding.

[0099] Thus, the polypeptides of the present invention include, without limitation, conjugates of VpreB sequences to heterogeneous amino acid sequences, provided that they retain the ability to bind a desired target. The binding of the VpreB sequence to the heterogeneous amino acid sequence can be either covalent or non-covalent, and may occur directly, or through a linker, including peptide linkers.

[0100] Specific examples of the polypeptide constructs herein include polypeptides in which a VpreB sequence, such as a VpreB1, VpreB2, or VpreB3 sequence, including fragments and variants of the native sequences, is conjugated to a .lamda.5 sequence, including fragments and variants of the native sequence. Representative fusions of this type are illustrated in FIGS. 2 and 11 and described in the Examples.

[0101] In a direct fusion, typically the C-terminus of a VpreB sequence (e.g. a VpreB1, VpreB2 or VpreB3 sequence) is fused to the N-terminus of a .lamda.5 sequence. While it is possible to fuse the entire length of a native VpreB sequence to a full-length .lamda.5 sequence (see, e.g. the first diagram in FIG. 3), typically the fusion takes place at or around a CDR3 analogous site in each of the two polypeptides. Such CDR3 analogous sites for VpreB1 and .lamda.5 are illustrated in FIG. 1, and a representative fusion construct is illustrated in FIG. 2. In this embodiment, the fusion may take place within, or at a location within about 10 amino acid residues at either side of the CDR3 analogous region. In a preferred embodiment, the fusion takes place between about amino acid residues 116-126 of the native human VpreB1 sequence (SEQ ID NO: 1) and between about amino acid residues 82 and 93 of the native human .lamda.5 sequence (SEQ ID NO: 5).

[0102] It is also possible to fuse the VpreB sequence to the CDR3 region of an antibody .lamda. light chain, as shown in FIG. 2. Further constructs, in which only one of VpreB and .lamda.5 is truncated are shown in FIG. 3. Similar constructs can be prepared using antibody .kappa. light chain sequences.

[0103] Further direct fusion structures are illustrated on the right side of FIG. 11. The structure designated "SLC fusion 1" is a tetramer, composed of two dimers, in which the fusion of a truncated V-preB1 sequence (lacking the characteristic "tail" at the C-terminus of native VpreB1) to a similarly truncated .lamda.5 sequence is non-covalently associated with an antibody heavy chain. The structure designated "SLC fusion 2" is a tetramer, composed of two dimers, in which the fusion of a truncated VpreB I sequence (lacking the characteristic "tail" at the C-terminus of native VpreB1) to an antibody light chain constant region is non-covalently associated with an antibody heavy chain. The structure designated "SLC fusion 3" is a tetramer, composed of two dimers, in which the fusion of an antibody light chain variable region to a truncated .lamda.5 sequence (lacking the characteristic "tail" at the N-terminus of native .lamda.5) is non-covalently associated with an antibody heavy chain.

[0104] As noted above, in addition to direct fusions, the polypeptide constructs of the present invention include non-covalent associations of a VpreB sequence (including fragments and variants of a native sequence) with a heterogeneous sequence, such as a X.5 sequence (including fragments and variants of the native sequence), and/or an antibody sequence. Thus, for example, a full-length VpreB sequence may be non-covalently associated with a truncated .lamda.5 sequence. Alternatively, a truncated VpreB sequence may be non-covalently associated with a full-length .lamda.5 sequence.

[0105] Surrogate light chain constructs comprising non-covalently associated VpreB1 and .lamda.5 sequences, in non-covalent association with an antibody heavy chain, are shown on the left side of FIG. 11. As the various illustrations show, the structures may include, for example, full-length VpreB1 and .lamda.5 sequences, a full-length VpreB1 sequence associated with a truncated .lamda.5 sequence ("Lambda 5dT"), a truncated V-reB1 sequence associated with a full-length .lamda.5 sequence (VpreB dT") and a truncated VpreB1 sequence associated with a truncated .lamda.5 sequence ("Short").

[0106] Although FIG. 11 illustrates certain specific constructs, one of ordinary skill will appreciate that a variety of other constructs can be made and used in a similar fashion. For example, the structures can be asymmetrical, comprising different surrogate light chain sequences in each arm, and/or having trimeric or pentameric structures, as opposed to the structures illustrated in FIG. 11. It is also possible to include different functionalities in various portions of the surrogate light chain constructs of the present invention, thereby producing multi-specific and/or multivalent constructs.

[0107] If desired, the constructs of the present invention can be engineered, for example, by incorporating or appending known sequences or sequence motifs from the CDR1, CDR2 and/or CDR3 regions of antibodies, including known therapeutic antibodies into the CDR1, CDR2 and/or CDR3 analogous regions of the surrogate light chain sequences. This allows the creation of molecules that are not antibodies, but will exhibit binding specificities and affinities very similar to those of a known therapeutic antibody.

[0108] All surrogate light chain constructs herein may be associated with antibody sequences. For example, as shown in FIG. 5, a VpreB-.lamda.5 fusion can be linked to an antibody heavy chain variable region sequence by a peptide linker. In another embodiment, a VpreB-.lamda.5 fusion is non-covalently associated with an antibody heavy chain, or a fragment thereof including a variable region sequence to form a dimeric complex. In yet another embodiment, the VpreB and .lamda.5 sequences are non-covalently associated with each other and an antibody heavy chain, or a fragment thereof including a variable region sequence, thereby forming a trimeric complex. Exemplary constructs comprising an antibody heavy chain are illustrated in FIG. 11.

[0109] While the constructs of the present invention are illustrated by reference to certain embodiments, one of ordinary skill will understand that numerous further embodiments obtained by various permutations of surrogate light chain and antibody sequences are possible, and are within the scope of the present invention. The present invention includes all constructs that comprise surrogate light chain sequences and have the ability to bind a desired target. In certain embodiment, the constructs also have the ability to associate with antibody heavy chain variable region sequences.

[0110] The constructs of the present invention may be used to build libraries of surrogate light chain sequences, which can be used for various purposes, similarly to antibody libraries, including selection of constructs with the desired binding specificities and affinities.

[0111] When the VpreB and .lamda.5 surrogate light chain sequences are non-covalently associated with each other, the free ends of one or both components (i.e. the C-terminal end of the VpreB sequence and/or the N-terminal end of the .lamda.5 sequence) are available for incorporating an additional diversity into the library of such sequences. For instance, a random peptide library can be appended or substituted to one of these free ends and panned for specific binding to a particular target. By combining the surrogate light chain identified to have the desired binding specificity with a heavy chain or heavy chain fragment from an antibody to the same target, a molecule can be created that has the ability to bind to the cognate target on two distinct places. This tandem binding, or "chelating" effect, strongly reinforces the binding to a single target, similarly to the avidity effects seen in dimeric immunoglobulins. It is also possible to use components binding to different targets. Thus, for example, the surrogate light chain component with the desired binding specificity can be combined with an antibody heavy chain or heavy fragment binding to a different target. For instance, the surrogate light chain component may bind a tumor antigen while the antibody heavy chain or heavy chain fragment may bind to effector cells. This way, a single entity with targeting and anti-tumor activity can be created. In a particular embodiment, the appendage or the polypeptide that connects the VpreB and .lamda.5 sequences can be an antibody or antibody fragments, such as a Fab or a scFv fragment. The incorporation of an antibody sequence will not only create a "chelating" effect but can also generate bispecificity in a single molecule, without the need of a second independent arm, such as that found in bispecific antibodies. The two specificities may be to different parts of the same target, to disparate targets, or to a target antibody complex. Similarly, multi-specific constructs can be made with any type of molecule, other than antibodies or antibody fragments, including peptides, proteins, enzymes, and the like. For example, the surrogate light chain component with the desired specificity can be combined with any therapeutic peptide or protein.

[0112] Preparation of Surrogate Light Chain Constructs

[0113] The surrogate light chain constructs of the present invention can be prepared by methods known in the art, including well known techniques of recombinant DNA technology.

[0114] Nucleic acid encoding surrogate light chain, e.g. VpreB and .lamda.5 polypeptides, can be isolated from natural sources, e.g. developing B cells and/or obtained by synthetic or semi-synthetic methods. Once this DNA has been identified and isolated or otherwise produced, it can be ligated into a replicable vector for further cloning or for expression.

[0115] Cloning and expression vectors that can be used for expressing the coding sequences of the polypeptides herein are well known in the art and are commercially available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and, a transcription termination sequence. Suitable host cells for cloning or expressing the DNA encoding the surrogate light chain constructs in the vectors herein are prokaryote, yeast, or higher eukaryote (mammalian) cells, mammalian cells are being preferred.

[0116] Examples of suitable mammalian host cell lines include, without limitation, monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line 293 (293 cells) subcloned for growth in suspension culture, Graham et al, J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0117] For use in mammalian cells, the control functions on the expression vectors are often provided by viral material. Thus, commonly used promoters can be derived from the genomes of polyoma, Adenovirus2, retroviruses, cytomegalovirus, and Simian Virus 40 (SV40). Other promoters, such as the .beta.-actin protomer, originate from heterologous sources. Examples of suitable promoters include, without limitation, the early and late promoters of SV40 virus (Fiers el al., Nature, 273: 113 (1978)), the immediate early promoter of the human cytomegalovirus (Greenaway el al., Gene, 18: 355-360 (1982)), and promoter and/or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell system.

[0118] Transcription of a DNA encoding a desired heterologous polypeptide by higher eukaryotes is increased by inserting an enhancer sequence into the vector. The enhancer is a cis-acting element of DNA, usually about from 10 to 300 bp, that acts on a promoter to enhance its transcription-initiation activity. Enhancers are relatively orientation and position independent, but preferably are located upstream of the promoter sequence present in the expression vector. The enhancer might originate from the same source as the promoter, such as, for example, from a eukaryotic cell virus, e.g. the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0119] Expression vectors used in mammalian host cells also contain polyadenylation sites, such as those derived from viruses such as, e.g., the SV40 (early and late) or HBV.

[0120] An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell.

[0121] The expression vectors usually contain a selectable marker that encodes a protein necessary for the survival or growth of a host cell transformed with the vector. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase thymidine kinase (TK), and neomycin.

[0122] Suitable mammalian expression vectors are well known in the art and commercially available. Thus, for example, the surrogate light chain constructs of the present invention can be produced in mammalian host cells using a pCI expression vector (Promega), carrying the human cytomegalovirus (CMV) immediate-early enhancer/promoter region to promote constitutive expression of a DNA insert. The vector can contain a neomycin phosphotransferase gene as a selectable marker.

[0123] The surrogate light chain constructs of the present invention can also be produced in bacterial host cells. Control elements for use in bacterial systems include promoters, optionally containing operator sequences, and ribosome binding sites. Suitable promoters include, without limitation, galactose (gal), lactose (lac), maltose, tryptophan (trp), .beta.-lactamase promoters, bacteriophage .lamda. and T7 promoters. In addition, synthetic promoters can be used, such as the tac promoter. Promoters for use in bacterial systems also generally contain a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the Fab molecule. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria.

[0124] The coding sequences of the individual chains within a multi-chain construct comprising antibody surrogate light chain sequences can be present in the same expression vector, under control of separate regulatory sequences, or in separate expression vectors, used to cotransfect a desired host cells, including eukaryotic and prokaryotic hosts. Thus, multiple genes can be coexpressed using the Duet.TM. vectors commercially available from Novagen.

[0125] The transformed host cells may be cultured in a variety of media. Commercially available media for culturing mammalian host cells include Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma). In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979) and Barnes et al., Anal. Biochem. 102:255 (1980) may be used as culture media for the host cells. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and are included in the manufacturer's instructions or will otherwise be apparent to the ordinarily skilled artisan.

[0126] Further suitable media for culturing mammalian, bacterial (e.g. E. coli) or other host cells are also described in standard textbooks, such as, for example, Sambrook et al., supra, or Ausubel et al., supra.

[0127] Purification can be performed by methods known in the art. In a preferred embodiment, the surrogate antibody molecules are purified in a 6.times. His-tagged form, using the Ni-NTA purification system (Invitrogen).

[0128] Libraries Comprise Surrogate Light Chain Sequences

[0129] The present invention further concerns various libraries of surrogate light chain sequences and constructs comprising such sequences. Thus, such libraries may comprise, consist essentially of, or consist of, displays of surrogate light chain sequences, such as the VpreB- and/or .lamda.5-containing constructs of the present invention, including, without limitation, those specifically described above, illustrated in the figures and/or described in the Examples.

[0130] The libraries of the present invention are preferably in the form of a display. Systems for displaying heterologous proteins, including antibodies and other polypeptides, are well known in the art. Antibody fragments have been displayed on the surface of filamentous phage that encode the antibody genes (Hoogenboom and Winter J. Mol. Biol., 222:381 388 (1992); McCafferty et al., Nature 348(6301):552 554 (1990); Griffiths et al. EMBO J., 13(14):3245-3260 (1994)). For a review of techniques for selecting and screening antibody libraries see, e.g., Hoogenboom, Nature Biotechnol. 23(9):1105-1116 (2005). In addition, there are systems known in the art for display of heterologous proteins and fragments thereof on the surface of Escherichia coli (Agterberg et al., Gene 88:37-45 (1990); Charbit et al., Gene 70:181-189 (1988); Francisco et al., Proc. Natl. Acad. Sci. USA 89:2713-2717 (1992)), and yeast, such as Saccharomyces cerevisiae (Boder and Wittrup, Nat. Biotechnol. 15:553-557 (1997); Kieke et al., Protein Eng. 10:1303-1310 (1997)). Other known display techniques include ribosome or mRNA display (Mattheakis et al., Proc. Natl. Acad. Sci. USA 91:9022-9026 (1994); Hanes and Pluckthun, Proc. Natl. Acad. Sci. USA 94:4937-4942 (1997)), DNA display (Yonezawa et al., Nucl. Acid Res. 31(19):e118 (2003)); microbial cell display, such as bacterial display (Georgiou et al., Nature Biotech. 15:29-34 (1997)), display on mammalian cells, spore display (Isticato et al., J. Bacterial. 183:6294-6301 (2001); Cheng et al., Appl. Environ. Microbial. 71:3337-3341 (2005) and co-pending provisional application Ser. No. 60/865,574, filed Nov. 13, 2006), viral display, such as retroviral display (Urban et al., Nucleic Acids Res. 33:e35 (2005), display based on protein-DNA linkage (Odegrip et al., Proc. Acad Natl. Sci. USA 101:2806-2810 (2004); Reiersen et al., Nucleic Acids Res. 33:e 10 (2005)), and microbead display (Sepp et al., FEBS Lett. 532:455-458 (2002)).

[0131] For the purpose of the present invention, the surrogate light chain-containing libraries may be advantageously displayed using any display technique, including phage display and spore display.

[0132] In phage display, the heterologous protein, such as a surrogate light chain polypeptide, is linked to a coat protein of a phage particle, while the DNA sequence from which it was expressed is packaged within the phage coat. Details of the phage display methods can be found, for example, McCafferty et al., Nature 348, 552-553 (1990)), describing the production of human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B-cell.

[0133] Phage display can be performed in a variety of formats; for their review see, e.g. Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3, 564-571 (1993). Several sources of heavy chain V-gene segments can be discovered through phage display. Clarkson et al., Nature 352, 624-628 (1991) isolated a diverse array of anti-oxazolone heavy chains and light chains from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of heavy and light chain V genes from unimmunized human donors can be constructed and recovered specific to a diverse array of antigens (including self-antigens) essentially following the techniques described by Marks et al., J. Mol. Biol. 222, 581-597 (1991), or Griffith et al., EMBO J. 12, 725-734 (1993). These, and other techniques known in the art, can be adapted to the display of any polypeptide, including polypeptides and other constructs comprising surrogate light chain sequences. Thus, for example, the surrogate light chain can be supplemented with a collection of heavy chains from either a naturally diverse source, such as lymphocytes, or a synthetically generated collection created entirely through techniques of molecular biology. These collections can be cloned, expressed and selected, by methods known in the art. The selected resulting SURROBODY.TM. can be used directly, expressed as multimeric a molecule, or further optimized through heavy chain optimization, or surrogate light chain optimization, for example, using random or nonrandom site specific or regional mutagenesis.

[0134] Spore display systems arc based on attaching the sequences to be displayed to a coat protein, such as a Bacillus subtilis spore coat protein. The spore protoplast (core) is surrounded by the cell wall, the cortex, and the spore coat. Depending on the species, an exosporium may also be present. The core wall is composed of the same type of peptidoglycan as the vegetative cell wall. Spore display, including surface display system using a component of the Bacillus subtilis spore coat (CorB) and Bacillus thuringiensis (Bt) spore display, is described in Isticato et al., J. Bacterial. 183:6294-6301 (2001); Cheng et al., Appl. Environ. Microbiol. 71:3337-3341 (2005), the entire disclosures of which is hereby expressly incorporated by reference. Various spore display techniques are also disclosed in U.S. Patent Application Publication Nos. 20020150594; 20030165538; 20040180348; 20040171065; and 20040254364, the entire disclosures are hereby expressly incorporated by reference herein.

[0135] An advantage of spore display systems is the homogenous particle surface and particle size of non-eukaryotic nature, which is expected to provide an ideal non-reactive background. In addition, the particle size of spores is sufficient to enable selection by flow cytometry that permits selectable clonal isolation, based upon interactions.

[0136] Leveraging on the stability of spores, it is possible to perform various post-sporulation chemical, enzymatic and/or environmental treatments and modification. Thus, it is possible to stabilize structural helical structures with chemical treatment using trifluoroethanol (TFE), when such structures arc displayed. In addition, oxidative stress treatments, such as treatments with Reactive Oxygen Species (e.g. peroxide) or reactive Nitrogen Species (e.g. nitrous acid) are possible. It is also possible to expose defined or crude populations of spore-displayed polypeptides to enzymatic treatments, such as proteolytic exposure, other enzymatic processes, phosphorylation, etc. Other possible treatments include, without limitation, nitrosylation by peroxynitrite treatment, proteolysis by recombinant, purified, or serum protease treatment, irradiation, coincubation with known chaperones, such as heat shock proteins (both bacterial and mammalian), treatment with folding proteins, such as protein disulfide isomerase, prolyl isomerase, etc., lyophilization, and preservative-like treatments, such as treatment with thimerosol. These treatments can be performed by methods well known in the art.

[0137] Similar techniques can be used in all spore display systems, including displays where the attachment is to a spore coat protein, including, for example, the spore display systems disclosed in

[0138] Uses of Surrogate Light Chain Sequences, Constructs and Libraries Containing Same

[0139] The libraries of the present invention can be used to identify surrogate light chain sequences and surrogate light chain constructs, such as fusions comprising surrogate light chain sequences, with desired properties. For example, in vitro or in vivo screening of the libraries herein can yield polypeptides comprising surrogate light chain sequences binding to desired targets with high binding specificity and affinity. Thus, the libraries herein can be used to identify molecules for therapeutic and diagnostic purposes, such as polypeptides comprising surrogate light chain sequences that bind to tumor markers or other molecular targets of therapeutic intervention. In addition, by the techniques described above, highly diverse libraries of surrogate light chain polypeptides can be engineered, including libraries comprising a collection of polypeptides binding to the same target, libraries of polypeptides binding to different targets, libraries of polypeptides with multiple specificities, and the like.

[0140] As a result of their ability to bind to any desired target, the antibody surrogate light chain constructs of the present invention can be used in analytical and diagnostic assays, to detect the presence of a desired target molecule, such as a tumor antigen or any polypeptide associated with a disease state or condition. In addition, the surrogate light chain constructs of the present invention can be used as therapeutic agents, such as, for example, in cancer therapy, to target tumor antigens that have been determined to associate with the development and/or spread of cancer.

[0141] Further details of the invention are provided in the following non-limiting Examples.

EXAMPLE 1

VpreB as a Binding Domain Protein and Fusions Containing it

[0142] To make a VpreB binding domain a single protein shown in FIG. 5 is created recombinantly. The SLC binding domain protein construct is comprised of the amino acids 20 to 121 from VpreB1 and the amino acids 87 to 105 from .lamda.5. If desired, to create novel and specific binding capabilities, the molecule is reengineered according to structural or sequence evidence. Additionally, or alternatively, a collection of variants is created either randomly, for example by error-prone PCR, or directly by single or multi-site specific mutagenesis with a collection of amino acids. The resulting clones or collections are then cloned in frame with pill for use in phage or phagemid display. This phagemid construct is transformed into TG1 cells. Next a single colony is propagated in Luria Broth (LB) supplemented with 50 .mu.g/ml Ampicillin and 2% glucose until it reached OD600 .about.0.3, and infected with MK307 helper phage at 37.degree. C. for 30 minutes without shaking. The cells are then pelleted and then resuspended in LB containing 50 .mu.g/ml ampicillin and 75 .mu.g/ml kanamycin and allowed to grow overnight with vigorous aeration at 30.degree. C. The next day the supernatant containing phagemid expressed SLC-HC fusion protein is used in Phage ELISA to determine targeted binding. Briefly the ELISA entails coating and blocking of an ELISA plate with human TNF-.alpha., followed by incubation of the SLC-HC phage for 2 hours at 4.degree. C., washing with PBS-Tween-20 (0.05%) and direct detection with anti-m13-HRP antibody. Alternatively binding is assessed by directly amplifying or eluting the bound phage and determining phage titers using XL-1Blue cells. This example describes a SLC binding domain fusion as a single clone, but this SLC can be recombinantly recombined with other heterologous sequences that recognize a common target and screened as a library. Furthermore, this SLC binding protein can be combined with a previously selected collection of heavy chains and screened directly on the same target of interest or a second target of interest to create a bispecific molecule. Alternatively this reinforced binding or bispecific binding can be discovered by screening in conjunction with unselected collections of heavy chains. In addition, while this example refers to antibody heavy chains, it should be understood that a complete heavy chain is not needed. Single-chain fusions comprising heavy chain variable region sequences, in the absence of a heavy chain constant region, or a complete heavy chain constant region, can be made in an analogous manner and are within the scope of this example.

EXAMPLE 2

VpreB Fusions as a Variable Heavy Chain (VH) Partner

[0143] A functional VpreB-.lamda.5 fusion protein shown in the second diagram of FIG. 5 (designated "VpreB protein fusion-dimeric complex") is recombinantly created. The VpreB-.lamda.5 fusion protein is comprised of an m13 gene III signal sequence, the amino acids 20 to 115 from VpreB1, and the amino acids 83 to 209 from .lamda.5. This construct is coexpressed with a variable heavy chain-CH1 fusion in frame with pIII for use in phage or phagemid display. As a VH coding sequence the VH coding sequence from the anti-TNF-.alpha. antibody, D2E7, is used, and CH1 is the CH1 region of human IgG1. This phagemid construct is transformed into TG1 cells. Next, a single colony is propagated in Luria Broth (LB) supplemented with 50 .mu.g/ml Ampicillin and 2% glucose until it reached 0D600 .about.0.3, and infected with MK307 helper phage at 37 degrees for 30 minutes, without shaking. The cells are then pelleted and then resuspended in LB containing 50 .mu.g/ml ampicillin and 75 .mu.g/ml kanamycin and allowed to grow overnight with vigorous aeration at 30 degrees. The next day the supernatant containing phagemid expressed SLC-HC fusion protein is used in Phage ELISA to determine targeted binding. Briefly the ELISA entails coating and blocking of an ELISA plate with human TNF-.alpha., followed by incubation of the SLC-HC phage for 2 hours at 4 degrees, washing with PBS-Tween-20 (0.05%) and direct detection with anti-m13-HRP antibody. Alternatively binding can be assessed by directly amplifying or eluting the bound phage and determining phage titers using XL-1Blue cells. This example describes a SLC fusion partnered with a heavy chain variable-CH1 fusion as a single clone, but this SLC can also be combined with a focused collection of heavy chain variable regions that recognize a common target and screened as a library. Furthermore, this SLC fusion can be combined with an unselected collection of heavy chains and screened directly on a target of interest. As a reasonable SLC fusion alternative, VH association can be reinforced by fusing the constant lambda region from a traditional antibody light chain instead of the .lamda.5 protein fragment.

EXAMPLE 3

VpreB and Lambda5 as an Associated Variable Heavy Chain (VH) Partner

[0144] The VpreB-.lamda.5 coexpressed protein shown in the third diagram of FIG. 5 (designated "VpreB and lambda 5-trimeric complex") is made of an m13 gene III signal sequence and the corresponding amino acids of the predicted mature, processed VpreB1 (amino acids 20 to 146) and lambda 5 (amino acids 31 to 209). These are coexpressed with a variable heavy chain-CH1 fusion in frame with pIII for use in phage or phagemid display. As a VH coding sequence the VH coding sequence from the anti-TNF-.alpha. antibody, D2E7, is used, and CH1 is the CH1 region from human IgG1. This phagemid construct is transformed into TG1 cells. Next a single colony is propagated in Luria Broth (LB) supplemented with 50 .mu.g/ml Ampicillin and 2% glucose until it reached OD600 .about.0.3, and is then infected with MK307 helper phage at 37 degrees for 30 minutes, without shaking. The cells are then pelleted and then resuspended in LB containing 50 .mu.g/ml ampicillin and 75 .mu./ml kanamycin and allowed to grow overnight with vigorous aeration at 30 degrees. The next day the supernatant containing phagemid expressed SLC HC trimeric protein complexes is used in Phage ELISA to determine targeted binding. Briefly the ELISA entails coating and blocking of an ELISA plate with human TNF-.alpha., followed by incubation of the SLC-HC phage for 2 hours at 4 degrees, washing with PBS-Tween-20 (0.05%) and direct detection with anti-m13-HRP antibody. Alternatively binding can be assessed by directly amplifying or eluting the bound phage and determining phage titers using XL-1Blue cells. This example describes a SLC partnered with a heavy chain variable-CH1 fusion as a single clone, but this SLC can be combined with a focused collection of heavy chain variable regions that recognize a common target and screened as a library. Furthermore, this SLC fusion can be combined with an unselected collection of heavy chains and screened directly on a target of interest.

EXAMPLE 4

[0145] Engineering Diversity into VpreB1 CDR3 Analogous Regions

[0146] As the CDR analogous regions of the surrogate light chain (SLC) will have similar functions to the CDR's of an antibody light chain, it is important to determine the fusion points between the VpreB and .lamda.5. According to one approach the most suitable fusion point for a particular purpose is determined starting with the CDR3 analogous site containing all VpreB amino acids and incrementally substituting amino acids position by position from .lamda.5 encoded in clonable oligonucleotides. This incremental substitution continues until the CDR analogous site is entirely composed of a .lamda.5 source sequence. At some point during this process, it might be desirable to add a complementary heavy chain and allow/facilitate its antigen binding and recognition. To further enhance or enable this complementation random diversity can be used in any of the CDR analogous sites, as well as diversity based upon matched CDR length analysis. Alternatively, or in addition, antibody V.lamda.5 sequences can be used to add diversity, as their CDR lengths match well with VpreB CDR analogous site lengths.

EXAMPLE 5

Adding Functionalities to SLC Components

[0147] As the SLC is comprised of two independent polypeptides this creates natural opportunities to append or embed secondary functionalities. In the present Example, in the first instance an anti-VEGF scFv is inserted to create a fusion protein linking VpreB and .lamda.5 (FIG. 9A). This resulting engineered SIX-constrained scFv is paired with the heavy chain of an anti-TNF-.alpha. antibody. The resulting construct is co-expressed with the heavy chain cloned in frame with pill for use in phage or phagemid display. This phagemid construct is transformed into TG1 cells and a single colony is propagated in Luria Broth (LB) supplemented with 50 .mu.g/ml Ampicillin and 2% glucose until it reached OD600 .about.0.3, and infected with MK307 helper phage at 37.degree. C. for 30 minutes without shaking. The cells are then be pelleted and then resuspended in LB containing 50 .mu.g/ml ampicillin and 75 .mu.g/ml kanamycin and allowed to grow overnight with vigorous aeration at 30.degree. C. The next day the supernatant containing phagemid expressed SLC-HC fusion protein is used in Phage ELISA to determine targeted binding. Briefly the ELISA entails coating and blocking of an ELISA plate with human TNF-.alpha. or human VEGF, followed by incubation of the SLC-HC phage for 2 hours at 4.degree. C., washing with PBS-Tween-20 (0.05%) and direct detection with anti-m13-HRP antibody.

[0148] Next a fusion of the anti-VEGF scFv to the C-terminus of VpreB is created, and the resulting tripartite protein complex construct assessed similarly to the phagemid ELISA described above.

[0149] Alternatively the an anti-ovalbumin scFv is fused to the amino terminus of .lamda.5 and the tripartite protein complex tested for binding to both TNF-.alpha. and ovalbumin.

[0150] Finally, these two fusion constructs (VpreB-antiVEGF scFv and the .lamda.5-anti-ovalbumin) are combined with the heavy chain of the anti-TNF-.alpha. antibody to create a trispecific molecule, which is then confirmed in phagemid ELISA as described above.

[0151] In the description scFv against disparate targets arc incorporated, however one can combine functional binders to the same target to create tandem "super-binders." These tandem binders can either provide reinforced binding or even in some instances cross-linking function. Fab cross-linking will be beneficial in instances where whole antibodies provide undesirable and prolonged cross-linking. For instance, it may be undesirable for whole immunoglobulin insulin receptor antibodies that act as insulin substitutes to require 3-4 weeks for scrum clearance. As insulin usually has a half-life of minutes, a Fab would be more in tune with this scale of half-life and the tandem functionality could appropriately address this application.

[0152] The above descriptions describe only antibodies as secondary functional groups, but one can also similarly incorporate relevant peptides (e.g., erythropoietin (EPO) mimetics), receptors (e.g., TNF-RI), binding proteins (e.g., IL-lra), and any therapeutic protein, such as interferons, to the appended and constrained constructs to create molecules of similar functions.

[0153] Also one might utilize the two sites to incorporate heterodimeric proteins, such as heavy and light chains to create a secondary Fab-like molecule.

[0154] Finally, we have described only singular instances, but the incorporation of combinatorially diverse phage antibody libraries and peptide diversity libraries is also included herein, to screen with SLC candidate antibodies against their directed and desired targets.

EXAMPLE 6

Expression of Surrogate Light Chain Constructs (SURROBODY.TM.) in Mammalian Cells

[0155] Coding sequences of the surrogate light chain components of the structures designated in FIG. 11 as "trimers" (also referred to as "SURROBODY.TM. variants") were cotransfected with a full-length IgG1 antibody heavy chain into CHO-K1 cells (ATCC CCL-61) to transiently produce surrogate light chain constructs for biochemical analysis. Specifically, full length human VpreB1 and .lamda.5 were cloned into the mammalian expression vector pCI (Promega, Madison Wis.). These constructs contained their native predicted secretion signals. In the case of VpreB1 the predicted signal peptide is amino acids 1-20 of SEQ ID NO: 1, for .lamda.5 the predicted signal sequence is amino acids 1-30 of SEQ ID NO: 5. For both of these proteins portions of their predicted nonstructural tails were deleted. For VpreB1 this included the C-terminal amino acids 122-146 of SEQ ID NO: 1 and for lambda 5 this included the N-terminal amino acids 30-86 of SEQ ID NO: 5.

[0156] The sequence of the truncated .lamda.5 sequence in the "trimer" designated in FIG. 11 as "Lambda 5 dT" is shown as SEQ ID NO: 7. The sequence of the truncated VpreB1 sequence in the "trimer" designated in FIG. 11 as "VpreB dT" is shown as SEQ ID NO: 8.

[0157] Each of the four combinatorial surrogate light chain possibilities were cotransfected with a known human anti-influenza heavy chain, containing a C-terminal hexahistidine (His6) tag (SEQ ID NO: 9), and expressed according to manufacturer's suggestions (Invitrogen, Carlsbad Calif.) in low serum media. After 3 days the supernatants were collected, filtered, and purified by nickel chelate chromatography (Qiagen, Germany). The purified proteins were then examined by western blot analysis with either anti-peptide rabbit serum (VpreB and .lamda.5) or anti-histidine antibodies (Scrotec, Raleigh N.C.). Detection of proteins was visualized following anti-rabbit HRP (VpreB and .lamda.5) or anti-mouse HRP (heavy chain) and colorimetric development with TMB substrate. (FIG. 12, lanes 1-4)

[0158] Additionally, surrogate light chain fusions (see FIG. 11) were created by engineering a chimeric protein composed of the VpreB1 gene and either the .lamda.5 gene or the light chain constant lambda domain. Specifically a recombinantly fused protein was produced that contained amino acids 1-87 from VpreB (SEQ ID NO: 1) to .lamda.5 protein amino acids 121-209 (SEQ ID NO: 5) (SEQ ID NO: 10). Additionally, a second fusion was made that contained amino acids 1-87 from VpreB (SEQ ID NO: 1) to the C-terminal 121 amino acid of the antibody .lamda. light chain constant (SEQ ID NO: 11). Each surrogate light chain fusion was transiently expressed, harvested, purified, and examined by western blot analysis, essentially as described above. Notably, as both fusions contained the epitope to the anti-VpreB1 anti-peptide serum, it was used for western blot analysis. (FIG. 12, lanes 5-6)

EXAMPLE 7

[0159] Expression of Surrogate Light Chain Constructs (SURROBODY.TM.) in E. coli

[0160] As recombinant proteins are often beneficially expressed in bacteria the ability of producing soluble surrogate light chain constructs in prokaryotic systems was tested. To address this, the surrogate light chain fusions designated as "dimers" in FIG. 11 were clones into E. coli expression/secretion systems. A plac repressible expression system was used, where the mature mammalian proteins were expressed and secreted into the periplasm by recombinant fusion to prokaryotic leader sequences. Specifically, the surrogate light chain fusions were directed to the periplasm by fusing the coding sequence of the mature protein to the C-terminus of the m13 gIII leader coding sequence (SEQ ID NOs: 12 and 13). The heavy chain was expressed by fusing the IgG1 heavy chain variable region and heavy chain constant region domain of an anti-influenza antibody to the C-terminus of the pelB leader sequence (SEQ ID NO: 14). The plasmids expressing both proteins were transformed into HB2151 E. coli cells (Stratagene) and expressed overnight in LB media containing 100 mcg/ml ampicillin, and 200 micromolar IPTG at 30 degrees. The cells were harvested and periplasmic lysates were prepared, following methods known in the art. The periplasmic lysates were tested directly by western blot analysis or purified as described above (FIG. 13, panel A).

[0161] As the surrogate light chain is traditionally a component of the membrane bound preB cell receptor, it is normally found paired with an IgM class heavy chain. For our utilitarian purposes we wished to compare the ability to pair a surrogate light chain fusion with an IgM versus a IgG based constant heavy domain 1 region. To examine this we substituted a .mu. constant heavy domain 1 (SEQ ID NO: 15) for the gamma constant heavy domain region of the anti-influenza antibody described above. We found, from western blot analysis of the periplasmic lysates that the IgG (.gamma.)-based constant heavy domain expressed better and purified to greater levels than a .mu.-based constant heavy domain based system (FIG. 13, panel B).

EXAMPLE 8

Expression of Surrogate Light Chain Constructs (SURROBODY.TM.) m13 Phagemid

[0162] As recombinant proteins are not only usefully expressed in bacteria but also individually and in diverse library collections on the surface of bacterial virus particles we wished to produce soluble surrogate light chain constructs on the surface of m13 phagemids. To address this, surrogate light chain fusions ("dimers" in FIG. 11) were clones into E. coli expression/secretion systems. For all systems a pLac repressible expression system described above was used. However, in this case we appended an E-tag epitope (GAPVPYPDPLEPR) (SEQ ID NO: 16) to the surrogate light chain fusions, as well as to a light chain control protein. The sequences of the geneIII VpreB1-lambda5-E tag fusion (Fusion 1) and the geneIII VpreB1-CI-E tag fusion (Fusion 2) are shown as SEQ ID NOs: 12 and 13, respectively. To anchor the heavy chain constructs to the m13 phagemid the heavy chain constructs were recombinantly cloned the variable heavy chains and gamma constant heavy domain 1 regions in frame with the m13 gene III product. Specifically the recombinant proteins contained an intervening, a hexahistidine peptide, the peptide epitope for the anti-c-myc antibody (GEQKLISLEEDL) (SEQ ID NO: 17), and amber stop codon. We examined the fidelity of protein expression and complex formation respectively by anti-histidine and anti-E capture ELISA.

[0163] Phagemid expression of antibodies and surrobodies were accomplished by standard methods well known in the art. Essentially, TG-1 cells transformed with expression plasmids were grown to mid log (OD 600 .about.0.3) in 2-YT media supplemented with 100 mcg/ml ampicillin and 2% glucose repression and then infected with m13K07 helper phage and then grown overnight in 2-YT media supplemented with 100 mcg ampicillin, 70 mcg/ml kanamycin, and 200 micromolar IPTG. Phage containing supernatants or precipitated and PBS resuspended phage were used for phage capture ELISA. The phage capture ELISA was accomplished by coating microtiter plates with either anti-histidine (Serotec) or anti-E antibodies(Abcam) and then detecting binding with anti-m13 peroxidase antibodies (Pharmacia), followed by colorimetric visualization with TMB substrate. In these instances we found specific capture of the phage by both methods, supporting high fidelity protein expression fusion to phage by the heavy chains and stable surrogate light chain association. The results are shown in FIG. 14.

EXAMPLE 9

Antigen Binding of Surrogate Light Chain Constructs Expressed in Mammalian Cells

[0164] As it appeared that the surrogate light chain variants formed readily detectable complexes following nickel chelate chromatography, their ability to bind the parent antigen of cognate heavy chain partner was tested. Transient expression and purification were performed as described above. Antigen binding was tested by ELISA. Briefly, microtiter wells were coated with inactivated H5N1 Vietnam 12-3/04 virus preparations (USFDA-CBER, antigen standard), blocked and then incubated with quantified serially diluted purified proteins. After washing, the complexes were detected with anti-human Fc peroxidase conjugated antibodies. Finally, binding was colorimetrially visualized and quantitated with TMB substrate development (see FIG. 15).

[0165] Additionally, supernatants from transient transfections were similarly tested undiluted for antigen binding and shown in FIG. 16. After washing, the surrogate light chain complexes were detected with either anti-VpreB1 anti-peptide sera and anti-rabbit peroxidase conjugated secondary or anti-human Fc peroxidase conjugated antibodies and then colorimetrically visualized and quantitated with TMB substrate development.

EXAMPLE 10

[0166] Antigen Binding of Surrogate Light Chain Constructs Expressed in E. coli

[0167] Because the surrogate light chain fusions appeared to form stable complexes we wanted to establish whether such fusions paired with a heavy chain from and anti-influenza antibody would bind the antibody's cognate virus. To test for binding periplasmic lysates were prepared as described above. The lysates were then subjected to ELISA antigen binding, essentially as described above, except binding was detected with either a monoclonal antibody to an appended hexahistidine epitope at the C-terminus of the heavy chain or to an appended E-tag at the C-terminus of the surrogate light chain fusion via polyclonal affinity purified antibodies. Epitope detection was accomplished by either anti-mouse or anti-rabbit peroxidase conjugated antibodies. Finally, binding was colorimetrically visualized and quantitated with TMB substrate development. The results are shown in FIG. 17.

EXAMPLE 11

Antigen Binding of Phage Displayed Surrogate Light Chain Constructs

[0168] Because it was possible to make surrogate light chain variants in heterologous systems, and as phage displayed collections are desirable to future protein discovery and engineering, we wanted to determine whether the surrogate light chain variants and/or fusions were readily displayed on the surface of m13 phage as gene III-associated complexes. The variants (SEQ ID NOs: 18-22) and previously described fusions (SEQ ID NOs: 12 and 13) were coexpressed with either of two anti-influenza antibody heavy chains (SEQ ID NOs: 19 and 14) as described above and binding followed essentially the conditions also described above. Briefly, microliter wells were coated with H5N 1 Vietnam 1203/04 virus and phagemid were allowed to bind and then washed and detected directly through anti-m13 peroxidase conjugated antibodies. Binding was quantitatively determined following colorimetric substrate product formation with TMB. The results are shown in FIGS. 18 and 19.

EXAMPLE 12

Phage Surrogate Light Chain Construct Library Constructions, Selection, and Clonal ELISA

[0169] An iterative approach using combinatorial antibody libraries was employed to generate and test surrogate light chain constructs that bound antigen. Briefly, combinatorial antibody libraries prepared from the bone marrow of H5N1 avian influenza survivors were created. These libraries were screened against H5N1 viral hemagglutinin protein for two rounds of selection. Next the phagemid plasmid was amplified and purified. Heavy chain variable regions isolated by restriction digest from this plasmid preparation and cloned in frame with the constant heavy domain 1 to form a recombinant fusion to the m 13 gene III coat protein for phagemid display. Importantly, we used two recipient plasmids that either coexpressed a surrogate light chain fusion comprised of VpreB1 and lambda 5 (SLC fusion 1) (SEQ ID NO: 10) or VpreB1 and a constant lambda domain (SEQ ID NO: 11) from the classical lambda light chain (SLC fusion 2). The fusion 1 library produced 3.84.times.10.sup.7 independent transformants, while the fusion 2 library produced 7.8.times.10.sup.7 transformants. Both libraries were screened independently through two rounds and both showed significant enrichment over background (Fusion 1=5x, Fusion 2=20x) that increased in a second round of panning (Fusion 1=97x, Fusion 2=48x).

[0170] To test by ELISA for clonal antigen binding phage from both rounds and both libraries were transferred into the HB2151 E. coli strain to produce soluble surrobody fusion proteins. Briefly, HB2151 clones were grown and induced to produce soluble surrobodies. Specifically, colonies were cultured overnight in 2-YT media supplemented with 100 mcg/ml ampicillin and 200 micromolar IPTG overnight at 30 degrees the periplasmic lysates, as described above. The resulting periplasmic lysates were tested by ELISA, essentially as outlined above.

[0171] The number of transformants and percent positive clones for the two fusions, at rounds 1 and 2 of panning are shown in FIG. 20, and the clonal analysis data for Rounds 1 and 2 of Fusion 1 and Fusion 2 library clones are shown in FIGS. 21 and 22.

EXAMPLE 13

Surrogate Light Chain Fusions to Increase Serum Half-Life

[0172] The half-life of an antibody fragment in vivo is extended considerably when it is part of a fusion to an intact and complete heavy chain that includes all heavy chain constant domains, not just those necessary to form a stable antigen binding fragment. In the case of IgG this means the inclusion of domains CH1, CH2, and CH3. In particular it is well established that CH2 and CH3 confer the majority of this effect in vivo. In fact fusion of these CH2 and CH3 domains to heterologous proteins is typically sufficient to improve the potencies and PK/PD of these chimeric molecules compared to the parent molecules. Similarly functional fusions to the either or both VpreB and .lamda.5 benefit by this association with the constant domains of the heavy chain.

[0173] For the treatment of type II diabetes administration of glucagon-like peptide 1 (or GLP-1) benefits individuals by inducing glucose-dependent insulin secretion in the pancreas, thereby improving glucose management in those patients. However, a long-lived GLP-1 peptide is a desirable goal. As the tails of the surrogate light chains are distinct and accessible, we could accomplish this goal by either recombinantly fusing the active GLP-1 moiety to either the C-terminus of the VpreB1 tail (SEQ ID NOs: 23 and 24) or the N-terminal tail of .lamda.5 (SEQ ID NOs: 25 and 26). In the case of a .lamda.5 fusion we may express this in the presence or absence of VpreB1 and even in the presence or absence of the Variable heavy domain, as depicted in FIG. 11. Fusions to VpreB1 can similarly be made in the presence or absence of .lamda.5, and possibly with or without the CH1 domain of the heavy chain. Similarly, other beneficial growth factor, cytokine, receptor, and enzyme fusions may be created. In all of these cases binding is not requisite of the surrogate light chain, or SURROBODY.TM. components, but rather may be conferred either entirely or in large part by the heterologous surrogate light chain fused element.

[0174] Although in the foregoing description the invention is illustrated with reference to certain embodiments, it is not so limited. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

EXAMPLE 14

Affinity Determination of Hemagglutinin-Binding Surrogate Light Chain Constructs

[0175] To determine the affinities of fusion surrogate light chain constructs (SURROBODIES.TM., see, FIG. 11) we overexpressed and purified various surrobodics in E. coli and compared them to the parental Fabs from which the heavy chains were first identified. Affinities were determined by Bio-Layer interferometry on a BioForte Octet essentially as follows. First, 100 .mu.g of purified hemagglutinin protein was biotinlyated at a 20:1 molar excess using Pierce No-Weigh PEO4 biotin (cat #21329) according to manufacturer's instructions, incubated at room temperature for 1-3 hours with intermittent mixing and then incubated overnight at 4 C. The excess biotin was removed by size exclusion spin column and exchanged into PBS. Next, HA binding surrogate light chain constructs and Fabs were purified by FPLC using Ni.sup.2+ affinity chromatography, desalted to remove excess imidazole, concentrated, and quantitated by quantitative light chain ELISAs (Bethel Labs, cat #E80-115-.kappa., and E80-116-.lamda.) are performed according to the manufacturer's instructions. Finally affinities were determined by analyzing a range of sample concentrations that are typically 15 nM-500 nM in serial 2 fold dilutions. The samples were incubated with biosensors coated with HA protein for up to 15 minutes, then incubated in sample diluent for up to 1 hour. All of these steps were done with sample plate rotation at 1500 RPM. Association was measured during the Fab incubation with the HA-coated biosensors and dissociation is measured in the sample diluent incubation following binding. Affinities are shown in the following Table

TABLE-US-00001 Fusion 1 Fusion 1 Clone VpreB1-Lambda5 VpreB1-constant lambda Fab F5 250-400 pM 150-270 pM 1 pM B11 31-180 pM Not determined 13 pM

[0176] All references cited throughout the specification, and the references cited therein, are hereby expressly incorporated by reference in their entirety.

Sequence CWU 1

1

351145PRTHomo sapiens 1Met Ser Trp Ala Pro Val Leu Leu Met Leu Phe Val Tyr Cys Thr Gly1 5 10 15Cys Gly Pro Gln Pro Val Leu His Gln Pro Pro Ala Met Ser Ser Ala 20 25 30Leu Gly Thr Thr Ile Arg Leu Thr Cys Thr Leu Arg Asn Asp His Asp 35 40 45Ile Gly Val Tyr Ser Val Tyr Trp Tyr Gln Gln Arg Pro Gly His Pro 50 55 60Pro Arg Phe Leu Leu Arg Tyr Phe Ser Gln Ser Asp Lys Ser Gln Gly65 70 75 80Pro Gln Val Pro Pro Arg Phe Ser Gly Ser Lys Asp Val Ala Arg Asn 85 90 95Arg Gly Tyr Leu Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala Met 100 105 110Tyr Tyr Cys Ala Met Gly Ala Arg Ser Ser Glu Lys Glu Glu Arg Glu 115 120 125Arg Glu Trp Glu Glu Glu Met Glu Pro Thr Ala Ala Arg Thr Arg Val 130 135 140Pro1452142PRTMus sp. 2Met Ala Trp Thr Ser Val Leu Leu Met Leu Leu Ala His Leu Thr Gly1 5 10 15Cys Gly Pro Gln Pro Met Val His Gln Pro Pro Ser Ala Ser Ser Ser 20 25 30Leu Gly Ala Thr Ile Arg Leu Ser Cys Thr Leu Ser Asn Asp His Asn 35 40 45Ile Gly Ile Tyr Ser Ile Tyr Trp Tyr Gln Gln Arg Pro Gly His Pro 50 55 60Pro Arg Phe Leu Leu Arg Tyr Phe Ser His Ser Asp Lys His Gln Gly65 70 75 80Pro Asp Ile Pro Pro Arg Phe Ser Gly Ser Lys Asp Thr Ala Arg Asn 85 90 95Leu Gly Tyr Leu Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala Val 100 105 110Tyr Tyr Cys Ala Val Gly Leu Arg Ser His Glu Lys Lys Arg Met Glu 115 120 125Arg Glu Trp Glu Gly Glu Lys Ser Tyr Thr Asp Leu Gly Ser 130 135 1403171PRTMus sp. 3Met Ala Trp Thr Ser Val Leu Leu Met Leu Leu Ala His Leu Thr Gly1 5 10 15Lys Gly Thr Leu Gly Val Gln Gly Phe Leu Ala Pro Pro Val Ala Leu 20 25 30Leu Cys Pro Ser Asp Gly His Ala Ser Ile Phe Ser Gly Cys Gly Pro 35 40 45Gln Pro Met Val His Gln Pro Pro Ser Ala Ser Ser Ser Leu Gly Ala 50 55 60Thr Ile Arg Leu Ser Cys Thr Leu Ser Asn Asp His Asn Ile Gly Ile65 70 75 80Tyr Ser Ile Tyr Trp Tyr Gln Gln Arg Pro Gly His Pro Pro Arg Phe 85 90 95Leu Leu Arg Tyr Phe Ser His Ser Asp Lys His Gln Gly Pro Asp Ile 100 105 110Pro Pro Arg Phe Ser Gly Ser Lys Asp Thr Ala Arg Asn Leu Gly Tyr 115 120 125Leu Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala Val Tyr Tyr Cys 130 135 140Ala Val Gly Leu Arg Ser His Glu Lys Lys Arg Met Glu Arg Glu Trp145 150 155 160Glu Gly Glu Lys Ser Tyr Thr Asp Leu Gly Ser 165 1704123PRTHomo sapiens 4Met Ala Cys Arg Cys Leu Ser Phe Leu Leu Met Gly Thr Phe Leu Ser1 5 10 15Val Ser Gln Thr Val Leu Ala Gln Leu Asp Ala Leu Leu Val Phe Pro 20 25 30Gly Gln Val Ala Gln Leu Ser Cys Thr Leu Ser Pro Gln His Val Thr 35 40 45Ile Arg Asp Tyr Gly Val Ser Trp Tyr Gln Gln Arg Ala Gly Ser Ala 50 55 60Pro Arg Tyr Leu Leu Tyr Tyr Arg Ser Glu Glu Asp His His Arg Pro65 70 75 80Ala Asp Ile Pro Asp Arg Phe Ser Ala Ala Lys Asp Glu Ala His Asn 85 90 95Ala Cys Val Leu Thr Ile Ser Pro Val Gln Pro Glu Asp Asp Ala Asp 100 105 110Tyr Tyr Cys Ser Val Gly Tyr Gly Phe Ser Pro 115 1205209PRTHomo sapiens 5Met Lys Leu Arg Val Gly Gln Thr Leu Gly Thr Ile Pro Arg Gln Cys1 5 10 15Glu Val Leu Leu Leu Leu Leu Leu Leu Gly Leu Val Asp Gly Val His 20 25 30His Ile Leu Ser Pro Ser Ser Ala Glu Arg Ser Arg Ala Val Gly Pro 35 40 45Gly Ala Ser Val Gly Ser Asn Arg Pro Ser Leu Trp Ala Leu Pro Gly 50 55 60Arg Leu Leu Phe Gln Ile Ile Pro Arg Gly Ala Gly Pro Arg Cys Ser65 70 75 80Pro His Arg Leu Pro Ser Lys Pro Gln Phe Trp Tyr Val Phe Gly Gly 85 90 95Gly Thr Gln Leu Thr Ile Leu Gly Gln Pro Lys Ser Asp Pro Leu Val 100 105 110Thr Leu Phe Leu Pro Ser Leu Lys Asn Leu Gln Pro Thr Arg Pro His 115 120 125Val Val Cys Leu Val Ser Glu Phe Tyr Pro Gly Thr Leu Val Val Asp 130 135 140Trp Lys Val Asp Gly Val Pro Val Thr Gln Gly Val Glu Thr Thr Gln145 150 155 160Pro Ser Lys Gln Thr Asn Asn Lys Tyr Met Val Ser Ser Tyr Leu Thr 165 170 175Leu Ile Ser Asp Gln Trp Met Pro His Ser Arg Tyr Ser Cys Arg Val 180 185 190Thr His Glu Gly Asn Thr Val Glu Lys Ser Val Ser Pro Ala Glu Cys 195 200 205Ser6213PRTHomo sapiens 6Met Arg Pro Gly Thr Gly Gln Gly Gly Leu Glu Ala Pro Gly Glu Pro1 5 10 15Gly Pro Asn Leu Arg Gln Arg Trp Pro Leu Leu Leu Leu Gly Leu Ala 20 25 30Val Val Thr His Gly Leu Leu Arg Pro Thr Ala Ala Ser Gln Ser Arg 35 40 45Ala Leu Gly Pro Gly Ala Pro Gly Gly Ser Ser Arg Ser Ser Leu Arg 50 55 60Ser Arg Trp Gly Arg Phe Leu Leu Gln Arg Gly Ser Trp Thr Gly Pro65 70 75 80Arg Cys Trp Pro Arg Gly Phe Gln Ser Lys His Asn Ser Val Thr His 85 90 95Val Phe Gly Ser Gly Thr Gln Leu Thr Val Leu Ser Gln Pro Lys Ala 100 105 110Thr Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala 115 120 125Asn Lys Ala Thr Leu Val Cys Leu Met Asn Asp Phe Tyr Pro Gly Ile 130 135 140Leu Thr Val Thr Trp Lys Ala Asp Gly Thr Pro Ile Thr Gln Gly Val145 150 155 160Glu Met Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser 165 170 175Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Arg Ser Arg Arg Ser Tyr 180 185 190Ser Cys Gln Val Met His Glu Gly Ser Thr Val Glu Lys Thr Val Ala 195 200 205Pro Ala Glu Cys Ser 2107158PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic construct" 7Met Arg Pro Gly Thr Gly Gln Gly Gly Leu Glu Ala Pro Gly Glu Pro1 5 10 15Gly Pro Asn Leu Arg Gln Arg Trp Pro Leu Leu Leu Leu Gly Leu Ala 20 25 30Val Val Thr His Gly Ser Val Thr His Val Phe Gly Ser Gly Thr Gln 35 40 45Leu Thr Val Leu Ser Gln Pro Lys Ala Thr Pro Ser Val Thr Leu Phe 50 55 60Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys65 70 75 80Leu Met Asn Asp Phe Tyr Pro Gly Ile Leu Thr Val Thr Trp Lys Ala 85 90 95Asp Gly Thr Pro Ile Thr Gln Gly Val Glu Met Thr Thr Pro Ser Lys 100 105 110Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro 115 120 125Glu Gln Trp Arg Ser Arg Arg Ser Tyr Ser Cys Gln Val Met His Glu 130 135 140Gly Ser Thr Val Glu Lys Thr Val Ala Pro Ala Glu Cys Ser145 150 1558119PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic construct" 8Met Ser Trp Ala Pro Val Leu Leu Met Leu Phe Val Tyr Cys Thr Gly1 5 10 15Cys Gly Pro Gln Pro Val Leu His Gln Pro Pro Ala Met Ser Ser Ala 20 25 30Leu Gly Thr Thr Ile Arg Leu Thr Cys Thr Leu Arg Asn Asp His Asp 35 40 45Ile Gly Val Tyr Ser Val Tyr Trp Tyr Gln Gln Arg Pro Gly His Pro 50 55 60Pro Arg Phe Leu Leu Arg Tyr Phe Ser Gln Ser Asp Lys Ser Gln Gly65 70 75 80Pro Gln Val Pro Pro Arg Phe Ser Gly Ser Lys Asp Val Ala Arg Asn 85 90 95Arg Gly Tyr Leu Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala Met 100 105 110Tyr Tyr Cys Ala Met Gly Ala 1159480PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic construct" 9Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Asp Ala Gln Met Gln Leu Gln Glu Ser Gly Pro Gly 20 25 30Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly 35 40 45Tyr Ser Phe Asp Ser Gly Tyr Tyr Trp Gly Trp Leu Arg Gln Pro Pro 50 55 60Gly Lys Gly Leu Glu Trp Ile Gly Ser Ile Tyr His Ser Arg Asn Thr65 70 75 80Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr 85 90 95Ser Lys Asn Gln Phe Ser Leu Gln Leu Ser Ser Val Thr Ala Ala Asp 100 105 110Thr Ala Val Tyr Tyr Cys Ala Arg Gly Thr Trp Tyr Ser Ser Asn Leu 115 120 125Arg Tyr Trp Phe Asp Pro Trp Gly Lys Gly Thr Leu Val Arg Val Ser 130 135 140Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser145 150 155 160Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 165 170 175Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 180 185 190Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 195 200 205Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 210 215 220Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp225 230 235 240Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 245 250 255Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 260 265 270Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 275 280 285Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 290 295 300Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg305 310 315 320Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 325 330 335Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 340 345 350Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 355 360 365Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 370 375 380Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe385 390 395 400Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 405 410 415Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 420 425 430Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 435 440 445Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 450 455 460Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly His His His His His His465 470 475 48010242PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic construct" 10Met Ser Trp Ala Pro Val Leu Leu Met Leu Phe Val Tyr Cys Thr Gly1 5 10 15Cys Gly Pro Gln Pro Val Leu His Gln Pro Pro Ala Met Ser Ser Ala 20 25 30Leu Gly Thr Thr Ile Arg Leu Thr Cys Thr Leu Arg Asn Asp His Asp 35 40 45Ile Gly Val Tyr Ser Val Tyr Trp Tyr Gln Gln Arg Pro Gly His Pro 50 55 60Pro Arg Phe Leu Leu Arg Tyr Phe Ser Gln Ser Asp Lys Ser Gln Gly65 70 75 80Pro Gln Val Pro Pro Arg Phe Ser Gly Ser Lys Asp Val Ala Arg Asn 85 90 95Arg Gly Tyr Leu Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala Met 100 105 110Tyr Tyr Cys Ala Met Gly Ala Arg Ser Ser Val Thr His Val Phe Gly 115 120 125Ser Gly Thr Gln Leu Thr Val Leu Ser Gln Pro Lys Ala Thr Pro Ser 130 135 140Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala145 150 155 160Thr Leu Val Cys Leu Met Asn Asp Phe Tyr Pro Gly Ile Leu Thr Val 165 170 175Thr Trp Lys Ala Asp Gly Thr Pro Ile Thr Gln Gly Val Glu Met Thr 180 185 190Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu 195 200 205Ser Leu Thr Pro Glu Gln Trp Arg Ser Arg Arg Ser Tyr Ser Cys Gln 210 215 220Val Met His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Ala Glu225 230 235 240Cys Ser11242PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic construct" 11Met Ser Trp Ala Pro Val Leu Leu Met Leu Phe Val Tyr Cys Thr Gly1 5 10 15Cys Gly Pro Gln Pro Val Leu His Gln Pro Pro Ala Met Ser Ser Ala 20 25 30Leu Gly Thr Thr Ile Arg Leu Thr Cys Thr Leu Arg Asn Asp His Asp 35 40 45Ile Gly Val Tyr Ser Val Tyr Trp Tyr Gln Gln Arg Pro Gly His Pro 50 55 60Pro Arg Phe Leu Leu Arg Tyr Phe Ser Gln Ser Asp Lys Ser Gln Gly65 70 75 80Pro Gln Val Pro Pro Arg Phe Ser Gly Ser Lys Asp Val Ala Arg Asn 85 90 95Arg Gly Tyr Leu Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala Met 100 105 110Tyr Tyr Cys Ala Met Gly Ala Arg Ser Ser Val Thr His Val Phe Gly 115 120 125Ser Gly Thr Gln Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser 130 135 140Val Thr Leu Phe Pro Pro Ser Ser Xaa Glu Leu Gln Ala Asn Lys Ala145 150 155 160Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val 165 170 175Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr 180 185 190Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu 195 200 205Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln 210 215 220Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Ala Glu225 230 235 240Cys Ser12256PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic construct" 12Val Lys Lys Leu Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr1 5 10 15Ser His Ser Ala Gln Pro Val Leu His Gln Pro Pro Ala Met Ser Ser 20 25 30Ala Leu Gly Thr Thr Ile Arg Leu Thr Cys Thr Leu Arg Asn Asp His 35 40 45Asp Ile Gly Val Tyr Ser Val Tyr Trp Tyr Gln Gln Arg Pro Gly His 50 55 60Pro Pro Arg Phe Leu Leu Arg Tyr Phe Ser Gln Ser Asp Lys Ser Gln65 70 75 80Gly Pro Gln Val Pro Pro Arg Phe Ser Gly Ser Lys Asp Val Ala Arg 85 90 95Asn Arg Gly Tyr Leu Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala 100 105 110Met Tyr Tyr Cys Ala Met Gly Ala Arg Ser Ser Val Thr His Val Phe 115 120 125Gly Ser Gly Thr Gln Leu Thr Val Leu Ser Gln Pro Lys Ala Thr Pro 130 135 140Ser Val Thr

Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys145 150 155 160Ala Thr Leu Val Cys Leu Met Asn Asp Phe Tyr Pro Gly Ile Leu Thr 165 170 175Val Thr Trp Lys Ala Asp Gly Thr Pro Ile Thr Gln Gly Val Glu Met 180 185 190Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr 195 200 205Leu Ser Leu Thr Pro Glu Gln Trp Arg Ser Arg Arg Ser Tyr Ser Cys 210 215 220Gln Val Met His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Ala225 230 235 240Glu Cys Ser Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg 245 250 25513256PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic construct" 13Val Lys Lys Leu Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr1 5 10 15Ser His Ser Ala Gln Pro Val Leu His Gln Pro Pro Ala Met Ser Ser 20 25 30Ala Leu Gly Thr Thr Ile Arg Leu Thr Cys Thr Leu Arg Asn Asp His 35 40 45Asp Ile Gly Val Tyr Ser Val Tyr Trp Tyr Gln Gln Arg Pro Gly His 50 55 60Pro Pro Arg Phe Leu Leu Arg Tyr Phe Ser Gln Ser Asp Lys Ser Gln65 70 75 80Gly Pro Gln Val Pro Pro Arg Phe Ser Gly Ser Lys Asp Val Ala Arg 85 90 95Asn Arg Gly Tyr Leu Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala 100 105 110Met Tyr Tyr Cys Ala Met Gly Ala Arg Ser Ser Val Thr His Val Phe 115 120 125Gly Ser Gly Thr Gln Leu Thr Val Leu Arg Gln Pro Lys Ala Ala Pro 130 135 140Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys145 150 155 160Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr 165 170 175Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Ala Gly Val Glu Thr 180 185 190Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr 195 200 205Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Lys Ser Tyr Ser Cys 210 215 220Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr225 230 235 240Glu Cys Ser Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg 245 250 25514269PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic construct" 14Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly 20 25 30Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45Phe Pro Phe Ser Ser Tyr Val Met Ile Trp Val Arg Gln Val Pro Gly 50 55 60Lys Gly Leu Glu Trp Val Ser Ala Ile Gly Gly Ser Gly Gly Ser Thr65 70 75 80Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Asp Asp 100 105 110Thr Ala Val Tyr Tyr Cys Val Leu Ser Pro Lys Ser Tyr Tyr Asp Asn 115 120 125Ser Gly Ile Tyr Phe Asp Phe Trp Gly Lys Gly Thr Leu Val Arg Val 130 135 140Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser145 150 155 160Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 165 170 175Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 180 185 190Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 195 200 205Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 210 215 220Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val225 230 235 240Asp Lys Arg Val Glu Pro Lys Ser Cys Ala Ala Ala His His His His 245 250 255His His Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 260 26515271PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic construct" 15Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly 20 25 30Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45Phe Pro Phe Ser Ser Tyr Val Met Ile Trp Val Arg Gln Val Pro Gly 50 55 60Lys Gly Leu Glu Trp Val Ser Ala Ile Gly Gly Ser Gly Gly Ser Thr65 70 75 80Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Asp Asp 100 105 110Thr Ala Val Tyr Tyr Cys Val Leu Ser Pro Lys Ser Tyr Tyr Asp Asn 115 120 125Ser Gly Ile Tyr Phe Asp Phe Trp Gly Lys Gly Thr Leu Val Arg Val 130 135 140Ser Ser Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys145 150 155 160Glu Asn Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala 165 170 175Gln Asp Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp Lys Tyr Lys Asn 180 185 190Asn Ser Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly 195 200 205Gly Lys Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val 210 215 220Met Gln Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn225 230 235 240Gly Asn Lys Glu Lys Asn Val Pro Leu Pro Val Ala Ala Ala His His 245 250 255His His His His Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 260 265 2701613PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 16Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg1 5 101712PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 17Gly Glu Gln Lys Leu Ile Ser Leu Glu Glu Asp Leu1 5 1018159PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic construct" 18Val Lys Lys Leu Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr1 5 10 15Ser His Ser Ala Gln Pro Val Leu His Gln Pro Pro Ala Met Ser Ser 20 25 30Ala Leu Gly Thr Thr Ile Arg Leu Thr Cys Thr Leu Arg Asn Asp His 35 40 45Asp Ile Gly Val Tyr Ser Val Tyr Trp Tyr Gln Gln Arg Pro Gly His 50 55 60Pro Pro Arg Phe Leu Leu Arg Tyr Phe Ser Gln Ser Asp Lys Ser Gln65 70 75 80Gly Pro Gln Val Pro Pro Arg Phe Ser Gly Ser Lys Asp Val Ala Arg 85 90 95Asn Arg Gly Tyr Leu Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala 100 105 110Met Tyr Tyr Cys Ala Met Gly Ala Arg Ser Ser Glu Lys Glu Glu Arg 115 120 125Glu Arg Glu Trp Glu Glu Glu Met Glu Pro Thr Ala Ala Arg Thr Arg 130 135 140Val Pro Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg145 150 15519120PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic construct" 19Val Lys Lys Leu Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr1 5 10 15Ser His Ser Ala Gln Pro Val Leu His Gln Pro Pro Ala Met Ser Ser 20 25 30Ala Leu Gly Thr Thr Ile Arg Leu Thr Cys Thr Leu Arg Asn Asp His 35 40 45Asp Ile Gly Val Tyr Ser Val Tyr Trp Tyr Gln Gln Arg Pro Gly His 50 55 60Pro Pro Arg Phe Leu Leu Arg Tyr Phe Ser Gln Ser Asp Lys Ser Gln65 70 75 80Gly Pro Gln Val Pro Pro Arg Phe Ser Gly Ser Lys Asp Val Ala Arg 85 90 95Asn Arg Gly Tyr Leu Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala 100 105 110Met Tyr Tyr Cys Ala Met Gly Ala 115 12020133PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic construct" 20Val Lys Lys Leu Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr1 5 10 15Ser His Ser Ala Gln Pro Val Leu His Gln Pro Pro Ala Met Ser Ser 20 25 30Ala Leu Gly Thr Thr Ile Arg Leu Thr Cys Thr Leu Arg Asn Asp His 35 40 45Asp Ile Gly Val Tyr Ser Val Tyr Trp Tyr Gln Gln Arg Pro Gly His 50 55 60Pro Pro Arg Phe Leu Leu Arg Tyr Phe Ser Gln Ser Asp Lys Ser Gln65 70 75 80Gly Pro Gln Val Pro Pro Arg Phe Ser Gly Ser Lys Asp Val Ala Arg 85 90 95Asn Arg Gly Tyr Leu Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala 100 105 110Met Tyr Tyr Cys Ala Met Gly Ala Gly Ala Pro Val Pro Tyr Pro Asp 115 120 125Pro Leu Glu Pro Arg 13021198PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic construct" 21Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala1 5 10 15Thr Val Ala Gln Ala Ala Leu Leu Arg Pro Thr Ala Ala Ser Gln Ser 20 25 30Arg Ala Leu Gly Pro Gly Ala Pro Gly Gly Ser Ser Arg Ser Ser Leu 35 40 45Arg Ser Arg Trp Gly Arg Phe Leu Leu Gln Arg Gly Ser Trp Thr Gly 50 55 60Pro Arg Cys Trp Pro Arg Gly Phe Gln Ser Lys His Asn Ser Val Thr65 70 75 80His Val Phe Gly Ser Gly Thr Gln Leu Thr Val Leu Ser Gln Pro Lys 85 90 95Ala Thr Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 100 105 110Ala Asn Lys Ala Thr Leu Val Cys Leu Met Asn Asp Phe Tyr Pro Gly 115 120 125Ile Leu Thr Val Thr Trp Lys Ala Asp Gly Thr Pro Ile Thr Gln Gly 130 135 140Val Glu Met Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala145 150 155 160Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Arg Ser Arg Arg Ser 165 170 175Tyr Ser Cys Gln Val Met His Glu Gly Ser Thr Val Glu Lys Thr Val 180 185 190Ala Pro Ala Glu Cys Ser 19522143PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic construct" 22Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala1 5 10 15Thr Val Ala Gln Ala Ala Ser Val Thr His Val Phe Gly Ser Gly Thr 20 25 30Gln Leu Thr Val Leu Ser Gln Pro Lys Ala Thr Pro Ser Val Thr Leu 35 40 45Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val 50 55 60Cys Leu Met Asn Asp Phe Tyr Pro Gly Ile Leu Thr Val Thr Trp Lys65 70 75 80Ala Asp Gly Thr Pro Ile Thr Gln Gly Val Glu Met Thr Thr Pro Ser 85 90 95Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr 100 105 110Pro Glu Gln Trp Arg Ser Arg Arg Ser Tyr Ser Cys Gln Val Met His 115 120 125Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Ala Glu Cys Ser 130 135 14023175PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic construct" 23Met Ser Trp Ala Pro Val Leu Leu Met Leu Phe Val Tyr Cys Thr Gly1 5 10 15Cys Gly Pro Gln Pro Val Leu His Gln Pro Pro Ala Met Ser Ser Ala 20 25 30Leu Gly Thr Thr Ile Arg Leu Thr Cys Thr Leu Arg Asn Asp His Asp 35 40 45Ile Gly Val Tyr Ser Val Tyr Trp Tyr Gln Gln Arg Pro Gly His Pro 50 55 60Pro Arg Phe Leu Leu Arg Tyr Phe Ser Gln Ser Asp Lys Ser Gln Gly65 70 75 80Pro Gln Val Pro Pro Arg Phe Ser Gly Ser Lys Asp Val Ala Arg Asn 85 90 95Arg Gly Tyr Leu Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala Met 100 105 110Tyr Tyr Cys Ala Met Gly Ala Arg Ser Ser Glu Lys Glu Glu Arg Glu 115 120 125Arg Glu Trp Glu Glu Glu Met Glu Pro Thr Ala Ala Arg Thr Arg Val 130 135 140Pro His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu145 150 155 160Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 165 170 17524149PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic construct" 24Met Ser Trp Ala Pro Val Leu Leu Met Leu Phe Val Tyr Cys Thr Gly1 5 10 15Cys Gly Pro Gln Pro Val Leu His Gln Pro Pro Ala Met Ser Ser Ala 20 25 30Leu Gly Thr Thr Ile Arg Leu Thr Cys Thr Leu Arg Asn Asp His Asp 35 40 45Ile Gly Val Tyr Ser Val Tyr Trp Tyr Gln Gln Arg Pro Gly His Pro 50 55 60Pro Arg Phe Leu Leu Arg Tyr Phe Ser Gln Ser Asp Lys Ser Gln Gly65 70 75 80Pro Gln Val Pro Pro Arg Phe Ser Gly Ser Lys Asp Val Ala Arg Asn 85 90 95Arg Gly Tyr Leu Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala Met 100 105 110Tyr Tyr Cys Ala Met Gly Ala His Ala Glu Gly Thr Phe Thr Ser Asp 115 120 125Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp 130 135 140Leu Val Lys Gly Arg14525243PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic construct" 25Met Arg Pro Gly Thr Gly Gln Gly Gly Leu Glu Ala Pro Gly Glu Pro1 5 10 15Gly Pro Asn Leu Arg Gln Arg Trp Pro Leu Leu Leu Leu His Ala Glu 20 25 30Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala 35 40 45Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Leu Ala Val Val 50 55 60Thr His Gly Leu Leu Arg Pro Thr Ala Ala Ser Gln Ser Arg Ala Leu65 70 75 80Gly Pro Gly Ala Pro Gly Gly Ser Ser Arg Ser Ser Leu Arg Ser Arg 85 90 95Trp Gly Arg Phe Leu Leu Gln Arg Gly Ser Trp Thr Gly Pro Arg Cys 100 105 110Trp Pro Arg Gly Phe Gln Ser Lys His Asn Ser Val Thr His Val Phe 115 120 125Gly Ser Gly Thr Gln Leu Thr Val Leu Ser Gln Pro Lys Ala Thr Pro 130 135 140Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys145 150 155 160Ala Thr Leu Val Cys Leu Met Asn Asp Phe Tyr Pro Gly Ile Leu Thr 165 170 175Val Thr Trp Lys Ala Asp Gly Thr Pro Ile Thr Gln Gly Val Glu Met 180 185 190Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr 195 200 205Leu Ser Leu Thr Pro Glu Gln Trp Arg Ser Arg Arg Ser Tyr Ser Cys 210 215 220Gln Val Met His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Ala225 230 235 240Glu Cys Ser26188PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic construct" 26Met Arg Pro Gly Thr Gly Gln Gly Gly Leu Glu Ala Pro Gly Glu Pro1 5 10 15Gly Pro Asn

Leu Arg Gln Arg Trp Pro Leu Leu Leu Leu Gly His Ala 20 25 30Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala 35 40 45Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Leu Ala Val Val 50 55 60Thr His Gly Ser Val Thr His Val Phe Gly Ser Gly Thr Gln Leu Thr65 70 75 80Val Leu Ser Gln Pro Lys Ala Thr Pro Ser Val Thr Leu Phe Pro Pro 85 90 95Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Met 100 105 110Asn Asp Phe Tyr Pro Gly Ile Leu Thr Val Thr Trp Lys Ala Asp Gly 115 120 125Thr Pro Ile Thr Gln Gly Val Glu Met Thr Thr Pro Ser Lys Gln Ser 130 135 140Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln145 150 155 160Trp Arg Ser Arg Arg Ser Tyr Ser Cys Gln Val Met His Glu Gly Ser 165 170 175Thr Val Glu Lys Thr Val Ala Pro Ala Glu Cys Ser 180 18527105PRTHomo sapiens 27Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro1 5 10 15Gly Gln Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly 20 25 30Asn Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys 35 40 45Leu Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg 50 55 60Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly65 70 75 80Leu Gln Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser 85 90 95Ser Leu Ser Ala Val Val Phe Gly Gly 100 10528114PRTHomo sapiens 28Gly Gly Thr Lys Leu Thr Val Leu Arg Gln Pro Lys Ala Ala Pro Ser1 5 10 15Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala 20 25 30Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val 35 40 45Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Ala Gly Val Glu Thr Thr 50 55 60Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu65 70 75 80Ser Leu Thr Pro Glu Gln Trp Lys Ser His Lys Ser Tyr Ser Cys Gln 85 90 95Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu 100 105 110Cys Ser 29105PRTHomo sapiens 29Gln Pro Val Leu Thr Gln Pro Ser Ser His Ser Ala Ser Ser Gly Ala1 5 10 15Ser Val Arg Leu Thr Cys Met Leu Ser Ser Gly Phe Ser Val Gly Asp 20 25 30Phe Trp Ile Arg Trp Tyr Gln Gln Lys Pro Gly Asn Pro Pro Arg Tyr 35 40 45Leu Leu Tyr Tyr His Ser Asp Ser Asn Lys Gly Gln Gly Ser Gly Val 50 55 60Pro Ser Arg Phe Ser Gly Ser Asn Asp Ala Ser Ala Asn Ala Gly Ile65 70 75 80Leu Arg Ile Ser Gly Leu Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys 85 90 95Gly Thr Trp His Ser Asn Ser Lys Thr 100 10530104PRTHomo sapiens 30Gln Ala Val Leu Thr Gln Pro Ala Ser Leu Ser Ala Ser Pro Gly Ala1 5 10 15Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Asn Val Gly Thr 20 25 30Tyr Arg Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Tyr 35 40 45Leu Leu Arg Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly Ser Gly Val 50 55 60Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn Ala Gly Ile65 70 75 80Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys 85 90 95Met Ile Trp His Ser Ser Ala Ser 10031104PRTHomo sapiens 31Gln Pro Val Leu Thr Gln Pro Pro Ser Ser Ser Ala Ser Pro Gly Glu1 5 10 15Ser Ala Arg Leu Thr Cys Thr Leu Pro Ser Asp Ile Asn Val Gly Ser 20 25 30Tyr Asn Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Arg Tyr 35 40 45Leu Leu Tyr Tyr Tyr Ser Asp Ser Asp Lys Gly Gln Gly Ser Gly Val 50 55 60Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn Thr Gly Ile65 70 75 80Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys 85 90 95Met Ile Trp Pro Ser Asn Ala Ser 10032113PRTHomo sapiens 32Gly Thr Lys Leu Thr Val Leu Arg Gln Pro Lys Ala Ala Pro Ser Val1 5 10 15Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr 20 25 30Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala 35 40 45Trp Lys Ala Asp Gly Ser Pro Val Lys Ala Gly Val Glu Thr Thr Thr 50 55 60Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser65 70 75 80Leu Thr Pro Glu Gln Trp Lys Ser His Lys Ser Tyr Ser Cys Gln Val 85 90 95Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys 100 105 110Ser 33107PRTHomo sapiens 33Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105346PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic 6xHis tag" 34His His His His His His1 53515PRTArtificial Sequencesource/note="Description of Artificial Sequence Synthetic peptide" 35Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 15

Claims

1-88. (canceled)

89. A trimeric polypeptide complex comprising (a) a VpreB sequence; a .lamda.5 sequence or an antibody light chain constant region sequence; and an antibody heavy chain sequence, or (b) a .lamda.5 sequence; an antibody light chain variable region sequence; and an antibody heavy chain sequence, wherein said sequences are non-covalently associated with each other; and wherein said trimeric polypeptide complex binds to a target.

90. The complex of claim 89, comprising a VpreB1 sequence.

91. The complex of claim 90, comprising all or part of a VpreB1 sequence of SEQ ID NO: 1.

92. The complex of claim 89, comprising all or part of a .lamda.5 sequence of SEQ ID NO: 6.

93. The complex of claim 91, comprising all or part of a .lamda.5 sequence of SEQ ID NO: 6.

94. The complex of claim 91, wherein at least part of the C-terminal tail of said VpreB1 sequence is removed.

95. The complex of claim 93, wherein at least part of the N-terminal tail of said .lamda.5 sequence is removed.

96. The complex of claim 93, wherein at least part of the C-terminal tail of said VpreB1 sequence and at least part of the N-terminal tail of said .lamda.5 sequence is removed.

97. The complex of claim 94, wherein the entire C-terminal tail of said VpreB1 sequence is removed.

98. The complex of claim 95, wherein the entire N-terminal tail of said .lamda.5 sequence is removed.

99. The complex of claim 98, comprising at least part of the C-terminal tail of said VpreB1 sequence.

100. The complex of claim 99, comprising the entire C-terminal tail of said VpreB1 sequence.

101. The complex of claim 96, wherein the entire C-terminal tail of said VpreB1 sequence and the entire N-terminal tail of said .lamda.5 sequence is removed.

102. The complex of claim 89 comprising a full-length VpreB1 sequence of SEQ ID NO: 1, and a .lamda.5 sequence of SEQ ID NO: 6 having at least part of its N-terminal tail removed.

103. The complex of claim 102 comprising a full-length VpreB1 sequence of SEQ ID NO: 1, and a .lamda.5 sequence of SEQ ID NO: 6 having its N-terminal tail removed.

104. The complex of claim 89 comprising a full-length .lamda.5 sequence of SEQ ID NO: 6, and a VpreB1 sequence of SEQ ID NO: 1 having at least part of its C-terminal tail removed.

105. The complex of claim 104 comprising a full-length .lamda.5 sequence of SEQ ID NO: 6, and a VpreB1 sequence of SEQ ID NO: 1 having its C-terminal tail removed.

106. The complex of claim 89 comprising a full-length VpreB1 sequence of SEQ ID NO: 1 and a full-length .lamda.5 sequence of SEQ ID NO: 6.

107. The complex of claim 89, comprising at least one additional functionality appended or substituted to a free end of the .lamda.5 sequence and/or a free end of the VpreB sequence.

108. The complex of claim 98, comprising at least one additional functionality appended or substituted to a free end of the .lamda.5 sequence and/or a free end of the VpreB1 sequence.

109. The complex of claim 89, wherein the complex comprises (a) a VpreB sequence, an antibody light chain constant region, and an antibody heavy chain sequence.

110. A library comprising a complex of any one of claims 89.

111. A library comprising the complex of claim 107.

112. The library of claim 111 comprising a random peptide library appended or substituted to said N-terminal or C-terminal tail present and panned for specific binding to a further target.

113. The library of claim 111 comprising an antibody or antibody fragment appended or substituted to said N-terminal or C-terminal tail present.

114. The library of claim 113 wherein said antibody or antibody fragment binds to the same target as said complex.

115. The library of claim 113 wherein said antibody or antibody fragment binds to a target different from the target to which said complex binds.

116. The library of claim 114 wherein said antibody or antibody fragment binds to a tumor antigen.

117. The library of claim 115 wherein said antibody or antibody fragment binds to a tumor antigen.

118. The library of claim 111 comprising a therapeutic peptide or therapeutic protein appended or substituted to said N-terminal or C-terminal tail present.