Title of Invention

"SECRETION OF ANTIBODIES WITHOUT SIGNAL PEPTIDES FROM BACTERIA"

Abstract A method for producing an antibody, antibody-fragment, or antibody-related polypeptide in a prokaryotic host cell without the need for a signal peptide, comprising the steps of: (a) Obtaining a prokaryotic host cell, characterized in that said host cell comprises a prl mutation-and at least one expression vector comprising a polynucleotide molecule encoding a heavy chain polypeptide of the antibody and a polynucleotide molecule encoding a light chain polypeptide of the antibody, further characterized in that at least one of the polynucleotide molecules does not encode a signal peptide, (b) Expressing the prl mutant protein and the polynucleotide encoding the heavy chain and the polynucleotide encoding the light chain polypeptide of the antibody, characterized in that at least one of the polypeptides lacks a signal peptide, and, (c) Forming at least one antibody by allowing the host cells containing expressed prl mutant protein and expressed heavy and light chain polypeptides to secrete the heavy chain polypeptide and the light chain polypeptide across a cytoplasmic membrane.
Full Text SECRETION OF ANTIBODIES WITHOUT SIGNAL PEFTIDES FROM
BACTERIA
CROSS-REFERENCES TO RELATED APPLICATION(S)
[0001] The present application claims the benefit of priority under 35 U.S.C. §119
from U.S. Provisional Patent Application Serial No. 60/701,902 entitled "SECRETION OF ANTIBODIES WITHOUT SIGNAL PEPTIDES FROM BACTERIA", filed on July 22, 2005, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.
FIELD OF THE INVENTION
[0002] The present invention is directed generally to methods for secreting
antibodies, antibody fragments, or antibody-related polypeptides from prokaryotes without the need for a signal peptide. In particular, the invention provides host cells and methods for secreting antibodies, antigen-binding antibody fragments, or antibody-related polypeptides from bacteria without the need for a signal peptide and provides diverse libraries of antibodies, antibody fragments, or antibody-related polypeptides resulting from such methods. The present invention is also directed at antibodies, antibody fragments, antibody-related polypeptides, and libraries of the same made by the methods of the invention.
BACKGROUND OF THE INVENTION
[0003] Most proteins destined for the periplasm or the outer membrane of gram
negative bacteria, such as enteric bacteria, are transported across the cytoplasrnic membrane by the general secretory pathway or Sec system, a complex of proteins, which identifies polypeptides for export and translocates them across the cytoplasrnic membrane. This system has been used to secrete mammalian proteins from the enteric bacteria E, coli, including antibody fragments, Natural prokaryotic secreted proteins and heterologous proteins such as mammalian proteins are directed to the secretory apparatus by the addition of a functional signal peptide, a sequence of typically between 13 and 30 amino acids at the N-terminus of the protein which has a hydrophobia core and additional sequences to direct the nascent polypeptide chain to the secretory apparatus and allow accurate removal of the signal peptide
after secretion. A number of prokaryotic signal peptides have been described which allow efficient secretion of at least some antibody fragments, including the signal peptidea from the Erwinia caratovora. pectate lyase B (PelB) protein, Escherichla coli heat-stable enterotoxin (Stfl) and the E. coli OmpA protein. Other ptokaryotes have similar secretory systems, and signal paptides have been described for many for these other prokaryotes,
[0004] However, secretion systems are highly variable in the efficiency with which
antibodies, antibody fragments or antibody-related polypeptides are secreted. The efficiency of secretion is dependent on the sequence of the variable regions of both the heavy and the light chains of antibodies, For example, Fab fragments containing murine V-regiona are poorly secreted if at all Further, human Fab fragments are secreted witti variable efficiency, depending cm the V-rogion sequence, andA>r VH subclass. Such variable secretion efficiency leads to bias in the sequences of antibodies which may be screened from a generated antibody library,
[0005] In some cases, mutations in the V-region can be introduced in order to
improve secretion. However, alterations in the ammo acid aequence of antibody V-regions may compromise antibody function and are not generally desirable,
[0006] Furthermore, cleavage of signal peptides from the secreted polypeptide is not
always efficient. For example, the signal peptide of E. coli OmpA or PhoA is not cleaved .from a fusion protein with.human interleulcin-1 beta (IL-1 beta) when 1he fusion protein is expressed hi-fi1, coli,
[0007J It is an object of the present invention to provide compositions and methods
for obtaining secretion of antibodies, antibody fragments, 01 antibody-related polypeptides from bacteria without the need for a signal sequence thereby removing any secretion constraints caused by variable region sequence,
SUMMARY OP THE INVENTION
J0008] The present invention, relates to methods for secreting antibodies, antibody
fragments, and/or antibody-related polypoptides in prokaryotea without the need for a signal peptide thereby overcoming limitations imposed by variable region sequences In one embodiment, the methods of the invention comprise expressing polymideotides encoding antibodies, antibody fragments, or antibody-relatedpolypeptides without a signal sequence in
a prolcaryotic noflt cell, followed by secretion of the antibodies, antibody fragments, or antibody-related polypeptides across the cytoptasmic membrane of the host cell. In an embodiment of the invention, antibodies, antibody 'fragments, or antibody-related polypeptides are secreted without a signal sequence by use of -A, prokaryotic host cell that contains one or more mutations in the gene(s) which encode the proteins of the cells secretory pathway(s), In another embodiment, the host cell is E, coli and the secretory mutant is a Protein-localization (prl) mutant.
[0009] The present invention also relates to libraries of antibodies or related
polypeptides (hat are made by the methods of the invention. In one embodiment, the libraries of the invention comprise antibody, antibody fragment, or antibody-related polypeptide clones that cannot be secreted or are difficult to secrete in prokaryotes when signal sequences are used to direct transport across the cytoplasmic membrane. In another embodiment, the antibody, antibody fragment, antibody-related polypeptide, or other polypeptide libraries of the invention have better representation of different VH and VL subclasses than libraries expressed with signal sequences, The antibodies and related polypep'tideB of the invention include intact iramunoglobulins, single chain antibodies, Fab, Fab', F(ab')2, Fv, camelid antibodies, antigen-binding scaffolds, antibody or antibody-related polypeptide fusion
proteins, and other polypeptides disclosed below.
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[0010] In another embodiment, the antibody-related polypeptides of the invention
include antigens that are recognized by the antibodies, antibody fragments, or antibody-related polypeptides of the invention. In an embodiment of the invention, these antigens are self-antigena,
[0011] In an embodiment of the invention, the methods of the invention allow for
expression, secretion, and assembly of multimeric proteins wherein one or more subunit of the rnultimeric protein lacks a functional signal sequence.
BRIEF DESCRIPTION OF THE FIGURES
[0012] For a better understanding of liifi nature and objects of some embodiments of
the invention, reference should be made to the following detailed description taken in conjunction with the accompanying drawings, in which;
[0013] Figure 1 provides the amino acid sequence of E. coli Sec Y protein from
mutant F286Y and I408N (prlA4), where the mutated amino acids ara shown underlined in bold (SEQ ID NO: 1).
[Q014J Figure 2 shows a plasmid map of a vector used for expression of heavy and
light chain genes for an antibody fragment without a signal peptide in E, coli. The plasmid has a chloramphenicol-acetyl transferase gane to confer resistance to chloratnplienicol and a ladq gene which expresses a lac represser for regulation of gene expression. The light and heavy chain genes are each under the control of a tac promoter and expression is inducjble by lactose or IPTG.
[OOJ5J Figure 3 shows a western blot analysis showing secretion of assembled Fab
1A8 in periplasmic fractions of SE6004 transformed with plasmid KB5246, Fab expression was induced by the addition of IPTG (SEQ ID NO:2) at the concentrations sbown (in uM), Samples were run on SDS-PAGB gel under non-reducing conditions, and probed using an anti-Human Kappa specific antibody conjugated to Horseradish peroxidase,
[0016] Figure 4 shows results of an ELISA demonstrating antigen-binding activity of
Fab Fragments present hi the periplasraic fraction of SE6004 cells. Serial dilutions of peripksmic extracts from cells transformed with plasmid KB5246 or a ten-fold dilution of the extract (5246 10%) ara analyzed for binding to pcrV antigen in comparison with a standard periplasraic extract (1150) or a ten-fold dilution (1150 10%) containing tmti-PcrV Fab expressed in a wild-type E, coli .strain with signal peptides to direct secretion, PciV binding is revealed as an increase in absorbance at 450nm as a result of enzymatic conversion of TMB substrate to a colored product by HRP-conjugated antibody.
[0017] Figure 5 is a comparison of secretion efficiency for Fab from SE6004 and
Topi OF' cells identified in periplasmic extracts, Fab was expressed using IPTG (SEQ ID NO:2) induction at the concentrations shown in SE6004 cells {prlA} without the use of signal peptides. Expression is compared with signal-dependent expression of the same Fab from fi wild-type strain (TOPIOF). Fab present in periplasmic extracts is detected by Western blot analysis using an anti-human kappa detection reagent.
[0018) Figure 6 is a Map of plasmid KB5282 for the over-expression of mutant SecY.
TheprlA4 mutant SecY gene is expressed from the pTrc promoter. The NPT2 gene confers resistance to kanamycin.
[0019J Figure 7 is a Western blot analysis comparing the secretion efficiency for Fab
1A8 from $&prlA4 mutant strain SE6004, Topi OF1 cells expressing wild-type SecY, and DH5D cells co-transformed with a prlA4 mutant SecY gene. Antibody-related proteins secreted into the medium ware detected using anti-human kappa antibody of blots from non-denaturing SDS-PAGE. Assembled Fab, light-chain dimers and light chain monomers were detected in the culture medium.
DETAILS DESCRIPTION OF THE INVENTION
DEFINITIONS
[0020] As used herein, "antigen" refers to substances that are capable, under
appropriate conditions, of reacting with specific antibodies, antibody fragments, or antibody-related polypeptides. Antigens can be soluble substances, such as toxins or foreign proteins, however, only the portion of the protein or autigenic molecule known as the antigenic determinant (epitope) combines with the antibody, antibody fragment, or antibody-related polypeptide. Mare broadly, the term "antigen" is used herein to refer to any substance to which an antibody binds, or for which antibodies we desired, regardless of whether Che substance is imraunogenic. For such antigens, antibodies can be identified by reoojnbmant methods, independently of any immune response.
[0021] As used herein, an "antibody" refers to a protein functionally defined as a
binding protein and structurally defined as comprising an ammo acid sequence that is recognized by one of skill as being derived from the variable region of an immunoglobulin. An antibody can consist of one or more polypeptides substantially encoded by immunoglobulin genes, fragments of immunoglobulin genes, hybrid immunoglobulin genes (made by combining the genetic information from different animals), or synthetic immunoglobulin genes. The recognized, native, immunoglobulin genes include the Icappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes and multiplo D-segments and J-segmenta. Light chains are classified aa either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively,
[0022] A typical antibody structural um't is known to comprise a tetramer. Each
tetrainer is composed of two identical pairs of polypeptide chains, each pair having one
"light" (about 25 IcD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region (V) of about 100 to 110 or more amino acids primarily responsible for antigen recognition.' The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
(0023) Antibodies exist as intact immunoglobulms, as a number of well characterized
fragments produced by digestion with various peptidases, or as a variety of fragments made by recombinant DNA technology, Thus, for example, papain digests antibodies into an antigen binding Fab fragment and a residual Fc fragment; pepsin digests an antibody below the disulfide linkages m the hinge region to produce F(ah')2, a dimar of Fab which itself is a light chain joined to VH-CHI by a disulfide bond. The F(ab')j inay be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the F(ab')2 dimer into an Fab' monomer. Ths Fab1 monomer is essentially a Fab with part of the hinge region ($ important point is that the two antibody chains in each Fab molecule assemble post-translationally and the dimer is incorporated into the phage particle via linkage of one of the chains to g3p (see, e.g., U.S. Patent No; 5,733,743, which is incorporated herein by reference in its entirety), The scFv antibodies and a number of other structures converting the naturally aggregated, but chemically separated light and heavy polypeptide chains from an antibody V region into a molecule that folds into a thtee dimensional structure substantially similar to the structure of an antigen-binding site are known to those of skill in the art (see e,g,, U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778). Particularly preferred antibodies include all those that have been displayed on phage (e.g.f acFv, Fv, Fab and disulfide linked Fv (Reiter et alt 1995, Protein Eng. 8; 1323-1331). Antibodies pan also include diantibodies and mint antibodies.
(0024] Antibodies can derive from multiple species. Far example, antibodies include
rodent (such as mouse and rat), rabbit, sheep, camel, and human antibodies. Antibodies can also include cbimeric antibodies, which join variable regions from one species to constant regions from another species. Likewise, antibodies can be humanized, that is constructed by recombinant DNA technology to produce iirimunoglobulins which have human framework regions from one species combined with complementarity determining regions (CDR's) from a another species' imraunoglobulin (see, e.g., EPO Publication No. 0239400), In the case of antibodies, the modules consist of "framework" and "CDR" modules, By creating separate framework and CDR modules, different combinatorial assembly possibilities are enabled. Moreover, if two or more artificial genea carry identical pairs of cleavage sites at the boundaries of each of the genetic sub-eleraenta, pre-built libraries of sub-elements can be inserted in these genes simultaneously, without any additional information related to any particular gene sequence. This strategy enables rapid optimization of, for example, antibody affinity, since DNA cassettes encoding libraries of genetic sub-elements can be (i), pre-built, stored and reused and (ii), inserted in any of these sequences at the right position without blowing the actual sequence or having to determine the sequence of the individual library member. Exemplary methods for generating synthetic libraries of antibodies are disclosed in, for example, U.S. Patent No. 5,885,793 and 6,300,064, which are incorporated herein by reference in their entirety.
[0025] Antibodies also include epitopo-focused antibodies, which have at least one
minimal essential binding specificity determinant from a heavy chain or light chain CDR3
from a reference antibody, methods for making fluch epitope-focusecj antibodies are described in U.S. Patent Application No. 11/040,159, which is incorporated herein by reference in its
entirety.
[0026J The term "cytoplasmic membrane" refers to a membrane that encloses the
cytoplasm of a cell and, in a bactorhvm, lies internal to the pariplaam and outer membrane in gram negative bacteria.
[0027] As used herein, the term "diversity" refers to the number of different specific
antigen binding antibodies or related polypeptides.
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[0028] An "expression vector" is a nucleic acid construct, generated recombinantly or
synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, vims, or nucleic acid fragment. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
[0029] As used herein, the term "framework region" refers to those portions of
immimoglobulin light and heavy chain variable regions that are relatively conserved (i.e., other than the CDRs) among different immunoglobulins in a single species, as definsd by Kabat. As used herein, a "human framework region" ie a framework region thai is substantially identical (about 85% or more) to Hie framework region of a naturally occurring human antibody.
[0030} As ijsed herein, the term "fusion antibody" refers to a molecule in which an
antibody is fiised to a non-antibody polypeptide at the N- or C- terminus of the antibody polypeptide. In one embodiment the antibody fragment may comprise one or more C-terminal peptide tags to facilitate detection and purification, fh another embodiment the antibody may be fused to a pepu'de ot polypeptide for display on the surface of a cell, spore or virus. For example one chain of the antibody fragment may be displayed as a fusion protein on the surface of a bacteriophage such as a filamentous phage.
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[0031] The term "host cell" refers to a cell that provides the cellular machinery for
expression and secretion of a polypeptide from an expression vector.

10032J Tne terra "humanized antibody" refers to antibodies constructed by
recombinant DNA technology to produce inanunoglobulins which have human framework regions from one species combined with complementarity datcrrrmrmg regions (CDR's) from another species' immunoglobuh'n (see, &g., EPO Publication No, 0239400),
{0033J As used herein, the term "hrnnunoglobulin" refers to tetrameric Antibodies as
wall as R variety of forms besides antibodies; including, for example, Fv, Fab, and $((&>% as well as bifunctional hybrid antibodies, fusion antibodies, chimeric antibodies, humanized antibodies, hurnaneered antibodies and single chain antibodies.
[O034J "Library11 means a collection of nucleotides sequences, e.g., DNA, encoding
antibodies or related polypeptides within clones; or a genetically diverse collection of antibodies or related polypeptidea.
[0035] A "multiraeric protein" as used herein refers to a globular protein containing
more than one separate polypeptide or protein chain associated with each other to fonn a single globular protein in vitro or in vivo. The niultimeric protein may consist of more than one polypepticle of the same kind to form a "homomultimer." Alternatively, the mulrimeric protein may also be composed of more than one polypeptide of distinct sequences to fonn a "heteromultimer," Thus, a "heterornultimer" is a molecule comprising at leaat a first polypeptide and ft second polypeptide, wherein the second polypeptide differs in amino acjd sequence from the first polypeptide by at least one ammo acid residue. The heteromulb'Tner can comprise a "hBterodimer" formed by the first and second polypeptide or can farm higher order tertiary structures where more than two polypoptidea are present Exemplary structures for the heteromulliroer include heterodimers (e.g. Fv and Fab fragments, diabodies, GABAg receptors 1 and 2 complexes), trimeric G-proieins, heteroterramers (e.g. F(ab')i fragments) and further oligomeric structures.
[0036J "Protein localization (prl) mutant" refers to a host cell with an alteration in its
secretory apparatus which rescues the secretion-defect in proteins containing a defective signal peptide and in proteins without a signal peptide,
[0037] The terms "secretion/secrete/secreting" refers to transport ftom the cytoplasm
of a cell across the cytoplasmfc membrane, including transport pathways that require a signal sequence and transport pathways that da not require a signal.
(0038] The terms "signal sequence" and "signal peptide" both refar to a peptide
sequence capable of aiding in tha secretion of a connected nascent peptide to the ovttside of Gie host cell.
{0039] The terms "VH and VL subclasses" refer in humans to the 7 recognised VH
sub-classes (VH1 - VH7) and 16 VL sub-classes (Vkappal - VkappaS and Vlambdai-VtaribAUO).
[0040] The term "vector" includes any nucleic acid suitable for cloning or for
expression of the nucleic acids of the invention in the host cells of the invention. Tlie vector may, for example, be in the form of a. plaamid, cosraid, viral particle, or phage. The vector may be self-regulating or may integrate into the host cell chromosome or other replication nucleic acid in the host cell. The vector may also be non-replicating or may poorly replicate, for example, in a transient expression system,
EXPRESSION SYSTEMS OP THE INVENTION
[0041] The invention provides rnsthods for secretion of antibodies, antibody
fragments, or antibody-related polypeptides from prokaiyotic host cells without the need for a signal peptide. In one aspect of the invention there is provided a new method for secretion of an sntibody or an antigen-binding fragment and its assembly into a functional antigsn binding molecule. The antibody is encoded by one or mora nucleic acids that comprise the coding sequences for the V region for an antibody. Antibodies of the invention may contain signal sequences, as described in U.S. Patent No. 6,204,023, which is incorporated herein by reference in its entirety. In some embodiments, the antibodies or multrnieric proteins of the invention are expressed from one or more polynucleotidas encoding polypeptides lacking a signal peptide. The antibody is encoded by one or move vector(s) capable of expressing an antibody, antibody fragment, or antibody-related polypeptide. If the antibody is formed from a heavy and a light chain, coding sequences for bolh, chains may be present on the same vector or the coding sequences may be present on different vectors within [0042] Generally, recombinant expression vectors will include at least one origin of
replication, phenotypic selectable markers permitting selection in host cells, e.g., the arnpiclllin resistance gene of £ call and Sacckaromyces cerevistae TRP1 gene, a functional promoter to direct transcription of a downstream structural sequence, aa well as suitable translation imitation and termination signals in operable reading frame. Suitable prakaryotic hosts for transformation include species in the family Enterobactertoceae such as E, coli, or Salmonella typhimwim, various speoias within the genexa Pseudomonas, Streptomyces, and Staphylacaccus, other species such as Bacillus subtllis, and other bacterial hosts may also be employed as a matter of choice,
J0043] There are many expression systems for producing the potypeptidea of the
invention that are well known to those of ordinary skill in the art. (See, e.g., Femandes and
Hoeffler, Eds,, 1999, Gene Expression Systems, Academic Press.) Larga numbers of suitable
vectors are Icnoxvn to those of skill in the art and are commercially available for generating
toe recombinant constructs of the present invention. The following vectors are provided as a
representative but nonlimitiug example; bacterial; pBs, pbagescript, PsiX174, pBluescript
SIC, pBs ICS, pNHSa, pNHlfia, pNHlSa, pNH46a (Stratagene); pTrc99A, pKK223-3,
pKK233-3, pDR540, pRITS (Pharmacia), pSKF, pET23D, k-phage derived vectors, p!5A-
based vectors (Rose, 19S8, Nucleic Acids Res. 16:355 and 356) and fusion expression
systems such as GST and LacZ, Some expression vectors for bacterial use can comprise a
selectable marker and bacterial origin of replication derived from commercially available
plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC
37017), Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine
Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotec, Madison, WI, USA). These
pBR322 "backbone" sections are combined with an appropriate promoter and the structural
sequence to be expressed, ,
[0044j Typically, the expression of tha polyrmcleotide that encades the polypeptide is
undsr the control of a promoter that is functional in the desired hast cell, A wide variety of promoters is available, end can be used in the expression vectors of the invention. Ordinarily, the selection of fee promoter depends upon the cell type iu which the promoter is to be used. Such promoters can be derived front operons encoding glycolytic enzymes such as acid phosphatase, or heat shock proteins, among others. Particular named bacterial promoters include lad, lacZ, T3, TV, gpt, lambda PR, tac and trc. Commonly used prokaryotic
promoters include the beta-lactamase (penicillinase) and lactose (lac} promoter systems (Change, et at., 1990, Nature 198: 1056), flie tryptaphan (trp) promoter system (Goaddel et al,t 1980, Nucleic Acids Res. 8: 4057), the tao promoter (DeBoer, et al, 1983, Proc, Natl Acad, Sci. V,S.A. 80:21-25); and the lambda-derived PL promoter and N-gene riboeome binding site (Shhnatake et al,, 1981, Nature 292:128), The particular promoter system is not critical to the invention, any available promoter that functions in prokaryotes can ba used. Selection of the appropriate vector and promoter is wall within the level of ordinary skill in the art.
J0045] For expression of polypeptidos in prokaryotio cells other than JE, coti,
regulatory sequences for transcription and translation that function in the particular prolcaryotic epecies ate required. Such promoters can be obtained from genee that have bean cloned from the species, or heterologous promoters can be used. For example, the hybrid trp-lac promoter fimctiona in Bacillus in addition to E. coli, These and other suitable bacterial promoters are well known in the art and are described, e.g.t in Sambrook et a/., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N,Y. and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Intsrscience, N.Y. Bacterial expression systems .for expressing the proteins of the invention are available in, e.g,, E, coli, Haoiltus sp.t and Salmonella (Palva et at., 1983, Gene 22:229-235; Moabach et al,, 1983, Nature 302:543-545). Kits for such expression systems are commercially available.
[0046] Either constitutive or regulated promoters can be used in Hie present invention.
Methods for regulating expression in £ coli are well known in the art and include the use of iuducible promoters such as the lac or (ate promoters which are inducible by IPTG, and arAbinose-inducibla promoters. Regulated promoters can be advantageous because tha concentration of heterologous protein in the boat cell can be controlled. An inducifale promoter is a promoter that directs expression of a gene where the level of expression is alterable by environmental or developmental factors such as, for example, temperature,, pH, atiaerobic or aerobic conditions, light, transcription factors and chemicals.
[0047] For E. coli and other bacterial host cells, inducible promoters are loiovra to
those of skill in the art. These include, for example, the lac promoter, the bacleriophage lambda PL promoter, the hybrid trp-lac promoter (Armmn et al.t 1983, Gene 25: 167; de Boer et al., 1983, Proa. Nat'I. Acad. Sci. USA 80: 21), and the bacteriophaee T7 promoter (Studier
et al, 1986, J, Mat Jtol; Tabor et alt 1985, Proa. Nat'l Acad. Set USA 82:1074-8). These promoters and their use are discussed in Sambrook at al., supra.
[0048] Inducible promoters for other organisms are also wall known to those of skill
m the art, These include, for example, the metallothionein promoter, the heat shock promoter, as well as many others.
'[0049] Other expression control sequences such as ribosome binding sites,
transcription termination sites, operators, and the like may also be included, DNA constructs that include one or more of these control sequences are termed "expression cassettes." Accordingly> the nucleic acids that encode the polypeptides are incorporated for the desired level of expression in a desired host cell,
{0050] A translation-initiation codon may be introduced djrectly upstream of the
mature antibody or antibody fragment coding sequence such that the antibody or antibody fragment polypepti.de is expressed with a methionyl (or N-fomiyl methionyl) residue at the N-terminus. Additional sequences may be included in the coding sequence of the antibody, for example to facilitate purification or detection of the antibody or for another purpose, Tha heterologous structural sequence is assembled in the appropriate translational reading frame and with the appropriate translation initiation and termination sequences. Optionally, we heterojogous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g,, stabilization or simplified purification of expressed recombinant product
[00S1J An ATG codon is present at the 5'and of the coding sequence such that the
expressed protein has an N-formyl-methiomne residue at the amino-terminus. Upon the expression of the coding sequence, the N-terminal a;nino acid may be retained or may be removed by proteases in the host cell. The antibodies, antibody fragments, or antibody-related porypeptidea generated from expression of the DNA coding sequence according to this aspect of the invention are capable of binding antigen. In the event mat the antibody fragment is formed from a heavy and a light chain, either or both chains may be expressed from a coding sequence with an ATG codon, Preferably both chains are expressed without a signal peptide.
[0052] Tianslational coupling may be used to anhance expression. The strategy uses
a short upatroam open translations! reading frame derived from, a highly expressed gene
native to the transtational system OT a. synthetical/ non-natural highly expressed open raading frame, which is placed downstream of the promoter and ribosome binding site and upstream of a termination codon. Just prior to the termination codon is a second ribosome binding site, and following liifi termination codon is the initiation aodon for the translation of the polypeptide to be expressed. The system allows for the efficient initiation of translation, See Squires, et, alt 1988, J. Bial. Chem, 263:16297-1(5302.
[0053] Secreted antibodies can be detected in or isolated from the culture medium
after a period of growth of the bacteria under conditions suitable for antibody expression. Methods for monitoring antibodies in the medium include Western blot analysis, SDS-PAGB and enzyme-linked iinmunosorptiou assays (ELISA), Secreted antibodies may alao be detected in or isolated from the periplasrn of the bacteria, Me&ods for disruption of the periplasm and release of antibody from the periplasrmc fraction are well known in the art and include the use of low pH (eg pH4,0) or osmotic shock.
[0054] To facilitate purification of the polypeptides of the invention, the nucleic acids
that encode fhe polypeptides can also include a coding sequence for an epitapa or "tog" for which an affinity "binding reagent is available. Such epitnpe tags include, e.g.t c-rnyc, HA-tug, maltose binding protein, VSV-G tag, emti-DYKDDDDK (SEQ ID N0:3) tag, or any such tag, a large number of which are well known to those of skill in the art Expression vectors useftil for recombhrant production of fusion polypeptides having these epitopes are commercially available (e.g., lovitrogen, Carlsbad, CA) vectors pcDNA3.1/Myc-His and pcDNA3.1AT5-His are suitable for expression in mammalian cells). Additional expression vectors suitable for attaching a tag to the fusion proteins of the invention, and corresponding detection systems are Iccown to those of skill in the ert, and several are commercially available (e.g., "FLAG" (Kodak, Rochester NY)). Another example of a suitable tag is a polyhistidine sequence, which is capable of binding to metal chelate affinity Uganda, Typically, six adjacent histidines are used, although one can use more or less than six. Suitable metal chelata affinity Uganda that can serve as the binding moiety for a polyhistidine tag include rritrilo-tri-acelic acid (NTA) (Hochuli, E., 1990, "Purification of recombiuant proteins with metal chelating adsorbents" in Genetic Engineering; Principles and Methods, J.K. Setlow, Ed., Plenum Press, NY; commercially available from Qiagen, Santa Clarita, CA).
[0055] One of skill would recognize that modifications can be made to the protein
domains without diminishing their biological activity, Some modifications may be made to facilitate the cloning, expression, or incorporation of a domain into a polypeptide. Such modifications are well known to those of skill in the art and include, for exatnpla, the addition of codons at either terminus of me polynuclaotida that encodes the binding domain to provide, for example, a methionme added at the arolno terminus to provide fin initiation site, or additional ammo acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences.
POLYPBFTfDES'
Antibodies
[0056] In one embodiment, the secreted polypeptides are antibodies. The basic
antibody structural unit is known to comprise a tetrainar. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 KDa), The araino-terminal portion of each chain includes a variable region of about 100 to 110 or more ammo acids primarily responsible for antigen recognition. The carboxy-tornrinal portion of each chain defines a constant region primarily responsible for effector function. The variable regions of each lighf heavy chain pair form (he antibody binding site. Thus, an intact antibody has two binding sites. Each chain has a constant region (C) and a variable region (V). Each chain is organized into a series of domains. The light chains have two domains, corresponding to the C region and the other to the V region, The heavy chains have four domains, one corresponding to tiie V region and three domains (1,2 and 3) in me C region, The antibody has two anus (each arm being a Fab region), each of which has a VL and & VH region associated wifli each other. It is this pair of V regions (VL and VH) that differ from one antibody to another (owing to amino acid sequence variations), and which together are responsible for recognizing the antigen and providing an antigen binding site, In even more detail, each V region is made up from three complementarity determining regions (CDR) separated by four framework regions (FR). The CDRs are the most variable part of ftje variable regions, and they perform the critical antigen binding function. The CDR regions are derived from many potential germ line sequences via. a complex process involving recombination, mutation and selection.
[0057] Light chains are classified as either kappa or lamibde Heavy chains are
classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively, Heavy chain subclasses in humans are designated VH1 - VH7. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids (see generally, 1993, Paul, W., ed., Fundamental Immunologyt 3rd ed, Raven Press, N.Y,, SH. 9 (incorporated by reference in its entirety for all purposes)).
[0058] From N-tennrnal to C-tertninal, both light and heavy chain variable regions
comprise alternating framework and complementarity determining regions (CDRs): FR. CDR. FR, CDR. FR, CDR and FR. The assignment of amino acids to each region is in accordance with the definitions of Kabat, 1987, and, 1991, supra, and/or Chothia & Leak, 1987, J. Mol Siol. 196: 901-917; Chothia et al., 1989, Nature 342; 878-883.
[0059] It has been shown that the function of binding antigens can be performed by
fragments of a whole Ewtibody, Exemplary binding fragments are (i) the Fab fragment consisting of the VL, VH, CL and CHI domains; (ii) the Fd fragment consisting of the VH and CHI domains; (iii) the Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) the dAb fragment (Ward, E.S. et al., 1989, Nature 341: 544-546) which consists of a VH domain; (v) isolated CDR regions; and (vi) F(ab')2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulphide bridge at the hinge region.
[0060] In various embodiments of the invention, the antibody or antibody fragment
may be a single-chain antibody or may be formed from a heavy and light chain. Exaraplary antibodies include intact rramunoglobma, single chain antibodies, scFv, dAB, VHH, Fab, Fob', F(ab')2, Fv, camelid antibodies, nanobodiea, antigen-binding scaffolds, and antibody or antibody-related polypeptide fusion proteins. If the antibody contains a heavy and a light chain, one or more chains is expressed without a signal peptide and preferably both chains are expressed without a signal peptide.
[0061] Two structures of IgGs constituting the imnumoglobulina (antibody
molecules) of camelids are known to exist: one a heterotetramer having heavy chains and light chains, and the other consisting of a heavy-chain dimer. The tetrameric structure is a common characteristic of IgGs among humans and most animals. On the other hand, the latter IgG having a heavy-chain dimer structure is considered characteristic of camelids,
JAN-10-2008 THU 07:19 AM

10062] Since the VH region of a heavy chain dimer IgG in a camelid does not have to
make hydrophobia interactions with a light chain, the region m the heavy chain that normally contacts a light chain is changed to hydtophilic amino acid residues in a camelid. Due to structural differences compared to VHs of normal heterotetrameiic IgGs, VH domains of the 5 heavy-chain dimer IgGs are called Variable domain of the heavy-chain of heavy-chain antibody (VHH).
[0063] VHH has excellent solubility due to its hydraphilic amino acid residues.
Amino acid substitutions are scattered throughout the primary structure (amino acid sequence) of VHH. Additionally, these hydrophilic amino acid residues form a cluster in the 10 space of the tertiary structure of VH corresponding to the Bite that interacts with the VL domain, Herein, the aforementioned space of the tertiary structure is specifically called former VL side. These amino acid substitutions are, for example, V37F 01 V37Y, G44E, L45R or L45C, and W47 are also mostly substituted with Gty. Such substitutions increase the hydtophilicity of the former VL. side of VHH.
[0064] Furthermore, VHHs derived from camels and llamaa have very high
thermostability compared to mouse heterotetrameric antibodies. The USE of VHH derived from these species can provide, for example, molecules that maintain their antigen binding ability even at 90DC (Van der Linden et at., 1999, Biochim. Biophys. Acta 1431(1): 37).
[0065] The diversity of antibody repertoire of carnelids is determined by the
complementary determining regions (CDR) 1, 1, and 3 in the VH or VHH regions,
Possession of three CDRa is in common with the IgGs of other animal species. However, the
CDR3 in the cainel VHH region is characterized by its relatively long length averaging 16
amino acids (Muyldermans et al, 1994, Protein Engineering 7(9): 1129). For example,
compared to the CDR3 of mouse VH having mi average of 9 amino acids, the CDR3 of camel
IgG is very long,
[0066] Libraries of camelid-dadved antibody variable regions, which maintain the in
vivo diversity of the variable regions of a camelid, can be made by, for example, the methods disclosed in United States Patent Application Set, No. 20050037421, published February 17, 2005, which is incorporated herein by reference in its entirety.
[0067] In another embodiment of the invention, the antibody related polypeptides are
scaffold polypeptides, Scaffold polypeptides are non-iimnunoglobulin binding polypcptidee

that exhibit selective binding activity toward a predetermined ligatid, The nou-immunoglobulin binding polypeptides are derived from an itnmunoglobulin-like domain containing scaffold that can be grafted with binding domains of a parent polypaptide to confer the binding specificity of the parent polypeptide onto the iromunoglobulin-like domain containing scaffold, The nqn-immunoglobulin binding polypeptides of the Invention have the advantages of being stable and modular in both the scaffold domain structures as well as in the ability to accept a broad range of haterologous polypeptide binding domains. Additionally, the inununoglobulin-like domain containing scaffolds can be readily obtainable from human sources so that their imtnunogenecity when used as a human therapeutic is negligible. The scaffolds of the invention also can be readily constructed to contain or omit naturally occurring polysaccharide chains or to include novel chains or other extra-scaffold moieties or polypeptide structures.
[0068] In one emboditneritj the invention is directed to non-immunogbbulin binding
polypeptides having antibody variable region complementarity determining regions (CDRs) inserted into a Thyl immunoglobulin-like domain containing scaffold, The CDRs are inserted into the loop regions of the Thyl polypeptide which allows the CDRs to fold into a similar confirmation as they would be in the tluee dimensional structure of the donor, or parent, antibody. The resulting hybrid, or chiroaric, antibody-related polypeptide exhibits similar binding characteristics compared to the parent antibody.
(0069] In another embodiment, me invention is directed to antibody-related
polypeplidea having altered immunoglobuu'n-lflce domain loops made by ammo acid substitution at some or all positions. The altered amino acid sequences in the loop domains confer selective binding activity toward a ligand omen: than that bound by the antibody-related polypeptide, The araino acid alterations can be made at the nucleic acid or
polypeptide level using a variety of methods lojown to those skilled in the art.
i
[0070] In yet another embodiment, the invention is directed to antibody-related
polypeptides derived from the ThyOx family of immunoglobulin-like domain containing polypeptides. The ThyOx polypeptides can be used as an immunoglobulin-like domain containing scaffold or as a carrier polypeptide to generate an antibody-related polypepticle of the invention. Scaffold polypeptides and libraries of such Scaffold polypeptides may be made by methods including, for example, those disclosed in U.S. Patent Application. No.
20040266993, published December 30,2004) which is incorporated harem by reference in its entirety.
[0071] As used herein, the term '4mrmmo globulin-like domain" or "Ig-like domain"
when used in reference to a scaffold is intended to refer to an art-recognised -sandwich
structural motif found in proteins of diverse function, including for example, extracellular
matrix proteins, muscle proteins, immune proteins, cell-surface receptors and enzymes, Ig-
lilce domain members have been divided into various superfamilies, including for example,
the iramunoglobulm, Fibronectin type III and cadherin Buperfamilies. Other euperfamilies
containing the Ig-Hlce domain structural motif include, for example, members of the PICD
domain, -galaotosidase/glucuTonidaae domain, transglutatnase two C-terminal domains,
actinoxanthin-like, CuZn superoxide dismutase-like, CBD94ike, lamin A/C globular tail
domain, clathrin adaptor appendage domain, integrin domains, PapD-like, purple acid
phosphataBe N-tenninal domain, BUperoxide leductaae-like, tbiolidiaulftda interchange
protein DsbD N-terminal domain and invasin/tntimin cell adhesion fragments suparfamilies.
Ig-like domain structural similarity ia maintained between members of different superfamilies
irrespective of significant sequence identity, The terra is intended to include Ig-like domain
members within and across each superfamily. Therefore, the term "immunoElobulhvlike (Ig-
like) domain containing supevfamily" is intended to refer to an Ig-like domain containing
member polypeptide within any of these superfamilies as well as others known in the art, A
description of the different Ig4ike domain containing superfamilies can be found, for
example, in Clarke et al, 1999, Structure Fold. Des. 7:1145-53 and within structural
databases such as at the URL pdb.weizmann.ac.il/soop/da- ta/6cop.b.c.b,htral.
[0072] As used herein, the term "ThyOx" or "ThyQx femily polypeptide" when used
in reference to a antibody-related polypeptide of the invention ia intended to mean a subclass of polypeptides within the immunoglobulin superfamily (IgSF) of immunoglobxilin-like domain containing polypeptides that are related by their common -sandwich structural motif and containing a scaffold framework structure similar to antibody variable region domains. Particular polypeptides within the ThyQx family of polypeptides include, for example, Thy-1, 0x2, OP40, Ox24ike protein and 0x2 homolog,
[0073] As used herein, the term "scaffold" ia intended to mean a supporting
polypeptide framework used to organize, orient and harbor heterologous binding domains or altered amino acid sequences conferring binding specificity to a ligand. A scaffold can be
structurally separable from the ammo acid sequences conferring binding specificity. The structurally separable portion of a scaffold can include a variety of different structural motifs including, for example, -sandwich, -sheet, -helk, v-banel, coil-coiled and other polypsptide secondary and tertiary stoctares well known in the art, A scaffold of the invention will also contain one or more regions that can be varied in amino acid sequence without substantially reducing the stability of the supporting framework structure, An exemplary region that can be varied includes a loop region segment that joins two strands of a -sandwich or -sheet. Amino acid residues corresponding to the structurally separated portion of a scaffold is referred to herein aa a scaffold framework, Immuno globulin-like domain containing scaffolds of the invention exhibit less than about 50% amino aoid identity to a human immunoglobulin variable heavy or light chain framework amino acid sequence, Generally, immunoglobulin-like domain containing scaffolds will exhibit, for example, amino acid sequence identity less than about 45%, about 40%, about 30%, about 20%, about 15% or about 10% compared to a human irannmoglobulin variable heavy or light chain framework amino acid sequence. Residues of a scaffold that can be varied are referred to herein with reference to its structural properties such as a loop region or with reference to its ability to accommodate altered residues. Therefore, a scaffold region that can be varied is referred to as a scaffold variable region, nmtabla region, exchange region, alterable region or changeable region, for example. Residues conferring secondary or tertiary structural properties can be retained, modified or conserved so long as the overall structure of the scaffold is maintained. Those skilled in the art know, or can determine, which residues firaction in structural stability of a polypeptide scaffold as well as the extent to which such residues can be modified.
[0074] Specific examples of scaffolds of the invention include immunoglobulin-like
domain containing superfamily members. These superfamily members contain a imimmaglobulin-like domain characterized as a -sandwich which can be used aa a scaffold of the invention. The P-sandwich consists of about 80-150 amino acid residues containing two layers of antiparallel -sheet in which the flat hydrophobic faces of the -sheets pack against each other. Each -sheet contains a loop region that can be varied in amino acid sequence BO as to confer unique binding specificity onto the scaffold palypeplide, Examples of Ig-like domain containing superfamily members include, for example, ThyOx family member polypeptidos os well as the various individual members wifliin the immunoglobulin-like domain containing superfamiliea described previously. Such individual members
include, for example, T cell receptor, CDS, CD4, CD2, class I MHC, claae H MHC, GDI, cytoldne receptor, GCSF receptor, GMCSP receptor, honnone receptors, growth hormone receptor, erythropoietin receptor, iuterferon receptor, interferon gamma receptor, prolactin receptor, NCAM, VCAM, ICAM, N-caderin, E-caderia, ilbroneotin, tanaacin, and I-flet containing domain polypeptides, or a runctian#l fragment thereof. Exemplary descriptions of these an other Ig-lilca domain containing superfmniiy members can be found in, for example, Isaclce and Horton, 2000, The Adhesion Molecule FactsBook, Second Ed., Academic Press, San Diego; Pitzgerald et al 2001, The Cytoktne FactsBook, Second Ed,, Academic PresBj San Diego; and Marsh et al.t 1999, The HLA FactsBook, Second Ed., Academic Press, San Diego,
(0075] The antibodies, antibody fragments or antibody-related potypeptidas of this
invention may be derived from a variety of sources, In various embodiments, the antibodies, antibody fragments or antibody-related polypeptides are derived from mammalian genes and raey be human, mouse, rabbit, sheep, rat, hamster chiraeric, humanized, hybrid, or apitope-focused. The antibodies of this invention may be monoclonal. Owing to then: high specificity for a given antigen, the advent of monoclonal antibodies (Kohler, G, and Milstein C, 1975, Nature 256; 495) represented a significant technical break-through with important consequences both scientifically and commercially.
[0076] Monoclonal antibodies are traditionally made by establishing an immortal
mammalian cell line which is derived from a single immunoglobulin producing cell secreting one form of a biologically functional antibody molecule with a particular specificity, Because the antibody-fiecreting mammalian cell line is immortal, the characteristics of the antibody are reproducible from batch to batch. The key properties pf monoclonal antibodies are their specificity for a particular antigen and the reproducibility with which they can be manufactured,
[0077] Early methods for producing monoclonals were laborious and time
consuming. An animal of choice, e.g., a mouse, was immunized with a desired antigen, antibody producing cells were harvested from the animal (usually by splenectorny) and fused to a suitable immortalized cells, e.g., myeloma cells, to make a hybridoma that clonally produced an antibody. Such hybridoma technology is disclosed, for example, in U.S. Patent Noa. 4,172,124 and 4,196,265; Zurawdd et al, 1980, Federation Proceedings 39:4922; Franlcel and Gerhnrd, 1979, Molecular Immunologyt 16:101-106.
[0078] The introduction of transgenvc animals that produce fully human antibodies
has permitted the selection of hybridomas which also produce My human antibodies. Such transgenic animals are disclosed, for example, in U.S. Patent NOB. 6,075,181 and 6,300,129, which are incorporated herein hy reference in their entirety.
[0079] Display technologies have also permitted the selection of monoclonal
antibodies that are fully human or other animal, chimeric, synthetic, and/or semi-synthetic. One example of such display technologies is phage display (examples are disclosed in U.S. Patent Nos. 5,565,332; 5,580,717; 5,821,047; 5,871,907; 5,885,793; 5,922,545; 5,403,484; 5,885,793; 6,172,197; 6,291,158; 6,291,650; and 6,387,627, which are incorporated herein by reference in their entirety) where a vectors for expression of fuaion antibodies in, which one or more antibody chain is fused at the N-tenninus of a phage protein are constructed. Such vectors can be introduced into prl mutant host strains of the present invention in order to express antibodies and isolate phage-antibodies in a signal-independent manner. The expression of phage-aivtibodies in. the host cells of the invention provides improved expression of poorly secreted antibodies and better representation of various sub-classes of antibodies present in libraries. The fusion proteins may also be expressed from polynucleotidas encoding antibody fusion proteins lacking a signal sequence. Methods for screening, purifying and analyzing phage antibodies are described in the above patents. Another example of such display technologies is yeast display (examples are disclosed in U.S. Patent No. 6,300,065, which is incorporated herein by reference in its entirety).
[0080] Phage-display technology has generally made use of the filamentous
bacteriophage Ml3 or the closely related phage fd. Thesephages are composed of circular, single-stranded DNA surrounded by a cylinder of coat proteins. Most of the viral capsid consists of the major protein pVHI, of which there are approximately 2,700 copies per plmge. At one end of the phage particle., there are five copies each of pill and pVI that are involved in host-cell binding and in the termination of the assembly process. At the other end, there are five copies each of pVII and pIX, hydrophobic peptides of 33 and 32 amino acids, respectively, required for the initiation of assembly and for maintenance of virion stability.
[0081] Embodiments of the invention include chiraeric antibodies and synthetic
antibodies. The early monoclonal technologies described above produced non-human antibodies. These antibodies ore potentially immunogemc in humans and this immunogenicity has severely hampered the development of therapeutic antibodies. The
production of BO called "chimeric antibodies," e,g,, variable regions fiom one species jorauu to constant regions from another species, has bean somewhat sucoess&l, but does not overcome the immunogenloity problem in many cases. Exemplary methods for chimBrizing antibodies are disclosed in, for example, U.S. Patent No. 4,816,567, which is incorporated herein by reference hi its entirety,
(0082] Recombinant DNA technology has been utilized to produce hiummoglobulins
which have human framework regions from one species combined with complementarity determining regions (CDR's) from another species' imrnunoglobulin (see, e.g., EPO Publication No. 0239400). Those new proteins are called "reshaped" or "humanized" (when the framework regions are human) immunoglobulins and the process by which the donor imrnunoglobulin is converted tato a human-lilce imrnunoglobulin by combining its CDR's with a human framework is called "humanization". Exemplary methods for humanization of antibodies are disclosed in, for example, U.S. Patent No, 6,180,370, which is incorporated herein by reference in its entirety.
[0083] Artificial antibodies and fragments thereof can be constructed baaed on known
antibody sequences, which reflect the structural properties of a whole group of homologous antibody genes. Therefore it is possible to reduce the number of different genes without any loss in the structural repertoire. This approach leads to a limited set of artificial genes, which can be synthesized de novo, thereby allowing introduction of cleavage sites and removing unwanted cleavages sites, Furthermore, Ms approach enables (i), adapting the codon usage of the genes to mat of highly expressed genes in any desired host cell and (ii), analyzing all possible pairs of antibody light (L) and heavy (H) chains in terms of interaction preference, antigen preference or recombinant expression titer, which is virtually impossible using the complete collection of antibody genes of an organism and all combinations thereof.
[0084] The use of a limited set of completely synthetic genes makes it possible to
create cleavage sites at the boundaries of encoded structural sub-elements. Therefore, each gene is built up from modules which represent structural sub-elements on the proteiny(poly)peptide level. In the case of antibodies, the modules consist of "framework" and "CDR" modules. By creating separate fimnework and CDR modules, different combinatorial assembly possibilities are enabled, Moreover, if two or more artificial genes carry identical pairs of cleavage sites at the boundaries of each of the genetic sub-elements, pre-built libraries of sub-elements can be inserted in these genes siniulta-neously, without any
additional information related to any particular gene sequence, This strategy enables rapid optimization of, for example, antibody affinity, since DNA cassettes encoding libraries of genetic sub-elements can be (i), pie-built, stored and reused and (ii), inserted in any of these sequences at the right position without knowing the actual sequence or having to determine the sequence of the individual library member, Exemplary methods for generating synthetic libraries of antibodies are disclosed in, for example, U.S. Patent No. 5,885,793 and 6,300,064, which are incorporated herein by reference in their entirety.
[0085] In one embodiment the antibodies are epitope-focused human antibodies
created by methods for engineering antibodies where the resulting antibodies retain epitope binding specificity and affinity while at the same lime having most of the non-human sequences replaced with human sequences, as described in patent application U.S. Patent Application Sei. No. 11/040,159, filed January 2.0, 2005, which ia incorporated herein by reference in its entirety. This is accomplished by transferring a BSD pair from the refeience antibody, e.g., & protein of a CDR3 pair (CDRSj). In antibodies that are affinity-matured, e.g,, the reference antibody, the heavy chain and light chain BSDs are in close contact with one another and are optimized for mutual stabilization of the combined antigen-binding conformation, hence, they form a unit, i.e., a BSD pair. The antigen-binding conformation is, of course, dependent on the support of the underlying frameworks of the V-iegions. When an affinity-matured BSD, e.g., that of the reference antibody, is combined with the structural diversity and stability of the complete human repertoire of heavy chain or light chain V-segment pairs, scaffolds that fully support the optimal antigen-binding conformation of the BSD are readily identified with the aid of selection systems including, but not limited to, phage display, cell viability, colony lift binding assays (CLBA),, or a variety of imnwnoassays, e.g., ELISA assays.
[0086] Further, transfer of a BSD pair to diverse germline V-segments often result in
selection of V-regions that that have affinities of greater than 50 nM. These selected V-regians can also be incorporated into the affinity maturation process of any antibody, V-aegment libraries are relatively small without CDR3 repertoires, thua selection of human V-regions can also be combined with limited mutagenic diversification of one or both BSDs in libraries of searchable size for many conventional selection systems.
[0087] The V-segment repertoire used in generating libraries to replace the heavy
and/or light chain V-segmdnt of the reference antibody can be from any source. The human
repertoires can be generated, e.g.s by polymeraae chain reaction (PCR.) amplification using primers appropriate for the desired segments from cDNA obtained from peripheral blood or spleen, in which case the repertoires are expected to contain clones with somatic mutations. Alternatively, the repertoires can be obtained by amplification of genomic DNA from non-immune system cells in order to obtain germline-encoded sequences,
10088] The human germline V- segment repertoire consists of 51 heavy chain V-
regions, 40 K light chain V-segroeuts, and 31 "k light chain V-segments, maldng a total of 3,621 germline V-region pairs. In addition, there are stable allelic variants for most of these V-segments, but the contribution of these variants to the structural diversity of the germline repertoire is limited. The sequences of all human germ-line V-segment genes are Icnown and can be accessed in me V-base database,- provided by the MRC Centre for Protein Engineering, Cambridge, United Kingdom (see, Chothia et alt 1992, J Mol Biot 227:776-798; Toralinson et [0089] Human V-region repertoires, both germline and affinity-matured, can bo
recovered, e.g.t from peripheral blood lymphocytes (PBL), often pooled from multiple (e,g., at least 10) healthy individuals, using conventional cDNA cloning methods (Sambrook and Russell, eda, Molecular Cloning: A Laboratory Manwt, 3rd Ed, vols. 1-3, Cold Spring Harbor Laboratory Press, 2001). Insofar as the germline frequency distribution is not uniform in expressed sequences, it is prudent to capture at least 103 independent clones for each of the three V-region isotypes (VH, Vic, and VX) to ensure optimal diversity of the repertoires. The PCR can be used to amplify V-region sequences during the cloning process. However, exponential amplification mechanisms are prone to random biases, and this may be compounded by the use of degenerate primers, which have variable priming efficiencies, resulting in a loss of diversity, Thus, when amplification is desired, it may be desirable,
where possible, to use a primer-independent linear amplification method, such as in vitro transcription (Sambiook awl Russell, eels, Molecular Cloning: A Laborcttoiy Manual, 3rd Ed, vols, 1-3, Cold Spring Harbor Laboratory Press, 2001),
[00901 • BSDs from the reference antibody are transferred to a library of V-segment sequences generated as described above. The BSDs can be incorporated into the expression vector before or after the population of V-segments is cloned into the expression vector. The BSD that is transferred can be a CDR3-FR4, a CDR3, a D segment (where the BSD is from the heavy chain), a MEBSD, or any other fragment of CDR3-FR4 that has binding specificity in combination with the complementing BSD from the other chain of the reference antibody. It ia understood that when transferring a BSD from a reference antibody to a different V-region, ths structure of the heavy or light chain V region is maintained in the resulting V-ragion. Thus, if the BSD from the reference antibody is a subregion of CDR3-FR4, the complete CDR3-FR4 structural length is maintained, i.e.t the remainder of the CDR3-FR4 residues that are not from the reference antibody are made up of other residues, typically human gennline residues,
[0091] As noted, the BSD can include Framework 4 regions, e.g,, from the reference
antibody, which are part of the J-segraents, but which are highly conserved among mammals, and are important for CDR3 structure. These sequences can, for example, be amplified by PCR willi primers containing restriction sites for in-frame ligation, to Framework 3, and other unique restriction sites downstream from the carboxyl terminus of Framework 4, e.g., for ligation to the C-region. Each CDR3-PR4 is then transferred into the appropriate sites of the V-region library construct. Alternatively, the desired sequence or mix of sequences for the CDR3-FR4 region can be synthesized as one continuous oligonucleotida or mix of oligonucelolides and can be joined to the V segment repertoire by primer extension using in vitro transcribed cRNA synthesized from the repertoire as a template for first-strand cDNA synthesis. Diversity can be introduced into & region, e.g., CDR3 and^or FR4.
[0092] The BSD can also be a sequence that is leas than the complete CDR3, e.g, the
D segment of a heavy chain CDR.3 or a MEBSD. As appreciated by one of skill in the art, when the reference antibody BSD is less than a complete CDR3, a complete CDR3 still results in the antibody expression library, as the remaining CDR3 residues are incorporated into the construct. For example, appropriate oligonucleotides can be designed to incorporate
human sequences, e.g., germUne J segments, to replace the CDR3 residues that are not part of the MEBSD.
[0093] The MEBSD is tha region within a GDR3 sequence or a pair of CDJUfl that is
required to retain the binding specificity of the reference antibody when combined with human sequences that re-constitute me remainder of CDR3 and the rest of the V^region. Tha MEBSD can be defined empirically or con be predicted from structural considerations.
[0094] The antibody library can be a library where the antibody is an IgG, an Fv, ft
Fab, a Fab', a F(ab')2, a single chain Fv, an IgG with, a deletion of one more domains, or any other antibody fragment that includes Hie V-region.
[0095] The antibodies can be displayed on l}ie surface of a virus, cell, spore or viros-
like particle, For this purpose, one or both chains of the antibody fragment are typically expressed as a fusion protein, for example as a fuaion to a phftge coat protein for display on tha surface of filamentous phage. Alternatively, the antibodies of the antibody library can be secreted from a host cell.
[0096] The following provides an exemplary description using secretion systems to
express the antibodies as Fab or Fab' fragments. It is readily apparent to those in the art, however, mat the axpression systems can be adapted for any library format For this general example, a library of complete V-regions is constructed by ligation of oligonucleotides encoding CDR3-FR4 segments to the V-segment repertoire as described above, The amplified sequences encoding complete V-regions are cloned into a suitable expression vector and can be fused to constant region sequences at this stage for expression of Fab or Fab' molecules. The antibody fragments can be secreted from prokaryotic or eukaryotic cells including bacteria, yeast, plant cells andmamnmlian ceils.
[0097] Filter screening methodologies have been described for detection of secreted
antibodies specific for a particular antigen. In one format, the secreted antibody fragments are trapped on a membrane which is probed with soluble antigen (Skerra et al., 1991, Anal Jitochem, 196:151-5). In this case, bacteria Harboring plasmid vectors that direct the secretion of Fab iragmonts into the bacterial peripkun are grown on a membrane 01 filter. The secreted fragments are allowed to diffuse to a second "capture" membrane coated with antibody which can bind the antibody fragments (e.g., witHmmunoglobulm antisentm) and the capture filter is probed with specific antigen. Antibody - enzyras conjugates can be used
to detect antigen-bindmg antibody fragments on the capture membrane as a colored spot, The colonies are re-grown on the first membrane and the clone expressing the desired antibody fragment recovered.
[0098] Colony lift binding assays have also been described in which the antibodies
are allowed to diffuse directly onto an antigen-coated membrane, Giovannoni et al have described auch a protocol for the screening of single-chain antibody libraries (Giovannoni et al, 2001 Nucleic Acids Research, Vol. 29, No, 5 e27).
[00991 Libraries of secreted antibody fragments can also be screened by ELISA,
either using pools of multiple clones or screening of individual clones each secreting a unique antibody sequence. One such method for screening individual clones is described by Watkiris et at, 1997, Anal Biochem. 253: 37-45. In this case, tnicrotiter wells were coated with anti-Fab antibody to capture Fab fragments secreted directly in the wells, The Fab samples were than probed with soluble biotinylated antigen followed by detection with streptavidin-alkalme phosphatase conjugates.
Fusion Antibodies
[00100] In. one embodiment, the polypeptides may be fusion antibodies. In one
embodiment, the antibody fragment may comprise one or more C-termiiwl peptide tags to facilitate detection and purification, m another embodiment the antibody may be fused to a peptide or polypeptide for display on the surface of a cell, spore or virus. For example one chain of the antibody fragment may be displayed as a fusion protein on the surface of a bacteriophage such as a filamentous phage. Methods for display of antibodies on phage ara well Imown in the art and include fusion to pHI and pVHI proteins of a filamentous phage. In a preferred embodiment at least one of the peptides comprising an antibody - ph'age protein fusion is expressed without a signal peptide in a prl strain of E. coli and is presented on the surface of a phage.
[00101] The secreted chains may retain the N-termmal methionine (or N-fornjyl-methiontne), Alternatively, and in some cases, depending on the sequence of the antibody, the initial methionine may be removed by proteolytic processing by the host cell.
100102) Embodiments also include other display technology, such as yeast cell display, bacterial cell display, ribosome display, and mammalian cell display, In one embodiment, screening is performed by screening pools of library members.
[00103] Fragment and subunit complementation systems can be used in me invention
to select/screen for antibodies having desired properties ("complementation system"). In general, fragment complementation systems are comprised of a responder that is fragmented or separated into two (or more) parts that must reassociate to mate a functional teeponder. The rragvnents/subumts of the responder are fused individually to members of a binding ensemble, and the reassembly of the reeponder is then driven by the direct or indirect interaction of the two binding ensemble members. In a preferred embodiment the binding ensemble is comprised of an antibody(s) and an antigen(s), Examples of fragmerd/subunit complementation systems mat may be used in the invention are disclosed in U.S. Patent Nos, 6,342,345, 6,270,964, 6,294,330, 5,503,977, 5,585,245, which are incorporated herein by reference in their entirety, PCX patent application WO 00/71702, and Fields et at., 1989, Nature 340:245-247; Bai et al,, 1996, Meth. EnxymoL 273:331-347, Luo et al, 1997, Biatechniques 22:350-352, which are hereby incorporated by reference in their entirety.
[00104] Reactivation-based molecular interaction systems (e,g., RAIR.TM.) can be used in the invention to select/screen for antibodies having desired properties ("reactivation system")- In general, reactivation-based molecular interaction systems are comprised of responders, inhibitors, reactivators, and binding ensembles of two or more members. The system has two complexes, one containing the responder, the inhibitor, and a binding ensemble member (the responder complex), and the other containing the raactivator and a binding ensemble member (the reactivator complex). The responder is inhibited in its complex, and docking of the reactivator complex to the responder complex by direct or indirect interaction of the binding ensemble members allows the reactivatoi to "reactivate" the responder by displacing Ihe inhibitor. Typically, a responder complex comprisea a responder molecule, an inhibitor of the reapondev, and a first binding ensemble member. The components of Ihe icsponder complex may be arranged in various configurations by covalent or non-covalent linkages. In a preferred embodiment the binding ensemble is comprised of an antibody(s) and an antigen(a),
100105] In a. preferred reactivation system, molecular interactions can be detected by a
process termed "reactivation of an auto-inhibited responder," or "RAIR." The RAJR systems
•comprise the following components; a tesponder complex and a reactivator complex. By avito-inlnbited, we mesa, that the responder is directly United to the responder so that the base state is automatically inhibited until the inhibitor is displaced and the responder activated by a reactivator complex. "Where this linkage is by a covalent bond, the covalant linkages may farther comprise a linker. A reactivate! complex comprises a reactivate molecule to displace the inhibitor and a second binding ensemble member. Like the components of the reapondei' complex, the raactivator and binding ensemble member may be linked either oovalently or non-covalently.
[003061 Molecular interaction between the fitst and the second ensemble members caii be detected by the following mechanism; the signal or activity of the raspondor in the responder complex is sequestered by the inhibitor present in me complex, i.e., the reaponder is auto-inhibited; when a reactivator complex is introduced, if the second ensemble member in the reactivator complex binds with sufficient affinity to the first ensemble member in the responder complex, the reactivate will be able to displace the inhibitor in the responder complex and lead to the so-called "reactivation of an auto4nhtbited tesponder," The detection of responder activity or signal indicates an interaction between the first and the second ensemble members.
[00107] Variations of the RAIR systems can be used for interaction mapping, improving the affinity of a first binding pair member, and isotropic selection of a plurality of binding molecules. In some variations, a third ensemble member may be used.
100108] Examples of reactivation systems are disclosed in U.S, Patent Application Ser. No. 10/208,730, which is incorporated herein by reference in its entirety.
(001091 Systems using molecular sensors activated by competition can also be used in the invention to select/screen for antibodies having desired properties. These systems are designated COMPACT.TM. In general, competitive activation systems are comprised of a binding ensemble, a vesponder, and an inhibitor. The responder is complexed with one binding ensemble member and the inhibitor is complexed to another binding ensemble member. The binding ensemble members, upon binding to one another, bring the Tesponder and inhibitor together so that the responder is inhibited. Antibodies of the invention that disrupt the binding ensemble or inhibit binding ensemble formation and thereby activate the responder can than be selected. In a preferred embodiment, the binding ensemble is an
antibody(s) and an antigan(s), and the "competitive activator" is an antibody. For example, the binding ensemble antibody might be a reference antibody, and the competitive activator may comprise a library of antibodies which compete with the reference for binding to the antigen. Examples of competitive activation systems that may be used in the invention are disclosed in U,S. patent application Ser, No, 10/076,845, which is incorporated herein by reference in its entirety.
[00110] Such a system may further employ a "mask" to control the sensitivity of the system. These systems are described, e.g., in co-pending U.S. application Ser, No. 10/076,845, filed February 14, 2002. A "mask", in the context of a competitive activation system, refers to a molecule that has low affinity for a reporter 01 inhibitor, such that the mask does not bind appreciably at working concentrations unless it is tethered covalently to the reporter or inhibitor. The mask does not affect reporter activity only the binding of the inhibitor and vice versa. Control of the system with Masks permits a high-affinity inhibitor to be uaed without fear of increasing the background inhibition because its association rate constant is greatly reduced by the Mask without affecting the dissociation rate constant of the reporter-inhibitor complex, thereby reducing the overall affinity while retaining the stability of the high-affinity reporter-inhibitor complex.
Libraries
[00111] In another embodiment of the invention, the secreted polypeptides may be a
diverse library of antibodies, antibody fragments, or antibody-related polypepticles with different binding characteristics expressed fiom a prokaryolic host cell such as a strain of E. coli expressing a prl mutation, wherein one or more antibody chain IB expressed without a signal peptide. In another embodiment, the secreted polypeptides are expressed in a signal-independent maianer. The libraries according to this aspect of the invention show broader representation of VH and VL subclasses,
[00112] It is known in the art liiat different antibodies are secreted at different levels
into the periplaam and that certain sub-classes of antibody are only poorly secreted in soluble correctly-folded form. In many cases the V-region sequences of the antibody can affect the ability of the antibody to be secreted from E. coli. Murine V-regions, for example, fold poorly in the periplasm and may lead to the accumulation of aggregated and inactive antibodies (Skerra and Pluckthun, 1991, Prot Eng, 4:971; Bcthmann and Pluckthun, 1998,
Nature Biotech 18; 376; Helle rf a/., 1995, Proc, Natt Acad. Sci. USA 92: 11907). Cbaperone proteins may improve the folding and expression of some antibody fragments (Bothmann and Pluckthun, 1998, Nature Biotech. \ 8:376; Bolhrnann and Pluclcthun, 2000, J. Bid, Chem 275:17100). Bias in secretion of some sub-classes of antibody from bacteria can lead to bias in the sequences of the antibodies which may be screened from ft library, An embodiment of the present invention addresses allows for higher yields of functional correctly folded antibodies and antibody fragments from bacterial secretion systems by secretion of antibody chains in a signal-independent manner.
[00113] Embodiments of this invention include naKve libraries and immunized
libraries. Naive libraries are made from the B-lympliocyteB of a suitable host which has not been challenged with any immunogea, nor which ie exhibiting symptoms of infection or inflammation. Immunized libraries are made from a mixture of B-cells and plasnaa cells obtained from a suitably "immunized" host, i.e., a host that lias been challenged with an hnmunogen. In one embodiment^ the mRNA from these cells is translated into cDNA using methods well known in the art (e.g., oligo-dT primers and reverse transcriptase). In an alternative embodiment, nucleic acids encoding antibodies from the host cells (mRNA or genomic DNA) are amplified by PCR with suitable primers. Primers for such antibody gene amplifications are well known in the art (e.g., U.S. Patent No. 6,096,551, which is incorporated herein by reference in its entirety, and PCT Patent Application WO 00/70023A1 disclose uuch primers). In a hybrid embodiment, the ,mRNA from the host cells is synthesized into cDNA and these cDNAs are then amplified in a PCR reaction with antibody specific primers (e,g., U.S. Patent No, 6,319,690, which is incorporated herein by reference in
I
its entirety, discloses such a hybrid method), Alternatively, the repertoires may be cloned by conventional cDNFA cloning technology (Sambrook and Russell, eds, Molecular Cloning,- A Laboratory Manual, 3rd Ed, vols. 1-3, Cold Spring Harbor Laboratory Press, 2001), without using PCR.
(00114) In one embodiment of the invention, a database of published antibody sequences of human origin is established where the antibody sequences are aligned to each other. The database is used to define subgroups of antibody sequences which show a high degree of similarity in both the sequence and the canonical fold of CDR bops (as determined by analysis of antibody structures). For each of the subgroups B consensus sequence is deduced which represents the members of mis subgroup; the complete collection of
consensus sequences represent therefore the complete structural repertoire of human antibodies.
[001J5] These artificial genee are then constructed, eg,, by total gene synthesis or by the use of synthetic genetic subunits, These genetic aubunits correspond to structural sub-elements on the (polyjpeptide level. On the DNA level, these genetic subunits are defined by cleavage sites at the start and the end of each of the sub-elements, which are unique in the vector system. All genes which are members of the collection of consensus sequences ara constructed such that they contain a similar pattern of corresponding genetic sub-sequences. Moat preferably, said (poly)peptides are or are derived from the HuCAL consensus genes: Vkl, Vk2, Vk3, Vk4. Vll, V12, V13, VH1A, VH1B, VH2, VH3, VH4S VH5, VH6, Ck, Cl, CHI cr any combination of said HwCAL consensus genes,
[00116J This collection of DNA molecules can then be used to create "synthetic libraries" of antibodies, preferably Fv, disulphide-linked Fv, single-chain Fv (scFv), Fab fragments, or .Fab' fragments which may be used as sources of specificities against new target antigens. U,S. Patent No. 6,300,064, which is incorporated herein by reference in its entirety, diacloaes methods for making synthetic libraries containing more than 10* transformants,
[00117] lu atiother embodiment, synthetic human antibodies have now been made by synthesis from defined V-gene elements. "Winter (EP 0368 684 Bl) lias provided a method for amplifying (by PCR), cloning, and expressing antibody variable region genes, Starting with these ganes he was able to create libraries of iunctianal antibody fragments by randomizing the CDR3 of the heavy and/or the light chain. This process is AinctionaUy equivalent to the natural process of VJ end VDJ recotnbination which occurs during the development of B-cellg in the immune system. For example, repertoires of human germ Ime VH gene segments can be rearranged in vitro by joining to synthetic "D-segments" of five random ammo acid residues and a J-segment, to create a synthetic third complementarity determining region (CDR) of eight residues. U.S. Patent No. 5,885,793, which is incorporated herein by reference in its entirety, discloses methods of making such antibody libraries such as these that create libraries containing 107 plmge clones.
[00118] The antibody fragments according to this aspect of the invention may be
soluble secreted antibody fragment's or may be presented as a fusion protein on the surface of a cell, spore or virus. Thus, for example, the library of antibodies may be a phage-display
library in which one or more chains of the antibody fragment ate expressed as a fusion protein with a phage protein in which at least one of the peptides comprising the fusion protein is expressed without a signal peptide. If tbe antibody fragment is comprised of a heavy and a light chain, it is preferred that both chains ate expressed without a signal peptide, In this aspect of tho invention the host strain is chosen to he suitahle for expression of the antibody library and may be a mutant strain such as prlA4 or may be a strain chosen for another purpose, for example a strain with high transformation frequency, in which the mutant prl protein is expressed from a plasmid expression vector,
[00119] Both pill and pVIII have been used to display peptide and antibody libraries, Display of nonimmune or "narve" antibody-phase libraries on pIII has been used to isolate human, antibodies against a variety of target antigens. Antibodies can be isolated in either scFv or Fab formats, the scFv or one of the Fab chains being fused at the N-terminus of the phage protein. In all cases described previously, a signal peptide is fused at the N-terminus of the antibody chain in order to direct secretion of the antibody-phage fusion protein. The other proteins of the phage coat have also been used to display antibody chains. pVTC and pDC have been used to display the antibody variable heavy-chain legion (VH) and variable light-chain region (VL), respectively. pIX display has also been used to construct a naive human antibody library based on the fusion of scFv to the N terminus of pIX using a PelB signal peptide for secretion (Gao et al, 2002, Proc. Natl Acad. Sci 99: 12612), Because of the high efficiency of phage transducticm, phage-displayed antibody libraries can be large, with diversities in excess of 109 antibody molecules or sometimes in excess of 10)0 or even 10U antibodies per library.
[00120] In some embodiments, the library can he a library of epitope-focuaed human antibodies as described in U.S. Patent Application Serial No. 11/040,159, filed January 20, 2Q05, which is incorporated herein by reference in its entirety. For example, such a library can comprise a plurality of nucleic acids that encode a diverse popvilation of heavy chain V segments, wherein the V segments are not linked to a CDR3, The invention also provides a library comprising nucleic acids that encode a diverse population of light chain V segments, wherein the V segments are not linked to a CDR3, The V segments of either OT both libraries can be, e.fi-,, human gennline. Libraries of epitope-focused hutnaii antibodies can range in size from 103 to 10s antibodies per library,

(001Z1] Antibody libraries may also be focused libraries comprising predominantly members of one or more sub-class of VH or VL gene segments. Thus, for example, in humans there are 1 recognized VH sub-classes (VH1 - VH7) and 16 VL sub-classes (Vkcappal - Vkappa6 and Vlambdal-VlambdalO) and a focused library may be constructed comprising members of one or more VH sub-class in combination with a diverse library of Vkappa chains or Vlambda light chains. Alternatively, a focused library of one or more VL sub-classes may be combined with a. diverse library of heavy chains. As a farther alternative, a. focused library maybe constructed comprising predominantly members of a single VL subclass and a single VH sub-class. Antibody fragments of the VH3 sub-class are typically expressed efficiently in E. colt when expressed in a signal-dependent fashion. Antibodies of other VH sub-classes are not efficiently secreted using signal peptides. Similarly, antibody fragments with murine V-regions are poorly secreted using signal peptides. The present invention allows improved representation of secreted antibodies of different sub-classes and allows efficient secretion of antibody libraries comprising murine V-regious.
[00122] In another embodiment, the invention provides a library comprising a
plurality of human antibody V-iegion pairs where a V-region pair comprises: i) an unselected heavy chain V-region comprising a human V segment and a heavy chain CDR.3 'from a reference antibody, and ii) an unselected light chain V-region comprising a human V segment and a light chain CDR3 from the reference antibody.
[001235 In other embodiments, the library is a library comprising nucleic acids encoding human antibody V-region pairs, where the VH and VL V segments are each linked to a MEBSD from a reference antibody of interest.
[00124] A library of the invention can also comprise nucleic acids encoding a'plurality of VH. or VL regions, wherein the VH or VL regions comprise V segments from one VH or VL subclass, wherein the V regions lack D and/or I segments. In one embodiment, the V segments of the VH regions are germline and/or the V segments of the VL regions are germline,
[00125] The invention also provides a library comprising & plurality of antibody V
region pairs, wherein a pair comprises: i) n. heavy-chain V region comprising a binding specificity determinant BSD from a heavy chain CDR3 from a reference antibody joined to a diversity of V segments, and ii) a light chain V region comprising a BSD from a light chain
CDB.3 from the reference antibody joined to a diversity of V segments, wherein at least one of the BSDs comprises lees than the reference antibody CDR3,
MI JLT1MERIC PROTEINS
[00126] Proper assembly of polypeptide Bxvbumts of a multiraedc protein to form a stable complex is required to ensure the biological function of the rnultimeric protein. An embodiment of the present invention enables expression, secretion and assembly of selected raoncitnsric polypeptidas to effect efficient production of heteroinultimers outside of the cytoplasm. One or more of the monomer polypeptides of the multimeric protein can be made without a signal sequence in the methods of the invention, and the other monomer polypeptides can be expressed with or without signal Bequence(s). Assembled multimeric proteins that may be produced by the present invention include antibodies, antibody fragments or antibody-related polypeptides,
NUCLEIC ACIDS
[00127] The rmcleic acid sequences that are useful in the methods of this invention, i.e., those that encode at least in part the individual peptides, polypeptides and proteins secreted in the method of the invention, or those expressed in or comprising the libraries of this invention, may be native, synthetic 01 a combination thereof. They may be mKNA, DMA or cDNA. In the preferred embodiment, the nucleic acids encode antibodies.
[00128] Recombinant DNA methodologies may be used to create antibody fragments that cannot be made by enzymatic digestion. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whale antibodies, or i those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al.} 1990, Nature 348:552-554), Nucleic acids encoding the polypeptides of the invention can be obtained using routine techniques in the field of recombinant genetics (see, e.g., Sambrook and Russell, eds, 2001, Molecular Cloning: A Laboratory Marnial, 3rd Ed, vols. 1-3, Cold Spring Harbor Laboratory Press; and Ausubel, ed,, 1997, Cwrent Protocols in Molecular Biology, John Wiley & Sons, Inc. New York).
[00129] Often, the nucleic acid sequences encoding the polypeptides of the invention
are cloned from cDNA or genomic DNA libraries by hybridization with probes, or isolated
using amplification techniques with oligonucleotide primers. Amplification techniques can bus vised to amplify and isolate sequences from DNA or UNA (see, e.g., Dieffbnbach & Dvekaler, 1995, PCR Primers: A Laboratory Manual). Alternatively, overlapping oligonucleotidea can be produced synthetically and joined to produce one or more of the domains. Nucleic acids encoding the component domains can also be isolated from expression libraries using antibodies as probes.
[00130] In an example of obtaining a nucleic acid encoding a polypeptide of the invention using PCR> the nucleic acid sequence or subsequence is PCK. amplified, using a sense primer containing one restriction site and an anu'sense primer containing another restriction site, This will produce a nucleic acid encoding the deaired polypeptide and having terminal restriction sites, This nucleic acid can then be easily Ugated into a vector having the appropriate corresponding restriction aitea, If the desired polypeptide is a fusion protein, (he domains can be directly joined or may be separated by a Inter, or other, protein sequence. Suitable PCR primers can be determined by one of skill in the art using the sequence information provided in GenBank or other sources. Appropriate restriction sites can also be added to the nucleic acid encoding the protein or protein subsequence by site-directed mutagenesis. The plasmid containing the polypeptide encoding sequence of the invention is cleaved with the appropriate restriction endonuoleaae and taen ligated into an appropriate vector for amplification and/or expression according to standard methods.
[00131] Examples of techniques sufficient to direct persons of sldll through in vitro amplification methods are found in Berger, Sarahrook, and Ausubel, as well as U.S. Patent No. 4,683,202, which is incorporated herein by reference in its entivaty; Innis et al,t eds, 1990, PCR Protocols A Guide to Methods and Application, Academic Press Inc. San Diego, CA; Amheim £ Levmson, 1990, C&EN 36-47, The Journal Of NIH Research 3: 81-04; Kwoh et al.t 1989, Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al, 1989, J. Clin. Chem., 35; 1826; Landegren et al., 1988, Science 241: 1077-1080; Van Brunt, 1990, Biotechnology 8: 291-294; Wu and Wallace, 1989, Gene*; 560; andBarringeref al, 1990, Gene 89:117*
100132] In some embodiments, it may be desirable to modify the polypeptides of the
invention. One of skill will recognize many ways of generating alterations in a given nucleic acid construct. Such welHcnown methods include site-directed mutagenesis, PCR amplification using degenerate oligonucleotides, exposure of cells containing the nucleic acid
to mutagenlo agents or radiation, chemical synthesis of a desired oligonucleovide (e.g., in conjunction with ligatton and/or cloning to generate large nucleic acids) and other well-known techniques. See, e.g, Giliman and Smith, 1979, Gene 8;81-97, Roberts et al, 1987, Nature 328; 731-734.
[00133] In some embodiments, the recombinant nucleic acids encoding the polypeptidee of the invention are modified to provide preferred codons which enhance translation of the nucleic acid in a selected organism (e.g, yeast preferred codons are substituted into a coding nucleic acid for expression in yeast),
[00134] The polynucleotidos of the invention also include polymicteotides including
rmcleotide sequences that are substantially equivalent to the polynucleotides of the invention. Polymicleotides according to the invention can have at least about 80%, more typically at least about 90%, and even more typically at least about 95%, sequence identity to a polynucleotide of the invention. The invention also provides the complement of the polynucleotides including anucleotide sequence that has at least about 80%, more typically at least about 90%, and even vnoie typically at least about 95%, sequence identity to a polynucleotide encoding a polypeptide recited above. The polynucleotide can be; DNA (genomic, cDNA, amplified, or synthetic) or RNA. Methods and algorithms for obtaining suchpolymicleotides are well known to those of alcill in 1ihe art and can include,' for example, methods for determining hybridization conditions which can routinely isolate polynucleotides of the desired sequence identities.
[00135] The nucleic acids useful in this invention may be naturally diverse, synthetic diversity may be introduced into those naturally diverse members, or the diversity may be entirely synthetic. For example, synthetic diversity can be introduced into one or more CDRs of antibody genes. Preferably, it is introduced into CDR1 and CDR2 of imunoglobulins. Preferably, natural diversity is captured in the CDR3 regions of the imnrnnoglobulin genes of this invention from B cells, Most preferably, the nucleic acids of this invention comprise a population of iromunoglobulin genes that comprise synthetic diversity in at least one, and wore preferably both of the CDR1 and CDR2 and diversity in CDR3 captured from B cells.
[00136] Nucleic acids which encode piotein analogs in accordance with this invention
(i.e., wherein one or more amino acids are designed to differ from the wild type polypeptide) may bo produced using site directed mutagenesis or PCR amplification in which the primer(s)
have the desired point mutations. For a detailed description of suitable rnutageuesis techniques, see gambrook et al, 1989, Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and/or Ausubel et al,, editors, 1994, Current Protocols in Molecular Biology, Green Publishers Inc, and Wiley and Sons, N.Y. Chemical synthesis using methods described by Engels et al, 1989, in Angew. Chem, Intl. Ed., Volume 28, pages 716-734, may also be used to prepare such nucleic acids.
[00137] "Recombinant variant" refers to any polypeptide differing from natirally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using recombinant D1S1A techniques. Guidance in determining which amino acid residues may be replaced, added, or deleted without abolishing activities of interest, such as enzymatic or binding activities, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology.
[00138] Preferably, amino acid "substitutions" are the result of replacing one amino
acid with another amino acid having similar structural and/or chemical properties, i.e, conservative amino acid replacements. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydiophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanme, tryptophan, and meuiionrae; polar neutral amino acids include glycine, serine, threonine, cysteme, tyrosine, asparagine, and glutatnine; positively charged (basic) amino acids include argmiue, lysine, and hisn'dine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
[00139] "Insertions" or "deletions" are typically in the range of about 1 to 5 amino acids. The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.
[00140] Alternatively, where alteration of function is desired, insertions, deletions or non-conservative alterations can be engineered to produce altered polypeptides or chimeric polypeptides. Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention. For example, such
alterations may change polypeptide characteristics such, as ligand-bmdmg affinities, interchain affinities, ot degradation/turnover rate. Further, such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells chosen for expression. For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate diaulfide bridges.
[00141] Alternatively, teoambinant variants encoding these same or similar polypeptides may be synthesized or selected by making vise of the "redundancy" in the genetic code. Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system. Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/tumover rate.
[00142J The nucleic acid sequences of the invention are further directed to sequences which encode variants of the described nucleic acids, These amino acid sequence variants may be prepared by methods known in the art by introducing appropriate micleotide changes into a native or variant polynucleotide. There are two variables in the construction of amino acid sequence variants; the location of the mutation and the nature of the mutation, The ammo acid sequence variants of the nucleic acids are preferably constructed by mutating the polynucleotide to give an amino acid sequence that does not occur in nature, These amino acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions .(constant regions). Sites at such locations will typically be modified in series, e.g., by substituting first with conservative choices (e,g., hydrophobic amino acid to a different hydrophobic ainino acid) and then with more distant choices (e.g., hydrophobic amino acid to a charged amino acid), and then deletions or insertions may be made at the target site.
(00143] Amino acid sequence deletions generally range from about ] to 30 residues, preferably about 1 to 10 residues, and are typically contiguous. Ainino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one to one hundred or more residues, as well as mtrasequence insertions of single or multiple ammo acid residues. Intrasequenca insertions may range generally from about 1 to 10 amino residues,
preferably from 1 to 5 residues. Examples of terminal insertions include the haterologous signal sequences necessary for intracellular targeting in different host cells.
[00144] In a preferred method, polynucleotides encoding the novel nucleic acids are changed via site-directed mutageneais. This method uses oligonuoleotide sequences that encode tha polynucleotide sequence of the desired amino acid variant, us well as a sufficient adjacent nucleotide on both sides of the changed arnino acid to form a stable duplex on either side of the site of being changed, In general, the tectoiques of site-directed rautagenesis are well Icnown to those of skill in the art and this technique is exemplified by publications such as, Edelraan et al,, 1983, DNA 2:183. A versatile and efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller and Smith, 1982, NudeicAcidsK.es. 10:6487-6500.
[00145] PCR may also be used to create amino acid sequence variants of the novel
nucleic acids, When small amounts of template DNA are used as starting material, primer(s) that differs slightly in sequence from the corresponding region in me template DNA can generate the desired amino acid variant. PCR amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the collagen at the position specified by the primer, The product DNA fragments replace the corresponding region in the plasraid and this gives the desired amino acid variant.
[00146] A further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells et ol, 1985, Gene 34:315; and other mutagenesis techniques well known in the art, such as, for example, the techniques in Sawbrook et «/., supra, and Current Protocols in Molecular Biology, Ausubele? al
[00147] Due to the inherent degeneracy of the genetic code, other DNA sequences
which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the invention for the cloning and expression of these novel nucleic acids. Such DNA sequences include those which are capable of hybridizing to the appropriate novel nucleic acid sequence under stringent conditions. In addition, nxicletc acids encoding the same amino acid sequence as that of the polypeptide of the invention but having very different nucleic acid sequences d.ue to the degeneracy of the genetic code are also encompassed within tha invention.
HOST CELLS
[00148] Another aspect of the invention provides a prokaryotio host cell allowing for
secretion of antibodies or antigen-binding antibody fragments or raultimeric proteins without the need for a signal peptide. Strains suitable for use in the invention do not have a generally increased permeability but selectively secrete proteins naturally destined for seoietion including the antibodies and antibody fragments of the invention. In a preferred embodiment, the prokEiryote is preferably a grain-negative bacterium, and most preferably is the bacterium E. coli.
[00149] Multiple pathways have been described in Gram-uegative bacteria for the
secretion of proteins from the cytoplasm either to the periplasim or through both the inner and outer membranes, traditionally grouped into four different systems, The Type III and Type IV systems are generally used for direct transfer of bacterial proteins to adjacent eukaryotic host cells. The Type I system forma a "tunnel" that links the outer and inner membranes such that proteins exported by this pathway are secreted directly in,to the extracellular medium. The Type II secretion system, also known as the general secretory pathway or Sec pathway, is responsible for the secretion of the majority of proteins through the inner membrane into tha periplasm. An additional secretion system, which also makes use of specific N-terramal signal peptidea 'to direct secretion of proteins via the periplasm, is the twin-arginine translocBtion (TAT) pathway. In contrast to the Sec system, which secretes loosely folded proteins to the periplaam where protein assembly takes place, the TAT pathway is used for the secretion of already folded enzymes (reviewed by Berks et. al., 2005, Current Opinion 8; 174-181).
[00150] . The Type II secretion system, has been widely used for the secretion of recombinant proteins from £. coli. In an embodiment of the invention, the host cells comprise a mutant gene(s) in the Type E, or Ssc, pathway. The addition of a short N-tenninal signal sequence to the recombinaivt protein serves to direct the recombinant protein to this secretion pathway and the signal peptide ia removed during the secretion process. The pathway has been used to express antibody fragments such as Fab fragments directed by signal sequences from bacterial proteins naturally secreted using this pathway, including OrnpA, PelB and PboA. The antibody heavy and light chains have been shown to assemble in the periplasm to form antigen-binding Fab fragments (e,g., Skerra and Pluckthun, 1991, Protein Eng. 4:971-979).
[00151] The translocation machinery of the Sec pathway is a well-studied enzyme complex, the ttanslocase, which consists of several integral membrane proteins and an associated ATPase to provide the energy for traualocation (reviewed in Fekkes and Driesseu, 1999, Microbiology and Molecular Biology Reviews, 63:161-173; van der Wollc et al., 1998, EMBO J. 17: 3631-3639). The core of this enzyme complex consists of the membrane-embedded hetarotrirner consisting of the SecY, SecE and SecG proteins (the SecYEG complex), and the peripheral homodimeric ATPase SecA, The SecD, SecF and YajC proteins form a separate heterotrimeric complex that associates with the SecYEG complex to form the complete tranalocase.
[00152] Newly synthesized precursor proteins are bound by the chaperone SecB which stabilizes the pro-protein in a loosely folded conformation competent for translocation, SecB and the signal sequence target the pre-protein to the membrane, and both associate with SecA which is bound with high affinity to the SecY siibunit of the SecYEG complex. As a result of the SecB-SecA interaction, the pre-proteiti is transferred to SecA, which binds both its signal
)
sequence and its mature domain. The release of SecB from the membrane requires the binding of ATP at one of the two ATP-binding sites of SacA. At this stage, a loop of the signal sequence and the N-teiminal region of the pre-protein are presented to the periplasmic face of the membrane, allowing cleavage of the signal sequence by leader peptidase.
[00153] The N-terminal signal sequence of pre-proteins has been regarded as important
for the initial targeting event, the recognition of toe pre-protein by SecA. Aberrant signal sequences are not efficiently recognized by the translocase resulting in. a deficiency in translocation.
[00154] The Type I secretion system has also been used to direct the secretion of
heterologous proteins from E. coli, In an embodiment of the invention, the hoat cell comprises a mutant gene in the Type I secretion system. The secretion signals recognized by the components of the Type I pathway are located at the carboxy terminus of the secreted protein and, in most cases, the secretion signals are not claaved off either during or after secretion. A well-characterized E. coli protein secreted via. the Type I secretion pathway is alpha-hemolysin. The signal sequence from this protein has been used to direct the secretion of a number of heterologous proteins including a scFv antibody fragment by fusion of the C-terminal region of hemolysin to the C-termirma of the scFv coding region (Fernandez and
Lorenzo, 2001, Mol Microbiology 40: 332-346)- In this case the C-termwml hemolyain signal peptide was retained in the secreted product.
[00155] Several mutations in the Sec system components are known which allow
efficient secretion of normally secreted E, coli proteins with defective or absent signal sequences. Such protein localization (prl) mutations have been identified in multiple components of the Sec system, including for example, SecY (prIA), SecE (p/'/G), SecG (prlH) and SecA (prlD) (Boat.and Belin, 1997, J. Biol Cheat. 272:4087-93, which ia incorporated herein by reference in its entirety).
[00156] It has also been found that prl mutations can rescue secretion of some
naturally secreted E. coli proteins in the complete absence of a signal peptide, Thus E. coli maltose binding protein and alkaline phosplmtase lacking a signal peptide have been secreted from prlA mutants (Derman el al., 1993, EMBO J. 12; 879). The bacteriophaga protein LamB can also be secreted without a signal sequence inprlA mutants (Flower at al, 1994, / Bacterlol 176; 18). However, heterologoiiB proteins such as eulcaryotic proteins have not bean previously shown, to be secreted without a signal sequence from prl strains, Wong et aL, 1988, Gene 68:193) achieved successful secretion of insulin-like growth factor-1 from^E. coli using the signal sequences from the LamB or OmpF genes and showed that the processing efficiency of LamB-IGF-1 and OmpF-IGF-1 was enhanced in a strain bearing the prlA4-mutation. Human CD4 iused to OmpA, PhoA, or OmpF signal peptides has also been shown to express efficiently in prlA mutants (Rockeribach et al, 1991, Appl Microbiol Biotechnal 35:32-7), Overexpresaion of SecY mutant proteins on a plasmid vector has also been explored for enhancing expression of a human protein from E. coli. Thus overexpression of the prlA4 mutant SecY protein together with secE increased secretion of human TL-6 fused to the OmpA signal peptide (Perez-Perez et at,,, 1994, BioTechnology 12: 178).
[00157] PrlA strains have also been used to improve diversity of peptides displayed on surface of filamentous phage fd using the pIll signal peptide (Peters et al, 1994, J. Bacterial. 176:4296) and to express bovine pancreatic trypsiu inhibitor as a phage fusion protein from a PhoA signal peptide (US Patent No. 5,223,409, which is incorporated herein by reference in its entirety; Ladner et al). Thus it appears that prlA mutant proteins can facilitate the expression of certain eukaiyotic proteins expressed in a signal-peptide-dependent manner,
comprise a mutant SecY or SecE gene expressed on a plasraid, Such a plasmid may be constructed using a constitutive 01 an inducible promoter allowing for induction of secretion of polypeptides without signal sequences only at desired times, Methods for regulating expression in E. coll are well known in the art and include the use of inducible promoters such as the lac, trc, or tac promoters which are inducible by IPTG, and arabinose-inducible promoters. The mutant Sec gene may be a mutant Sec gana from E. coli or ftom another gram-negative bacterium. Thus, for example, the prlA4 mutant form of SacY may be expressed from a plasmid in an E. coli host cell in order to permit secretion of B Fab fragment expressed without a signal peptide.
[00161] The host cell may be a wild type & coli strain such as W3110 or may be
another strain of E. coli. Suitable host strains include TOPIC, DH5, DHSalpha, Origami and HB101. The host cell may be chosen to provide mutations in other chaperones and genea which affect the folding, assembly and secretion of heterologous proteins. It has been demonstrated that a combination of molecular chaperones such as bacterial DnalC and GroE systems, can augment refolding df proteins that interact with the chaperoning yet fail to fold properly (Buchberger, A., Schroder, H,, Hesterkampj T., Sohonfeld, H. J., and Bulcau, B,, 1996, /, Mol Biol 261, 328-233, Petit, M. A., Bedale, W., Osipiuk, J., Lu, C., Rajagopalan, M., Mclnemey, P., Goodman, M. P., Echols, H,, 1994, J. Biol. Chen. 269, 23824-23829). DnaK also cooperates with Trigger Factor in folding of newly synthesized proteins.
100162] Library embodiments of mis invention may be expressed inprl mutant strains
in order to allow secretion for screening of antibody fragments in various functional assays. The identical vectors may also bo expressed in alternative strains of E. coli for expression within the cytoplasm without me need to re-engineer the antibody molecules, Intracellular expression may be used for efficient production of antibody fragments, for example using trxB gor mutants to provide an oxidizing cytoplasm to allow dimxlphide-bond formation. High level expression of correctly folded and assembled Fab fragments can be achieved in the cytoplasm of E. coli carrying mutations in the glutathione oxjdoreductase (gar) and the thioredaxin reductase (trxB1) genes (Venturi et al, 2002, Mol Biol. 315:1-8). Expression and assembly of correctly folded antibody fragments can be further enhanced using coexpression of molecular chaperones (Levy et al, 2001 Protein Expr Purif. 23: 338-47; Jurado et «/., 2002 J. Moimol 28: 320:1-10).
[00158] Proteins that ate normally secreted from calls often form insoluble inclusion bodies when reoombinantly expressed in E. coli without a signal sequence. For example, genes encoding antibody heavy and light chains or antibody fragments have been expressed without signal sequences in E. coli and ths proteins produced typically accumulate as insoluble prodiictG in inclusion bodies inaida the cell (Boss et al, 1984, A/we/. Acids Res 12: 3791; Cabilly et al,, 1984 Proc. Natl, Acad. Sci. USA 81:3273). Such proteins ars not available for transport across the oytoplasrnic membrane and do not form functional protein. Methods for re-folding antibody fragments from inclusion bodies are provided in US Patents 6,331,415 and 4,816,567- However, such methods for generating antibodies are inefficient and provide poor yields of functional antibody and cannot be used for library screening purposes.
[001S9] In a preferred embodiment of the invention, the prokaryotic host cell contains
a prl mutation in one or more components of the secretory apparatus which allows secretion of the antibody in the absence of an encoded signal peptide. The prl mutant is a mutation which permits secredon of signal-less monoraoric proteins and may comprise a mutation affecting the activity of a SecY, SecA, SecE or SecG gene or another gene, Prl mutants also permit secretion of proteins encoded with a signal peptide in a signal-independent fashion, One embodiment of the invention comprises secreting an antibody polypeptide with a signal sequence in a host strain with aprt mutation and achieving assembly of the antibody into a functional multimeric protein. This is an unexpected result since secretion and chain assembly aro, thought to be closely co-ordinated. Another embodiment carnpri6e.s secreting a polypeptide lacking a signal sequence in a host strain with uprl mutation. Preferably the prl mutation comprises a mutation in SecY (prtA) or SecE (prlG). Most preferably the prl mutant comprises one or more mutations in SecY such as the prlA4 mutant strain (Erar and Bassford, 1982, J. Biol Chem 257: 5852-5860), The prlA4 allele contains two missenge mutations in the seoY gene, resulting in the amino-acid substitutions F286Y and 140 8N. Most preferably, tlieprlA mutant comprises at least an 140 8N mutation in SecY. The sequence of faeprlAJ mutant SecY protein is shown in Figure 1,
(00160] The prl mutant may contain a prl mutation in the chromosome of the host
attain, such as the prlAA mutant strain of E. coli. Alternatively, the prl mutant may be derived by over-expression of an additional copy of a mutant Sec gene, for example, by expression in a plasmid-based expression vector. Thus, for instance, the prl mutant may
[00163] Another embodiment of the invention includes mutants in a second secretory pathway, the twin arginine tamslocation or TAT pathway. It is intended that all tat-dependent signal peptides are to be encompassed by the presant invention, Specific examples include but are not limited to the pnoD and the lipA sequences.
[00164] Another embodiment of the. invention includes mutants in a third secretory pathway, referred to as lie Type III secretion system. Type 01 secretion machinery is present in numerous gram-negative bacteria (including members of the species Shigella, Salmonella, Yersitita, Escherichta, Pseiidomonas, Xcaithomonas, ftalstonia, and Erwinia) that are pathogenic for man, animals, and plants, For example, the Sec-independent type HI secretion pathway is involved in secretion of Yersima anti-host proteins. In Salmonella and Shigella species, it is involved in the process of entry into epithelial cells. It is also implicated in EPEC signal transducing proteins, Pseudomonas aeruginosa toxins, and virulence factors of many plant pathogens, as well as in flagelhun assembly of bacteria such as S. typhimvrium and Bacillvn subtilis.
[00165] Features of this secretion pathway can include activation of secretion by
contact of the bacterium with host colls (Menard et a/., 1994, The secretion of me Shigella flexneri Ipa invasins is activated by epithelial cells and controlled by IpaB and IpaD, EMBO J., 13:5293-5302; Watarai et alt 1995, Contact of Shigella with host cells triggers release of Ipa invasine and is an essential function of invafiiveneBB, EMBO J,, 14:2461-2470; Zierler and Galan» 1995, Contact with cultures epithelial cells stimulates secretion of Salmonella typhimurium invasion proteins IrrvJ, Infect, Immun,, 63:4024-4028); that some of the secreted proteins are delivered into Hie cytoplasm of host cells (Rosqvist et al, 1994, Target cell contact triggers expression and polarized transfer of Yersinia YopE cytotoxin into mammalian cells, EMBO J., 13:964-972; Sory and Cornells, 1994, Translocatum of an hybrid YopE-adenylate-cyclase from Yerainia enterocoliu'Da into HeLa cells, Mol. Microbiol., 14:583-594; Wood et aL, 1996, SopE, a secreted protein of Salmonella dublin, is translocated into the target eukaryotio cell via a sip-dependent mechanism and promotes bacterial entry, Mol Microbiol, 22:327-338; Collazo and Galaii, 1997, The invasion-associated type HI system of Salmonella typhirourium directs the tranalocation of Sip proteins into the host cell, Mol. Microbiol, 24:747-756); and that transcription of genas encoding secreted proteins is controlled by secretion of regulatory proteins (Hughes et al., 1993, Sensing structural intermediates in bacterial flagellar assembly by export of a negative regulator, Science,
262:1277-1280; Pettersson et al., 1996, Modulation of virulence factor expression by pathogen target cell contact, Science, 273:1231-1233).
[00166] In another embodiment of this aspect of the invention, the host strain may be selected for other mutations impacting secretion. For this purposa, a secreted selectable marker protein is expressed without a signal peptide in the host call and mutants are selected which permit secretion of the marker protein, The host strain may be treated with a mutogen to increase the number of mutations or another method to introduce mutations may be used such as transposon mutagenesis. A suitable marker protein is bata-lactamaae, which confers resistance to beta-lactam antibiotics such as ampicillin, Beta-lactamase is expressed without a signal peptide and ampicilliu-resistant mutants are selected. These mutants are screened for the ability to secrete other proteins such as antibody fragments in the absence of signal peptides in order to identify prl mutations. By this means, a mutation allowing secretion without a signal sequence can he introduced into any desired strain of E. coti such as a wild-type W3110 strain or a strain with a high transformation frequency or a strain with mutations in other chaperone proteins.
[00167) Some embodiments of the invention use singly or multiply protease-deficiont
mutant hosts. Different proteins will be more or less sensitive to different proteases normally produced by the microorganisms. Strains may be used which are deficient in proteases such as ompT and degp, Protease HI, La Protease, ClpYQ, ClpXP and ClpAP.
[00168] This invention will be better understood from the Experimental Details which
follow. However, one skilled in flie art will readily appreciate that the specific methods and
results discussed are merely illustrative of the invention as described more folly in the claims
whi ch follow there after. ,
EXAMPLES
Example 1, Expression and secretion of human anti-PorV Fab fragment without sigjial
peptidas
[00169] The Fab fragment of human antibody 1A8 was expressed and secreted from E,
coli mutant prlA4 without signal peptides. Fab 1A8 is an engineered human antibody fragment which binds specifically to an epitope on the PcrV protein of Pseudomonas aeruginosa with high affinity. It competes for binding with a mouse antibody Mabl66
identified to the same epitope (Frank et al, 2002, J Infect Dis. 186:64-73). The light chain of 1A8 consists of a Vkl -kappa light chain and the Fd chain is a VH3 sub-class V-region fused to an IgGl CHI domain,
[00170] A signal-less expression vector for the expression of Fab 1A8 was derived from pGEX-4T-l (GE Healthcare) as follows. The Ampidllin-resistance gene in pGEX-4T-l was deleted by digestion with Aatn and AlwNI, and was replaced with a Chloraraphenicol resistance gene obtained by PCR amplification from plasmid pACYCDuet (Novagen) to form pGEX-CAT. A point mutation of I to A at position 256 was generated by PCR-mutagenesis to mtrodwce a unique Bstll07l restriction site in pGEX-CAT just before the translation initiation codoti downstream of the pTac promoter.
[00171] The pTac promoter of pGEX-CAT was used to express the light chain which was cloned between the Bstll07I and EcoRI sites by PCR using following primers to give vector KB-L, A T7 terminator sequence was incorporated in the Primer 2 before the EcoRI
site.
Primer 1: GGAAACAGTATACATGGACATCCAGTTGACCCAGTC (SEQ ID N0;4)
Primer 2: GCCAGTGAATTCAAACCCCTCAAGACCCGTTTAGAGGCCCCAA
GGGGTTATOCTAGTTAATCGATTTAACACTCTCCCCTGTTGAAGC TC(SEQIDNO:5)
This primer pair amplifies the mature light-chain coding sequence of 1A8 and adds a translation-initiation codon and an upstream sequence to provide an appropriate distance between the Shine-Dalgarno ribosome-binding sequence (AGGA (SEQ ID N0;6)) and the initiation codou of 9 nucleotidos. The predicted amino acid sequence of the N-terminus of the light chain (in single-letter amino-acid code) is:
MDIQLTQ (SEQ ID NO;7)
[00172] The heavy chain (Fd chain) of Fab 1A8 was cloned similarly by PCR, using primers 3 and 4, and introduced between the Bstll07I and NotI sites of pGEX-CAT to give vector KB-H.
Primer 3: GGAMCAGTATACATCGAGGTGCAGCTGGTGGAQTC (SEQ ID NO: 8)
Primer 4; CACGATGCGGCCOCTTAACAAGATTTGGGCTCAACTTTC (SEQ1D NO: 9)
This primer pair amplifies the mature Fd chain coding sequence and adds a translarion-initiation codon and sequences to provide a Shine-Dalgarno - ATG distance of 9 nucleotides. The predicted amino acid sequence of the N-tarminus of the heavy chain is:
MEVQLVE (SEQ ID NO: 10)
[00173] The pTac expression cassette of KB-H was then amplified by PCR. using
Primer 4 and 5 and cloned into KB-L between EcoRI and Notl sites to give vector KB-LH,
Primer 5: CGATGCGAATTCGACTCTAGCGCTGTGGTATGGCT GTGCAGGTCG (SEQ ID NO; 11)
[00174] The final signal-less expression vector for expression of Fab 1AS was constructed by cloning the EcoRI and Fspl fragrnant (138 nucleotides) of pUC19 (Fermentas) into KB-LH between EcoRI and Afel sites to provide a spacer between 2 pTac expression cassettes. A map of the plasim'd. KB5246 is shown in Figure 2.
[00175] E, coli strain SE6004, containing theprlA4 mutation (Emr et al, 1982, J. Biol.
Chem 257: 5852; Wong et a/., 1988, Gote 68: 193), was obtained from the Netherlands Culture Collection of Bacteria (NCCB catalog number 2976).
[00176] Plasmid KB5246 was introduced into SE6004 by electroporation. Electro-
competent cells were prepared using standard techniques as described in Short Protocols in Molecular Biology (3rd edition), Auaubel et at., (John Wilay and Sons foe). Electropration was carried out using a Biorad E. coli Pulser elactroporation apparatus according to the manufacturer's man-actions with a 1.8 kV pulse and a 5 ms time constant. Electroporation cuvettes were from BTX. Transfonnants selected on 34 µg/ml chloramphenicol were cultured in 2xYT medium and expression of the heavy and light chains of Fab 1 AS was induced using isopropyl-beta-D-thiogalactopyranoside (JPTG) at concentrations up to 1 mM. Induction was carried out. for 3 hours for analysis of Fab expression in the periplasm, or cultured for 16 hours for analysis of Fab released into the medium.
[00177] For analysis of Fab secreted across the cytoplasmic membrane into the periplasm, cells were fractionated as follows. The bacterial cell pallet from a 1 liter culture was resuspeuded in I Oral of TES buffer (0.2M Tria pH 8.0, 17.12% sucrose trad O.5mM EDTA) and incubated at 4"C for 15 minutes. After the addition of 12.Sral of TES / H2O at a ratio of 1 / 4, the cell mixture was incubated at 4"C for a further 15 minutes, The cells were pelleted by centrifugation at 7000 rpm in a Sorvall banch-top centrifuge for 15 minutes and the superaatant was kept. The pellet was then resuspended in 10ml TES supplemented with 15mM Mg2S04 and incubated at 4"C for 10 minutes followed by ropefletwg at 7000 rpm and retention of the supernatant.
[00178] 10 µl of periplasmic extract was run on an SDS-PAGE gel under naiwrediidng
conditions, transferred to PVDF membrane and western blotted using an anti-Human Kappa specific antibody conjugated .to Horseradish peroxidase (Zymed labs). The Peroxidase substrate ECL plus (GE Healthcare) was use to produce luminescent signal which was then detected on radiographic film to detect Fab secretion. Figure 3 shows a representative Western blot demonstrating secretion of assembled 1A8 Fab detected in the periplasm. A small amount of immuiioglobulm-related protein of lower molecular weight is also detectable. These bands are consistent with the secretion of light-chain dimera and moncroeric light chain as typically found on secretion of Fab fragments from E. coli in other, signal-dependent secretion systems.
[00179] Fab 1A8 secreted either into lie periplasin or into the medium was analyzed
for antigen-binding activity using a specific antigen-based enzyme-linked immunasarbent assay (ELISA). For this purpose, recombinant PcrV antigen, closed as a fusion protein in frame with an amino terminal glutathione S-transferase (GST) purification tag, was used as described previously (Frank et at., 2002, J. Infectious Diseases 186: 64-73). The PcrV coding sequence is cloned in the expression vector pGEX 2TK (GE Healthcare) to generate the GST-PcrV fusion protein.
[00180] For production of antigen for use in ELISA for the detection of functional anti-PcrV Fabs, GST-PcrV fusion protein was expressed from JS. coli (BL21) transformed witli pGEX 2TK-PcrV and purified as follows. 4 liter liquid culture batches of JS. coli expressing GST-PcrV were grown in 2xYT medium to an optical density of 0.6 at 60Qmn before induction of protein expression with 0.5 mM IPTG and a farther 3 hours growth. The bacterial cells were pelleted by centrifiigation and lysed in a solution of Bug Buster
(Novagen) supplemented with lU/ml iLysozyrae (Novagen) and a protease inlribitor cocktail (Sigma-Aldrich) diluted to the mamifecturer's insn-uctions, After clearing the lyaate by centrifugation and filtration, it was passed over a glutathione sepharose column (GSTrap FF, GE Healthcare), washed and the pure GST-PcrV was eluted in lOmM Glutathione. Tlie antigen was desalted back into PBS.
[00181] Antigen-binding ELISAs for detection of anli-PcrV Fab in periplftsm fractions
i
or in medium samples were carried out as follows. ELISA plates (Costar ElA / R1A) were coated with 100 ng/well GST-PcrV in PBS (see above) by incubating at 4°C for 16 hours and blocking for 1 hour with a 5% solution of non-fat dry railk in PBS 0.1% Tween 20 (PBST). periplasmic fraction samples were diluted in a 1 fold series and applied to the ELISA plate for Ihour at 33DC. After washing with PBST, antibody fragments binding to the antigen were detected with goat anti-human. kappa-ERP. conjugate (US Biological) at a dilution of 1/1000 hi PBST. Antibody binding was revealed using the peroxidase substrate Tetramethyl benzidine (TMB) (lOOjjl / well), and the reaction was stopped with the addition of lOOul 2N HaSO* and read by a standard plate-reader.
[00182] Antigen-binding ELISA confirmed the presence of functional Fab 1 AS in the periplasm (see Figure 4) and released into the medium of SE6QQ4 transfarmants containing plasmid ICB5246. Figure 4 demonstrates secretion of significant amounts of Fab fragment capable of binding to PcrV in. comparison with a standard preparation of Fab fragment in the periplaam of cells expressing Fab 1A8 in a signal-dependent manner (preparation 1150 in Figure 4),
i
[00183] Thus the heavy and light chains of Fab 1A8 are secreted form prlA4 mutant E. coli without the need for a signal peptide on either chain. The two chains assemble to form Fab fragment which can be detected in the periplasm and released into the culture medium as functional antigen-binding molecules.
Example 2. Detection of antigen-binding Fabs by Colony-Lift Binding Assay (CLBA)
[00184] Libraries of antibody Fab fragments cloned in plasmid KB5246 and
transformed into SE6004 are plated onto 2YT agar (Becton, Dickinson Difco™ 2xYT yeast extract tryptone medium) containing -the appropriate antibiotic (chlorampheniool at 34µg/ml). The plating efficiency is adjusted so the resulting bacterial colonies are discreet but dense enough to maximize the area of the plate. Various sizes of plate are used depending on the
number of clonal colonies to be screened, Thus, at optimal density a 10cm diameter plate contains 4000 colonies, a 15cm diameter plate contains 10000 colonies and a 25cm square plate contains 50,000 colonies,
[00185] Nitrocellulose filters (Schlmcher & Schuell BA85) of diameter 8,2cm, 13.2cm or 20cm square are pre-coated with antigen in. Phosphate Buffered Saline (PBS) at an empirically determined concentration (usually between 0.5 and 20µg/ml). Tha volume of coating solution depends upon the filter size, 4ml, 8ml or 20ml can be used for the various filter sizes listed above. Filters are placed face down in a pool of the antigen and capillary action evenly distributes the antigen. The filters are coated for 2-3 hours at 33°C with occasional agitation. Hie filters.are then rinsed once with excess PBS and blocked with a 5% solution of non-fat dry milk in PBS for an additional 2 hours at 25°C with agitation. The filters are then drained and rinsed once in PBS supplemented with 0.1% Tweeii 20 (PBST) and twice in excess 2YT liquid media supplemented with antibiotic selection (34 µg/ml chlommphenicol) and transcriptional tnducer (IPTG). The IPTG concentration can be optimized for each library but is typically in the range 0.01 - 0.1 mM. After allowing tha filters to drain, they are placed on a 2YT-agar plate supplemented with the same concentration of antibiotic and inducer (the expression plate).
[00186] Un-coatedj dry nitrocellulose membrane is placed face-down on the plates of colonies containing the antibody-fragment library. Once the filters are visibly wet (~20sec) ' and in one movement, the filters are lifted and placed colony side up onto the coated filter which is already on the expression platfl. A sterile needle is used pierce Ilia filters in a pattern which will allow alignment.
[00187] The expression plate with the nitrocellulose filter sandwich is placed at 33DC for 12-16 hours. During this time, tha antibody fragments are secreted and diffuse through the first nitrocellulose membrane to the second, antigen-coated membrane. If the antibody fragment ftom a given bacterial colony has antigen binding activity, it is retained on the antigen filter and is subsequently detected,
[00188] After the 12-16 hour expression period, the colony filter is removed from the
expression, plate and stored at 4°C on a 2YT-agar plate with antibiotic selection but no trgnscriptional inducer.
[00189] The antigen-coated filter is removed and washed throe times (5 minute washes) in excess PBST followed by blocking with a 5% solution of non-fat dry milk in PBST for 1.5 hours at 25°C. The antibody fragments retained on the antigen filter are then detected by first incubating with one of the following alternative primary antibodies: Goat anti-human Kappa-HRP conjugate (US Biological) is used to reveal binding. After four 10-mhiuta washes, the filters are incubated in peroxidase substrate solution (EGL plus, GE Healthcare) and used to expose light-sensitive photographic film. Alternatively, antibodies conjugated with fluorescent labels may be used. In this case a flatbed excitation scanner such as the Typhoon (GE Healthcare), FX-Pro (Biorad) or Odyssey (Licor) can be used to visualize the positive spots,
[00190] Using a light box for back illumination, the pattern of spots on the photographic film or digital image is aligned with the colony filter (the filter can be removed from the 2YT-agur plate and placed on a plastic transparency for this process). The identified positive colonies are picked and used to inoculate ft 2YT liquid mini-culture. Bacteria from the primary screen are then re-plated at a lower density and picked for subsequent analysis to ensure that a clonal population is expanded.
Example 3. Detection of anti-PcrV Fab secreted froro prlA4 cells without signal peirtides using CLBA
[00191] For Fab fragments expressed without signal peptides in plasmid KB 5246, transformed cells were plated on 2YT expression plates containing chloramphemcol (34|J-g/ml) and 10µM IPTG. Cells were induced for 16 hours and antibody fragments binding to GST-PcrV on the antigen-coated filter were detected as described in Example 2, using a goat ant-Miwnan kappa antibody - Horseradish peroxidase conjugate (US Biological) at a dilution of 1/5000 in PBST. After four 15-mmute washes and the application of ECL Plus (GE Healthcare), the filters were weed to expose autoradiographic film (Hyperfihn from GE Healthcare),
[00192] Plasmid KB5246, expressing FablAB, was transformed into SE6004 cells,
which have a mutant SecY gene (containing the prlA44 mutation), and into TOPIC cells which contain a wild type SecY gene. Positive colonies secreting FablA8 were detected in the PcrV antigen-CLBA only from SE6004 tranaformants; the TOP 10 tamsformants did not secrete detectable amounts of FablA8. This result indicates that the prlA4 mutant strain is
able to secrete Fab fragment without the need for a signal peptide on either the heavy or light chain, The heavy and light chains assemble and are capable of forming Mly ftinetional Fab fragment capable of binding the cognate antigen coated onto a nitrocellulose filter.
Example 4, Screening for binders to specific antigens of Fabs eecretad without signal peptidea
[00193] A second human Fab FB42-8, specific for a human cytakine, was expressed without signal peptides in SE6004 by cloning the appropriate V-regtons sequences into KB5246 in place of the FablA8 V-regions, Cells expressing the two Fabs (FB42-8 and FablAS) were mixed in a 50/50 ratio and plated on 2xYT agar, A CLBA was performed as described in Example 2, with the coated, antigen being either PCRV or the cytoldne antigen specific for FB42-8. Duplicate CLBA, detection and alignment showed that Fabs specific for each antigen could ba picked from, a mixture of the two tranafonnants.
(00194] A library of diverse Fabs can be screened for binders to a specific antigen in
the same manner.
Example 5. Efficiency of secretion of Fabs without signal peptidas
[00195] A Fab was cloned into either a typical bacterial expression plaatnid with
bacterial secretion leader-peptides (KB 1150) or into KB5246 hi place of the Fab 1A8 coding sequences, These two constructs were compared for efficiency of expression and secretion at various IPTG induction conditions. Cultures were grown until OD0.6 at 600nm and then induced, Growth was continued for lolirs, Fab secreted into the culture medium was detected on a western blot by an aflti-kappa-HRP polyclonal, as in Example 1 (see figure 5).
[00196] Fabs containing murins V-regions are known to be difficult to express to high yields in bacteria. In this experiment the Fabs secreted, more efficiently without signal peptides from SE6004 than when expressed with signal peptides in the wild-type TOP 10 straia Indeed, Fab secretion was undetectable using signal-peptide mediated secretion in TOP10F' cells and was readily detectable in the medium when the SE6004 strain was used for secretion of signal-less Fab. Thus Fabs and other antibodies which are poorly expressed in E. coli may advantageously be produced by secretion in the absence of signal peptides from appropriate mutant strains such as the prlA4 mutant SE60Q4,
Example 6. Construction of an expression vector for a orlA4 mutant SacY gene
[00197] An expression vector, plSA, for expression of genes in bacterial cells under the control of a strong bacterial promoter, Ifte trc promoter, was constructed as follows.
[00198] Plasmid pACYC!77 (Fermantas) was digested with BanI and partially digested with Stul. The 2386 bp DNA fragment was then blunt-ended using Klenow fragment of DNA polymerase I. The pTrc promoter was PCR amplified from the plasmid pfixHis-GFP (Clontech) with the following primara:
Primer 1: TCTTCCAGGCCTGAGCTCGAGCTGTTGACAATTAATCA (SEQ ID NO: 12)
Primer 2: CAGTTACAGGCCTGGTACCTCACCGGCCGTTAAACCCCCCAT GGriTATTCC (SEQ ID NO: 13)
The PCR product was then digested with Still, and ligatod with the 2386 DNA fragment of pACYC!77 to give vector plSA, which has Ncol and Kpnl sitee after the pTrc promoter.
[00199] The prlA4 mutant SecY gene was cloned from SE6004 cells by PCR
amplification using the following primers:
Prirner3; ACGGAATTCACCATGGCTAAACAACCGGGATTAGATTTTC (SEQ ID NO: 14)
Primer4: CAGTTACGGTACCrfATCGGCCGTAGCCTTTCAGGTTC (SEQ ID NO: 15)
[00200] The PCR product was then digested with Ncol and Kpnl and cloned into vector pi 5A between the same two sites to give KB5282 which expresses the mutant SecY gene under the control of the bacterial trc promoter (pTrc; see Figure 6). Transformation of E. coli strains with KB5282 confers theprW phenotypa on the host cell and allows secretion via Hie periplasm of heterologous proteins such as antibodies from coding sequences which do not encode signal peptides.
[00201] Electro-competent DHS-alplia cells were transformed with plasmid KB5282
by elesclroporation and transformants were selected uauig 35 |ig/ml kanamycin in 2xYT medium.
[00202] The expression of FablA8 in DH5 cells in the presence of oveiexpressed mutant SocY was assessed as follows. DH5 cells were co-transformed, by electropor&tion with plasmids KB5246 (expressing the Fab without signal sequences) and KB5282 (expressing mutant SecY). Transformants selected on ohloratnphenicol and Kanamycm were cultured in 2xYT medium and expression of the heavy and light chains of Fab 1 AS was induced using isopropyl-bata--thiogalactopyratioside (IPTG) at a concentration of 20^iM or 200uM. Expression was continued for 16 houra at 33°C with shaldng. The levels of expressed and secreted intact Fab fragments from DH5 cells with the co-transformation of mutant SecY was compared with expression of Fab from tiffiprlAJ strain SE6004 using the same concentrations of IPTG, as described in Example 1. Western blots using a detection antibody specific for human Kappa chains were carried out on expression madia ran on SDS-PAGE under non-reducing conditions (Figure 7). High levels of secreted Fab ware detected in the media of DH5α cells expressing mutant SecY. Indeed these cells secreted higher levels of Fab when induced using 20 µM IPTG than SE6004 cells. In contrast, no detectable secretion of Fab was observed when 1CB5246 was transformed into TOPIC F' cells, a strain which expresses wild-type SecY, (see Figure 7).
Bxarnple 7. Expression of antibody fragments with signal sequences in prl mutant K call strains
[00203] Expression vectors which encode antibody polypeptides including signal
peptides can also be expressed in a prl mutant E. coli strain as follows, A signal peptide is introduced at the N-terminus of the heavy chain coding sequence, the light chain coding sequence or both in order to secrete assembled and fanctional Fab or Fab' fragments from the prl mutant strain.
[00204] To generate a convenient prl mutant strain for the expression of antibody
fragments containing signal peptides, plasmid ICB5282 (Example 6) is used to transform DH5-alpha cells. The kananrychvvesistant transformants have the prlA phenotype and can secrete Fab fragments lacking signal peptides as described in Example 6. In this case the KB5282 DH5-alpha Irarisformants ate subsequently transformed by electroporatiou with an expression vector expressing antibody heavy and light chains in which one or both of the chains is expressed with a signal peptide. Electrocorapetent cells are prepared according to standard techniques as described in Short Protocols in Molecular Biohgy (3rd edition),
Auaubel et al (John Wiley and Sana Inc.) and elactroporation is carried out as described in Example 1.
[00205) Functional Fab or Fab' fragments are identified and may be isolated from the peripksmic fraction or the culture medium as desoribod in Examples 1,2 and 3 above.
SECRETION OF ANTIBODIES WITHOUT SIGNAL PEFTIDES FROM
BACTERIA
CROSS-REFERENCES TO RELATED APPLICATION(S)
[0001] The present application claims the benefit of priority under 35 U.S.C. §119
from U.S. Provisional Patent Application Serial No. 60/701,902 entitled "SECRETION OF ANTIBODIES WITHOUT SIGNAL PEPTIDES FROM BACTERIA", filed on July 22, 2005, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.
FIELD OF THE INVENTION
[0002] The present invention is directed generally to methods for secreting
antibodies, antibody fragments, or antibody-related polypeptides from prokaryotes without the need for a signal peptide. In particular, the invention provides host cells and methods for secreting antibodies, antigen-binding antibody fragments, or antibody-related polypeptides from bacteria without the need for a signal peptide and provides diverse libraries of antibodies, antibody fragments, or antibody-related polypeptides resulting from such methods. The present invention is also directed at antibodies, antibody fragments, antibody-related polypeptides, and libraries of the same made by the methods of the invention.
BACKGROUND OF THE INVENTION
[0003] Most proteins destined for the periplasm or the outer membrane of gram
negative bacteria, such as enteric bacteria, are transported across the cytoplasrnic membrane by the general secretory pathway or Sec system, a complex of proteins, which identifies polypeptides for export and translocates them across the cytoplasrnic membrane. This system has been used to secrete mammalian proteins from the enteric bacteria E, coli, including antibody fragments, Natural prokaryotic secreted proteins and heterologous proteins such as mammalian proteins are directed to the secretory apparatus by the addition of a functional signal peptide, a sequence of typically between 13 and 30 amino acids at the N-terminus of the protein which has a hydrophobia core and additional sequences to direct the nascent polypeptide chain to the secretory apparatus and allow accurate removal of the signal peptide
after secretion. A number of prokaryotic signal peptides have been described which allow efficient secretion of at least some antibody fragments, including the signal peptidea from the Erwinia caratovora. pectate lyase B (PelB) protein, Escherichla coli heat-stable enterotoxin (Stfl) and the E. coli OmpA protein. Other ptokaryotes have similar secretory systems, and signal paptides have been described for many for these other prokaryotes,
[0004] However, secretion systems are highly variable in the efficiency with which
antibodies, antibody fragments or antibody-related polypeptides are secreted. The efficiency of secretion is dependent on the sequence of the variable regions of both the heavy and the light chains of antibodies, For example, Fab fragments containing murine V-regiona are poorly secreted if at all Further, human Fab fragments are secreted witti variable efficiency, depending cm the V-rogion sequence, andA>r VH subclass. Such variable secretion efficiency leads to bias in the sequences of antibodies which may be screened from a generated antibody library,
[0005] In some cases, mutations in the V-region can be introduced in order to
improve secretion. However, alterations in the ammo acid aequence of antibody V-regions may compromise antibody function and are not generally desirable,
[0006] Furthermore, cleavage of signal peptides from the secreted polypeptide is not
always efficient. For example, the signal peptide of E. coli OmpA or PhoA is not cleaved .from a fusion protein with.human interleulcin-1 beta (IL-1 beta) when 1he fusion protein is expressed hi-fi1, coli,
[0007J It is an object of the present invention to provide compositions and methods
for obtaining secretion of antibodies, antibody fragments, 01 antibody-related polypeptides from bacteria without the need for a signal sequence thereby removing any secretion constraints caused by variable region sequence,
SUMMARY OP THE INVENTION
J0008] The present invention, relates to methods for secreting antibodies, antibody
fragments, and/or antibody-related polypoptides in prokaryotea without the need for a signal peptide thereby overcoming limitations imposed by variable region sequences In one embodiment, the methods of the invention comprise expressing polymideotides encoding antibodies, antibody fragments, or antibody-relatedpolypeptides without a signal sequence in
a prolcaryotic noflt cell, followed by secretion of the antibodies, antibody fragments, or antibody-related polypeptides across the cytoptasmic membrane of the host cell. In an embodiment of the invention, antibodies, antibody 'fragments, or antibody-related polypeptides are secreted without a signal sequence by use of -A, prokaryotic host cell that contains one or more mutations in the gene(s) which encode the proteins of the cells secretory pathway(s), In another embodiment, the host cell is E, coli and the secretory mutant is a Protein-localization (prl) mutant.
[0009] The present invention also relates to libraries of antibodies or related
polypeptides (hat are made by the methods of the invention. In one embodiment, the libraries of the invention comprise antibody, antibody fragment, or antibody-related polypeptide clones that cannot be secreted or are difficult to secrete in prokaryotes when signal sequences are used to direct transport across the cytoplasmic membrane. In another embodiment, the antibody, antibody fragment, antibody-related polypeptide, or other polypeptide libraries of the invention have better representation of different VH and VL subclasses than libraries expressed with signal sequences, The antibodies and related polypep'tideB of the invention include intact iramunoglobulins, single chain antibodies, Fab, Fab', F(ab')2, Fv, camelid antibodies, antigen-binding scaffolds, antibody or antibody-related polypeptide fusion
proteins, and other polypeptides disclosed below.
i
[0010] In another embodiment, the antibody-related polypeptides of the invention
include antigens that are recognized by the antibodies, antibody fragments, or antibody-related polypeptides of the invention. In an embodiment of the invention, these antigens are self-antigena,
[0011] In an embodiment of the invention, the methods of the invention allow for
expression, secretion, and assembly of multimeric proteins wherein one or more subunit of the rnultimeric protein lacks a functional signal sequence.
BRIEF DESCRIPTION OF THE FIGURES
[0012] For a better understanding of liifi nature and objects of some embodiments of
the invention, reference should be made to the following detailed description taken in conjunction with the accompanying drawings, in which;
[0013] Figure 1 provides the amino acid sequence of E. coli Sec Y protein from
mutant F286Y and I408N (prlA4), where the mutated amino acids ara shown underlined in bold (SEQ ID NO: 1).
[Q014J Figure 2 shows a plasmid map of a vector used for expression of heavy and
light chain genes for an antibody fragment without a signal peptide in E, coli. The plasmid has a chloramphenicol-acetyl transferase gane to confer resistance to chloratnplienicol and a ladq gene which expresses a lac represser for regulation of gene expression. The light and heavy chain genes are each under the control of a tac promoter and expression is inducjble by lactose or IPTG.
[OOJ5J Figure 3 shows a western blot analysis showing secretion of assembled Fab
1A8 in periplasmic fractions of SE6004 transformed with plasmid KB5246, Fab expression was induced by the addition of IPTG (SEQ ID NO:2) at the concentrations sbown (in uM), Samples were run on SDS-PAGB gel under non-reducing conditions, and probed using an anti-Human Kappa specific antibody conjugated to Horseradish peroxidase,
[0016] Figure 4 shows results of an ELISA demonstrating antigen-binding activity of
Fab Fragments present hi the periplasraic fraction of SE6004 cells. Serial dilutions of peripksmic extracts from cells transformed with plasmid KB5246 or a ten-fold dilution of the extract (5246 10%) ara analyzed for binding to pcrV antigen in comparison with a standard periplasraic extract (1150) or a ten-fold dilution (1150 10%) containing tmti-PcrV Fab expressed in a wild-type E, coli .strain with signal peptides to direct secretion, PciV binding is revealed as an increase in absorbance at 450nm as a result of enzymatic conversion of TMB substrate to a colored product by HRP-conjugated antibody.
[0017] Figure 5 is a comparison of secretion efficiency for Fab from SE6004 and
Topi OF' cells identified in periplasmic extracts, Fab was expressed using IPTG (SEQ ID NO:2) induction at the concentrations shown in SE6004 cells {prlA} without the use of signal peptides. Expression is compared with signal-dependent expression of the same Fab from fi wild-type strain (TOPIOF). Fab present in periplasmic extracts is detected by Western blot analysis using an anti-human kappa detection reagent.
[0018) Figure 6 is a Map of plasmid KB5282 for the over-expression of mutant SecY.
TheprlA4 mutant SecY gene is expressed from the pTrc promoter. The NPT2 gene confers resistance to kanamycin.
[0019J Figure 7 is a Western blot analysis comparing the secretion efficiency for Fab
1A8 from $&prlA4 mutant strain SE6004, Topi OF1 cells expressing wild-type SecY, and DH5D cells co-transformed with a prlA4 mutant SecY gene. Antibody-related proteins secreted into the medium ware detected using anti-human kappa antibody of blots from non-denaturing SDS-PAGE. Assembled Fab, light-chain dimers and light chain monomers were detected in the culture medium.
DETAILS DESCRIPTION OF THE INVENTION
DEFINITIONS
[0020] As used herein, "antigen" refers to substances that are capable, under
appropriate conditions, of reacting with specific antibodies, antibody fragments, or antibody-related polypeptides. Antigens can be soluble substances, such as toxins or foreign proteins, however, only the portion of the protein or autigenic molecule known as the antigenic determinant (epitope) combines with the antibody, antibody fragment, or antibody-related polypeptide. Mare broadly, the term "antigen" is used herein to refer to any substance to which an antibody binds, or for which antibodies we desired, regardless of whether Che substance is imraunogenic. For such antigens, antibodies can be identified by reoojnbmant methods, independently of any immune response.
[0021] As used herein, an "antibody" refers to a protein functionally defined as a
binding protein and structurally defined as comprising an ammo acid sequence that is recognized by one of skill as being derived from the variable region of an immunoglobulin. An antibody can consist of one or more polypeptides substantially encoded by immunoglobulin genes, fragments of immunoglobulin genes, hybrid immunoglobulin genes (made by combining the genetic information from different animals), or synthetic immunoglobulin genes. The recognized, native, immunoglobulin genes include the Icappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes and multiplo D-segments and J-segmenta. Light chains are classified aa either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively,
[0022] A typical antibody structural um't is known to comprise a tetramer. Each
tetrainer is composed of two identical pairs of polypeptide chains, each pair having one
"light" (about 25 IcD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region (V) of about 100 to 110 or more amino acids primarily responsible for antigen recognition.' The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
(0023) Antibodies exist as intact immunoglobulms, as a number of well characterized
fragments produced by digestion with various peptidases, or as a variety of fragments made by recombinant DNA technology, Thus, for example, papain digests antibodies into an antigen binding Fab fragment and a residual Fc fragment; pepsin digests an antibody below the disulfide linkages m the hinge region to produce F(ah')2, a dimar of Fab which itself is a light chain joined to VH-CHI by a disulfide bond. The F(ab')j inay be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the F(ab')2 dimer into an Fab' monomer. Ths Fab1 monomer is essentially a Fab with part of the hinge region ($ important point is that the two antibody chains in each Fab molecule assemble post-translationally and the dimer is incorporated into the phage particle via linkage of one of the chains to g3p (see, e.g., U.S. Patent No; 5,733,743, which is incorporated herein by reference in its entirety), The scFv antibodies and a number of other structures converting the naturally aggregated, but chemically separated light and heavy polypeptide chains from an antibody V region into a molecule that folds into a thtee dimensional structure substantially similar to the structure of an antigen-binding site are known to those of skill in the art (see e,g,, U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778). Particularly preferred antibodies include all those that have been displayed on phage (e.g.f acFv, Fv, Fab and disulfide linked Fv (Reiter et alt 1995, Protein Eng. 8; 1323-1331). Antibodies pan also include diantibodies and mint antibodies.
(0024] Antibodies can derive from multiple species. Far example, antibodies include
rodent (such as mouse and rat), rabbit, sheep, camel, and human antibodies. Antibodies can also include cbimeric antibodies, which join variable regions from one species to constant regions from another species. Likewise, antibodies can be humanized, that is constructed by recombinant DNA technology to produce iirimunoglobulins which have human framework regions from one species combined with complementarity determining regions (CDR's) from a another species' imraunoglobulin (see, e.g., EPO Publication No. 0239400), In the case of antibodies, the modules consist of "framework" and "CDR" modules, By creating separate framework and CDR modules, different combinatorial assembly possibilities are enabled. Moreover, if two or more artificial genea carry identical pairs of cleavage sites at the boundaries of each of the genetic sub-eleraenta, pre-built libraries of sub-elements can be inserted in these genes simultaneously, without any additional information related to any particular gene sequence. This strategy enables rapid optimization of, for example, antibody affinity, since DNA cassettes encoding libraries of genetic sub-elements can be (i), pre-built, stored and reused and (ii), inserted in any of these sequences at the right position without blowing the actual sequence or having to determine the sequence of the individual library member. Exemplary methods for generating synthetic libraries of antibodies are disclosed in, for example, U.S. Patent No. 5,885,793 and 6,300,064, which are incorporated herein by reference in their entirety.
[0025] Antibodies also include epitopo-focused antibodies, which have at least one
minimal essential binding specificity determinant from a heavy chain or light chain CDR3
from a reference antibody, methods for making fluch epitope-focusecj antibodies are described in U.S. Patent Application No. 11/040,159, which is incorporated herein by reference in its
entirety.
[0026J The term "cytoplasmic membrane" refers to a membrane that encloses the
cytoplasm of a cell and, in a bactorhvm, lies internal to the pariplaam and outer membrane in gram negative bacteria.
[0027] As used herein, the term "diversity" refers to the number of different specific
antigen binding antibodies or related polypeptides.
i
[0028] An "expression vector" is a nucleic acid construct, generated recombinantly or
synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, vims, or nucleic acid fragment. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
[0029] As used herein, the term "framework region" refers to those portions of
immimoglobulin light and heavy chain variable regions that are relatively conserved (i.e., other than the CDRs) among different immunoglobulins in a single species, as definsd by Kabat. As used herein, a "human framework region" ie a framework region thai is substantially identical (about 85% or more) to Hie framework region of a naturally occurring human antibody.
[0030} As ijsed herein, the term "fusion antibody" refers to a molecule in which an
antibody is fiised to a non-antibody polypeptide at the N- or C- terminus of the antibody polypeptide. In one embodiment the antibody fragment may comprise one or more C-terminal peptide tags to facilitate detection and purification, fh another embodiment the antibody may be fused to a pepu'de ot polypeptide for display on the surface of a cell, spore or virus. For example one chain of the antibody fragment may be displayed as a fusion protein on the surface of a bacteriophage such as a filamentous phage.
i
[0031] The term "host cell" refers to a cell that provides the cellular machinery for
expression and secretion of a polypeptide from an expression vector.

10032J Tne terra "humanized antibody" refers to antibodies constructed by
recombinant DNA technology to produce inanunoglobulins which have human framework regions from one species combined with complementarity datcrrrmrmg regions (CDR's) from another species' immunoglobuh'n (see, &g., EPO Publication No, 0239400),
{0033J As used herein, the term "hrnnunoglobulin" refers to tetrameric Antibodies as
wall as R variety of forms besides antibodies; including, for example, Fv, Fab, and $((&>% as well as bifunctional hybrid antibodies, fusion antibodies, chimeric antibodies, humanized antibodies, hurnaneered antibodies and single chain antibodies.
[O034J "Library11 means a collection of nucleotides sequences, e.g., DNA, encoding
antibodies or related polypeptides within clones; or a genetically diverse collection of antibodies or related polypeptidea.
[0035] A "multiraeric protein" as used herein refers to a globular protein containing
more than one separate polypeptide or protein chain associated with each other to fonn a single globular protein in vitro or in vivo. The niultimeric protein may consist of more than one polypepticle of the same kind to form a "homomultimer." Alternatively, the mulrimeric protein may also be composed of more than one polypeptide of distinct sequences to fonn a "heteromultimer," Thus, a "heterornultimer" is a molecule comprising at leaat a first polypeptide and ft second polypeptide, wherein the second polypeptide differs in amino acjd sequence from the first polypeptide by at least one ammo acid residue. The heteromulb'Tner can comprise a "hBterodimer" formed by the first and second polypeptide or can farm higher order tertiary structures where more than two polypoptidea are present Exemplary structures for the heteromulliroer include heterodimers (e.g. Fv and Fab fragments, diabodies, GABAg receptors 1 and 2 complexes), trimeric G-proieins, heteroterramers (e.g. F(ab')i fragments) and further oligomeric structures.
[0036J "Protein localization (prl) mutant" refers to a host cell with an alteration in its
secretory apparatus which rescues the secretion-defect in proteins containing a defective signal peptide and in proteins without a signal peptide,
[0037] The terms "secretion/secrete/secreting" refers to transport ftom the cytoplasm
of a cell across the cytoplasmfc membrane, including transport pathways that require a signal sequence and transport pathways that da not require a signal.
(0038] The terms "signal sequence" and "signal peptide" both refar to a peptide
sequence capable of aiding in tha secretion of a connected nascent peptide to the ovttside of Gie host cell.
{0039] The terms "VH and VL subclasses" refer in humans to the 7 recognised VH
sub-classes (VH1 - VH7) and 16 VL sub-classes (Vkappal - VkappaS and Vlambdai-VtaribAUO).
[0040] The term "vector" includes any nucleic acid suitable for cloning or for
expression of the nucleic acids of the invention in the host cells of the invention. Tlie vector may, for example, be in the form of a. plaamid, cosraid, viral particle, or phage. The vector may be self-regulating or may integrate into the host cell chromosome or other replication nucleic acid in the host cell. The vector may also be non-replicating or may poorly replicate, for example, in a transient expression system,
EXPRESSION SYSTEMS OP THE INVENTION
[0041] The invention provides rnsthods for secretion of antibodies, antibody
fragments, or antibody-related polypeptides from prokaiyotic host cells without the need for a signal peptide. In one aspect of the invention there is provided a new method for secretion of an sntibody or an antigen-binding fragment and its assembly into a functional antigsn binding molecule. The antibody is encoded by one or mora nucleic acids that comprise the coding sequences for the V region for an antibody. Antibodies of the invention may contain signal sequences, as described in U.S. Patent No. 6,204,023, which is incorporated herein by reference in its entirety. In some embodiments, the antibodies or multrnieric proteins of the invention are expressed from one or more polynucleotidas encoding polypeptides lacking a signal peptide. The antibody is encoded by one or move vector(s) capable of expressing an antibody, antibody fragment, or antibody-related polypeptide. If the antibody is formed from a heavy and a light chain, coding sequences for bolh, chains may be present on the same vector or the coding sequences may be present on different vectors within [0042] Generally, recombinant expression vectors will include at least one origin of
replication, phenotypic selectable markers permitting selection in host cells, e.g., the arnpiclllin resistance gene of £ call and Sacckaromyces cerevistae TRP1 gene, a functional promoter to direct transcription of a downstream structural sequence, aa well as suitable translation imitation and termination signals in operable reading frame. Suitable prakaryotic hosts for transformation include species in the family Enterobactertoceae such as E, coli, or Salmonella typhimwim, various speoias within the genexa Pseudomonas, Streptomyces, and Staphylacaccus, other species such as Bacillus subtllis, and other bacterial hosts may also be employed as a matter of choice,
J0043] There are many expression systems for producing the potypeptidea of the
invention that are well known to those of ordinary skill in the art. (See, e.g., Femandes and
Hoeffler, Eds,, 1999, Gene Expression Systems, Academic Press.) Larga numbers of suitable
vectors are Icnoxvn to those of skill in the art and are commercially available for generating
toe recombinant constructs of the present invention. The following vectors are provided as a
representative but nonlimitiug example; bacterial; pBs, pbagescript, PsiX174, pBluescript
SIC, pBs ICS, pNHSa, pNHlfia, pNHlSa, pNH46a (Stratagene); pTrc99A, pKK223-3,
pKK233-3, pDR540, pRITS (Pharmacia), pSKF, pET23D, k-phage derived vectors, p!5A-
based vectors (Rose, 19S8, Nucleic Acids Res. 16:355 and 356) and fusion expression
systems such as GST and LacZ, Some expression vectors for bacterial use can comprise a
selectable marker and bacterial origin of replication derived from commercially available
plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC
37017), Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine
Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotec, Madison, WI, USA). These
pBR322 "backbone" sections are combined with an appropriate promoter and the structural
sequence to be expressed, ,
[0044j Typically, the expression of tha polyrmcleotide that encades the polypeptide is
undsr the control of a promoter that is functional in the desired hast cell, A wide variety of promoters is available, end can be used in the expression vectors of the invention. Ordinarily, the selection of fee promoter depends upon the cell type iu which the promoter is to be used. Such promoters can be derived front operons encoding glycolytic enzymes such as acid phosphatase, or heat shock proteins, among others. Particular named bacterial promoters include lad, lacZ, T3, TV, gpt, lambda PR, tac and trc. Commonly used prokaryotic
promoters include the beta-lactamase (penicillinase) and lactose (lac} promoter systems (Change, et at., 1990, Nature 198: 1056), flie tryptaphan (trp) promoter system (Goaddel et al,t 1980, Nucleic Acids Res. 8: 4057), the tao promoter (DeBoer, et al, 1983, Proc, Natl Acad, Sci. V,S.A. 80:21-25); and the lambda-derived PL promoter and N-gene riboeome binding site (Shhnatake et al,, 1981, Nature 292:128), The particular promoter system is not critical to the invention, any available promoter that functions in prokaryotes can ba used. Selection of the appropriate vector and promoter is wall within the level of ordinary skill in the art.
J0045] For expression of polypeptidos in prokaryotio cells other than JE, coti,
regulatory sequences for transcription and translation that function in the particular prolcaryotic epecies ate required. Such promoters can be obtained from genee that have bean cloned from the species, or heterologous promoters can be used. For example, the hybrid trp-lac promoter fimctiona in Bacillus in addition to E. coli, These and other suitable bacterial promoters are well known in the art and are described, e.g.t in Sambrook et a/., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N,Y. and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Intsrscience, N.Y. Bacterial expression systems .for expressing the proteins of the invention are available in, e.g,, E, coli, Haoiltus sp.t and Salmonella (Palva et at., 1983, Gene 22:229-235; Moabach et al,, 1983, Nature 302:543-545). Kits for such expression systems are commercially available.
[0046] Either constitutive or regulated promoters can be used in Hie present invention.
Methods for regulating expression in £ coli are well known in the art and include the use of iuducible promoters such as the lac or (ate promoters which are inducible by IPTG, and arAbinose-inducibla promoters. Regulated promoters can be advantageous because tha concentration of heterologous protein in the boat cell can be controlled. An inducifale promoter is a promoter that directs expression of a gene where the level of expression is alterable by environmental or developmental factors such as, for example, temperature,, pH, atiaerobic or aerobic conditions, light, transcription factors and chemicals.
[0047] For E. coli and other bacterial host cells, inducible promoters are loiovra to
those of skill in the art. These include, for example, the lac promoter, the bacleriophage lambda PL promoter, the hybrid trp-lac promoter (Armmn et al.t 1983, Gene 25: 167; de Boer et al., 1983, Proa. Nat'I. Acad. Sci. USA 80: 21), and the bacteriophaee T7 promoter (Studier
et al, 1986, J, Mat Jtol; Tabor et alt 1985, Proa. Nat'l Acad. Set USA 82:1074-8). These promoters and their use are discussed in Sambrook at al., supra.
[0048] Inducible promoters for other organisms are also wall known to those of skill
m the art, These include, for example, the metallothionein promoter, the heat shock promoter, as well as many others.
'[0049] Other expression control sequences such as ribosome binding sites,
transcription termination sites, operators, and the like may also be included, DNA constructs that include one or more of these control sequences are termed "expression cassettes." Accordingly> the nucleic acids that encode the polypeptides are incorporated for the desired level of expression in a desired host cell,
{0050] A translation-initiation codon may be introduced djrectly upstream of the
mature antibody or antibody fragment coding sequence such that the antibody or antibody fragment polypepti.de is expressed with a methionyl (or N-fomiyl methionyl) residue at the N-terminus. Additional sequences may be included in the coding sequence of the antibody, for example to facilitate purification or detection of the antibody or for another purpose, Tha heterologous structural sequence is assembled in the appropriate translational reading frame and with the appropriate translation initiation and termination sequences. Optionally, we heterojogous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g,, stabilization or simplified purification of expressed recombinant product
[00S1J An ATG codon is present at the 5'and of the coding sequence such that the
expressed protein has an N-formyl-methiomne residue at the amino-terminus. Upon the expression of the coding sequence, the N-terminal a;nino acid may be retained or may be removed by proteases in the host cell. The antibodies, antibody fragments, or antibody-related porypeptidea generated from expression of the DNA coding sequence according to this aspect of the invention are capable of binding antigen. In the event mat the antibody fragment is formed from a heavy and a light chain, either or both chains may be expressed from a coding sequence with an ATG codon, Preferably both chains are expressed without a signal peptide.
[0052] Tianslational coupling may be used to anhance expression. The strategy uses
a short upatroam open translations! reading frame derived from, a highly expressed gene
native to the transtational system OT a. synthetical/ non-natural highly expressed open raading frame, which is placed downstream of the promoter and ribosome binding site and upstream of a termination codon. Just prior to the termination codon is a second ribosome binding site, and following liifi termination codon is the initiation aodon for the translation of the polypeptide to be expressed. The system allows for the efficient initiation of translation, See Squires, et, alt 1988, J. Bial. Chem, 263:16297-1(5302.
[0053] Secreted antibodies can be detected in or isolated from the culture medium
after a period of growth of the bacteria under conditions suitable for antibody expression. Methods for monitoring antibodies in the medium include Western blot analysis, SDS-PAGB and enzyme-linked iinmunosorptiou assays (ELISA), Secreted antibodies may alao be detected in or isolated from the periplasrn of the bacteria, Me&ods for disruption of the periplasm and release of antibody from the periplasrmc fraction are well known in the art and include the use of low pH (eg pH4,0) or osmotic shock.
[0054] To facilitate purification of the polypeptides of the invention, the nucleic acids
that encode fhe polypeptides can also include a coding sequence for an epitapa or "tog" for which an affinity "binding reagent is available. Such epitnpe tags include, e.g.t c-rnyc, HA-tug, maltose binding protein, VSV-G tag, emti-DYKDDDDK (SEQ ID N0:3) tag, or any such tag, a large number of which are well known to those of skill in the art Expression vectors useftil for recombhrant production of fusion polypeptides having these epitopes are commercially available (e.g., lovitrogen, Carlsbad, CA) vectors pcDNA3.1/Myc-His and pcDNA3.1AT5-His are suitable for expression in mammalian cells). Additional expression vectors suitable for attaching a tag to the fusion proteins of the invention, and corresponding detection systems are Iccown to those of skill in the ert, and several are commercially available (e.g., "FLAG" (Kodak, Rochester NY)). Another example of a suitable tag is a polyhistidine sequence, which is capable of binding to metal chelate affinity Uganda, Typically, six adjacent histidines are used, although one can use more or less than six. Suitable metal chelata affinity Uganda that can serve as the binding moiety for a polyhistidine tag include rritrilo-tri-acelic acid (NTA) (Hochuli, E., 1990, "Purification of recombiuant proteins with metal chelating adsorbents" in Genetic Engineering; Principles and Methods, J.K. Setlow, Ed., Plenum Press, NY; commercially available from Qiagen, Santa Clarita, CA).
[0055] One of skill would recognize that modifications can be made to the protein
domains without diminishing their biological activity, Some modifications may be made to facilitate the cloning, expression, or incorporation of a domain into a polypeptide. Such modifications are well known to those of skill in the art and include, for exatnpla, the addition of codons at either terminus of me polynuclaotida that encodes the binding domain to provide, for example, a methionme added at the arolno terminus to provide fin initiation site, or additional ammo acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences.
POLYPBFTfDES'
Antibodies
[0056] In one embodiment, the secreted polypeptides are antibodies. The basic
antibody structural unit is known to comprise a tetrainar. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 KDa), The araino-terminal portion of each chain includes a variable region of about 100 to 110 or more ammo acids primarily responsible for antigen recognition. The carboxy-tornrinal portion of each chain defines a constant region primarily responsible for effector function. The variable regions of each lighf heavy chain pair form (he antibody binding site. Thus, an intact antibody has two binding sites. Each chain has a constant region (C) and a variable region (V). Each chain is organized into a series of domains. The light chains have two domains, corresponding to the C region and the other to the V region, The heavy chains have four domains, one corresponding to tiie V region and three domains (1,2 and 3) in me C region, The antibody has two anus (each arm being a Fab region), each of which has a VL and & VH region associated wifli each other. It is this pair of V regions (VL and VH) that differ from one antibody to another (owing to amino acid sequence variations), and which together are responsible for recognizing the antigen and providing an antigen binding site, In even more detail, each V region is made up from three complementarity determining regions (CDR) separated by four framework regions (FR). The CDRs are the most variable part of ftje variable regions, and they perform the critical antigen binding function. The CDR regions are derived from many potential germ line sequences via. a complex process involving recombination, mutation and selection.
[0057] Light chains are classified as either kappa or lamibde Heavy chains are
classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively, Heavy chain subclasses in humans are designated VH1 - VH7. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids (see generally, 1993, Paul, W., ed., Fundamental Immunologyt 3rd ed, Raven Press, N.Y,, SH. 9 (incorporated by reference in its entirety for all purposes)).
[0058] From N-tennrnal to C-tertninal, both light and heavy chain variable regions
comprise alternating framework and complementarity determining regions (CDRs): FR. CDR. FR, CDR. FR, CDR and FR. The assignment of amino acids to each region is in accordance with the definitions of Kabat, 1987, and, 1991, supra, and/or Chothia & Leak, 1987, J. Mol Siol. 196: 901-917; Chothia et al., 1989, Nature 342; 878-883.
[0059] It has been shown that the function of binding antigens can be performed by
fragments of a whole Ewtibody, Exemplary binding fragments are (i) the Fab fragment consisting of the VL, VH, CL and CHI domains; (ii) the Fd fragment consisting of the VH and CHI domains; (iii) the Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) the dAb fragment (Ward, E.S. et al., 1989, Nature 341: 544-546) which consists of a VH domain; (v) isolated CDR regions; and (vi) F(ab')2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulphide bridge at the hinge region.
[0060] In various embodiments of the invention, the antibody or antibody fragment
may be a single-chain antibody or may be formed from a heavy and light chain. Exaraplary antibodies include intact rramunoglobma, single chain antibodies, scFv, dAB, VHH, Fab, Fob', F(ab')2, Fv, camelid antibodies, nanobodiea, antigen-binding scaffolds, and antibody or antibody-related polypeptide fusion proteins. If the antibody contains a heavy and a light chain, one or more chains is expressed without a signal peptide and preferably both chains are expressed without a signal peptide.
[0061] Two structures of IgGs constituting the imnumoglobulina (antibody
molecules) of camelids are known to exist: one a heterotetramer having heavy chains and light chains, and the other consisting of a heavy-chain dimer. The tetrameric structure is a common characteristic of IgGs among humans and most animals. On the other hand, the latter IgG having a heavy-chain dimer structure is considered characteristic of camelids,
JAN-10-2008 THU 07:19 AM

10062] Since the VH region of a heavy chain dimer IgG in a camelid does not have to
make hydrophobia interactions with a light chain, the region m the heavy chain that normally contacts a light chain is changed to hydtophilic amino acid residues in a camelid. Due to structural differences compared to VHs of normal heterotetrameiic IgGs, VH domains of the 5 heavy-chain dimer IgGs are called Variable domain of the heavy-chain of heavy-chain antibody (VHH).
[0063] VHH has excellent solubility due to its hydraphilic amino acid residues.
Amino acid substitutions are scattered throughout the primary structure (amino acid sequence) of VHH. Additionally, these hydrophilic amino acid residues form a cluster in the 10 space of the tertiary structure of VH corresponding to the Bite that interacts with the VL domain, Herein, the aforementioned space of the tertiary structure is specifically called former VL side. These amino acid substitutions are, for example, V37F 01 V37Y, G44E, L45R or L45C, and W47 are also mostly substituted with Gty. Such substitutions increase the hydtophilicity of the former VL. side of VHH.
[0064] Furthermore, VHHs derived from camels and llamaa have very high
thermostability compared to mouse heterotetrameric antibodies. The USE of VHH derived from these species can provide, for example, molecules that maintain their antigen binding ability even at 90DC (Van der Linden et at., 1999, Biochim. Biophys. Acta 1431(1): 37).
[0065] The diversity of antibody repertoire of carnelids is determined by the
complementary determining regions (CDR) 1, 1, and 3 in the VH or VHH regions,
Possession of three CDRa is in common with the IgGs of other animal species. However, the
CDR3 in the cainel VHH region is characterized by its relatively long length averaging 16
amino acids (Muyldermans et al, 1994, Protein Engineering 7(9): 1129). For example,
compared to the CDR3 of mouse VH having mi average of 9 amino acids, the CDR3 of camel
IgG is very long,
[0066] Libraries of camelid-dadved antibody variable regions, which maintain the in
vivo diversity of the variable regions of a camelid, can be made by, for example, the methods disclosed in United States Patent Application Set, No. 20050037421, published February 17, 2005, which is incorporated herein by reference in its entirety.
[0067] In another embodiment of the invention, the antibody related polypeptides are
scaffold polypeptides, Scaffold polypeptides are non-iimnunoglobulin binding polypcptidee

that exhibit selective binding activity toward a predetermined ligatid, The nou-immunoglobulin binding polypeptides are derived from an itnmunoglobulin-like domain containing scaffold that can be grafted with binding domains of a parent polypaptide to confer the binding specificity of the parent polypeptide onto the iromunoglobulin-like domain containing scaffold, The nqn-immunoglobulin binding polypeptides of the Invention have the advantages of being stable and modular in both the scaffold domain structures as well as in the ability to accept a broad range of haterologous polypeptide binding domains. Additionally, the inununoglobulin-like domain containing scaffolds can be readily obtainable from human sources so that their imtnunogenecity when used as a human therapeutic is negligible. The scaffolds of the invention also can be readily constructed to contain or omit naturally occurring polysaccharide chains or to include novel chains or other extra-scaffold moieties or polypeptide structures.
[0068] In one emboditneritj the invention is directed to non-immunogbbulin binding
polypeptides having antibody variable region complementarity determining regions (CDRs) inserted into a Thyl immunoglobulin-like domain containing scaffold, The CDRs are inserted into the loop regions of the Thyl polypeptide which allows the CDRs to fold into a similar confirmation as they would be in the tluee dimensional structure of the donor, or parent, antibody. The resulting hybrid, or chiroaric, antibody-related polypeptide exhibits similar binding characteristics compared to the parent antibody.
(0069] In another embodiment, me invention is directed to antibody-related
polypeplidea having altered immunoglobuu'n-lflce domain loops made by ammo acid substitution at some or all positions. The altered amino acid sequences in the loop domains confer selective binding activity toward a ligand omen: than that bound by the antibody-related polypeptide, The araino acid alterations can be made at the nucleic acid or
polypeptide level using a variety of methods lojown to those skilled in the art.
i
[0070] In yet another embodiment, the invention is directed to antibody-related
polypeptides derived from the ThyOx family of immunoglobulin-like domain containing polypeptides. The ThyOx polypeptides can be used as an immunoglobulin-like domain containing scaffold or as a carrier polypeptide to generate an antibody-related polypepticle of the invention. Scaffold polypeptides and libraries of such Scaffold polypeptides may be made by methods including, for example, those disclosed in U.S. Patent Application. No.
20040266993, published December 30,2004) which is incorporated harem by reference in its entirety.
[0071] As used herein, the term '4mrmmo globulin-like domain" or "Ig-like domain"
when used in reference to a scaffold is intended to refer to an art-recognised -sandwich
structural motif found in proteins of diverse function, including for example, extracellular
matrix proteins, muscle proteins, immune proteins, cell-surface receptors and enzymes, Ig-
lilce domain members have been divided into various superfamilies, including for example,
the iramunoglobulm, Fibronectin type III and cadherin Buperfamilies. Other euperfamilies
containing the Ig-Hlce domain structural motif include, for example, members of the PICD
domain, -galaotosidase/glucuTonidaae domain, transglutatnase two C-terminal domains,
actinoxanthin-like, CuZn superoxide dismutase-like, CBD94ike, lamin A/C globular tail
domain, clathrin adaptor appendage domain, integrin domains, PapD-like, purple acid
phosphataBe N-tenninal domain, BUperoxide leductaae-like, tbiolidiaulftda interchange
protein DsbD N-terminal domain and invasin/tntimin cell adhesion fragments suparfamilies.
Ig-like domain structural similarity ia maintained between members of different superfamilies
irrespective of significant sequence identity, The terra is intended to include Ig-like domain
members within and across each superfamily. Therefore, the term "immunoElobulhvlike (Ig-
like) domain containing supevfamily" is intended to refer to an Ig-like domain containing
member polypeptide within any of these superfamilies as well as others known in the art, A
description of the different Ig4ike domain containing superfamilies can be found, for
example, in Clarke et al, 1999, Structure Fold. Des. 7:1145-53 and within structural
databases such as at the URL pdb.weizmann.ac.il/soop/da- ta/6cop.b.c.b,htral.
[0072] As used herein, the term "ThyOx" or "ThyQx femily polypeptide" when used
in reference to a antibody-related polypeptide of the invention ia intended to mean a subclass of polypeptides within the immunoglobulin superfamily (IgSF) of immunoglobxilin-like domain containing polypeptides that are related by their common -sandwich structural motif and containing a scaffold framework structure similar to antibody variable region domains. Particular polypeptides within the ThyQx family of polypeptides include, for example, Thy-1, 0x2, OP40, Ox24ike protein and 0x2 homolog,
[0073] As used herein, the term "scaffold" ia intended to mean a supporting
polypeptide framework used to organize, orient and harbor heterologous binding domains or altered amino acid sequences conferring binding specificity to a ligand. A scaffold can be
structurally separable from the ammo acid sequences conferring binding specificity. The structurally separable portion of a scaffold can include a variety of different structural motifs including, for example, -sandwich, -sheet, -helk, v-banel, coil-coiled and other polypsptide secondary and tertiary stoctares well known in the art, A scaffold of the invention will also contain one or more regions that can be varied in amino acid sequence without substantially reducing the stability of the supporting framework structure, An exemplary region that can be varied includes a loop region segment that joins two strands of a -sandwich or -sheet. Amino acid residues corresponding to the structurally separated portion of a scaffold is referred to herein aa a scaffold framework, Immuno globulin-like domain containing scaffolds of the invention exhibit less than about 50% amino aoid identity to a human immunoglobulin variable heavy or light chain framework amino acid sequence, Generally, immunoglobulin-like domain containing scaffolds will exhibit, for example, amino acid sequence identity less than about 45%, about 40%, about 30%, about 20%, about 15% or about 10% compared to a human irannmoglobulin variable heavy or light chain framework amino acid sequence. Residues of a scaffold that can be varied are referred to herein with reference to its structural properties such as a loop region or with reference to its ability to accommodate altered residues. Therefore, a scaffold region that can be varied is referred to as a scaffold variable region, nmtabla region, exchange region, alterable region or changeable region, for example. Residues conferring secondary or tertiary structural properties can be retained, modified or conserved so long as the overall structure of the scaffold is maintained. Those skilled in the art know, or can determine, which residues firaction in structural stability of a polypeptide scaffold as well as the extent to which such residues can be modified.
[0074] Specific examples of scaffolds of the invention include immunoglobulin-like
domain containing superfamily members. These superfamily members contain a imimmaglobulin-like domain characterized as a -sandwich which can be used aa a scaffold of the invention. The P-sandwich consists of about 80-150 amino acid residues containing two layers of antiparallel -sheet in which the flat hydrophobic faces of the -sheets pack against each other. Each -sheet contains a loop region that can be varied in amino acid sequence BO as to confer unique binding specificity onto the scaffold palypeplide, Examples of Ig-like domain containing superfamily members include, for example, ThyOx family member polypeptidos os well as the various individual members wifliin the immunoglobulin-like domain containing superfamiliea described previously. Such individual members
include, for example, T cell receptor, CDS, CD4, CD2, class I MHC, claae H MHC, GDI, cytoldne receptor, GCSF receptor, GMCSP receptor, honnone receptors, growth hormone receptor, erythropoietin receptor, iuterferon receptor, interferon gamma receptor, prolactin receptor, NCAM, VCAM, ICAM, N-caderin, E-caderia, ilbroneotin, tanaacin, and I-flet containing domain polypeptides, or a runctian#l fragment thereof. Exemplary descriptions of these an other Ig-lilca domain containing superfmniiy members can be found in, for example, Isaclce and Horton, 2000, The Adhesion Molecule FactsBook, Second Ed., Academic Press, San Diego; Pitzgerald et al 2001, The Cytoktne FactsBook, Second Ed,, Academic PresBj San Diego; and Marsh et al.t 1999, The HLA FactsBook, Second Ed., Academic Press, San Diego,
(0075] The antibodies, antibody fragments or antibody-related potypeptidas of this
invention may be derived from a variety of sources, In various embodiments, the antibodies, antibody fragments or antibody-related polypeptides are derived from mammalian genes and raey be human, mouse, rabbit, sheep, rat, hamster chiraeric, humanized, hybrid, or apitope-focused. The antibodies of this invention may be monoclonal. Owing to then: high specificity for a given antigen, the advent of monoclonal antibodies (Kohler, G, and Milstein C, 1975, Nature 256; 495) represented a significant technical break-through with important consequences both scientifically and commercially.
[0076] Monoclonal antibodies are traditionally made by establishing an immortal
mammalian cell line which is derived from a single immunoglobulin producing cell secreting one form of a biologically functional antibody molecule with a particular specificity, Because the antibody-fiecreting mammalian cell line is immortal, the characteristics of the antibody are reproducible from batch to batch. The key properties pf monoclonal antibodies are their specificity for a particular antigen and the reproducibility with which they can be manufactured,
[0077] Early methods for producing monoclonals were laborious and time
consuming. An animal of choice, e.g., a mouse, was immunized with a desired antigen, antibody producing cells were harvested from the animal (usually by splenectorny) and fused to a suitable immortalized cells, e.g., myeloma cells, to make a hybridoma that clonally produced an antibody. Such hybridoma technology is disclosed, for example, in U.S. Patent Noa. 4,172,124 and 4,196,265; Zurawdd et al, 1980, Federation Proceedings 39:4922; Franlcel and Gerhnrd, 1979, Molecular Immunologyt 16:101-106.
[0078] The introduction of transgenvc animals that produce fully human antibodies
has permitted the selection of hybridomas which also produce My human antibodies. Such transgenic animals are disclosed, for example, in U.S. Patent NOB. 6,075,181 and 6,300,129, which are incorporated herein hy reference in their entirety.
[0079] Display technologies have also permitted the selection of monoclonal
antibodies that are fully human or other animal, chimeric, synthetic, and/or semi-synthetic. One example of such display technologies is phage display (examples are disclosed in U.S. Patent Nos. 5,565,332; 5,580,717; 5,821,047; 5,871,907; 5,885,793; 5,922,545; 5,403,484; 5,885,793; 6,172,197; 6,291,158; 6,291,650; and 6,387,627, which are incorporated herein by reference in their entirety) where a vectors for expression of fuaion antibodies in, which one or more antibody chain is fused at the N-tenninus of a phage protein are constructed. Such vectors can be introduced into prl mutant host strains of the present invention in order to express antibodies and isolate phage-antibodies in a signal-independent manner. The expression of phage-aivtibodies in. the host cells of the invention provides improved expression of poorly secreted antibodies and better representation of various sub-classes of antibodies present in libraries. The fusion proteins may also be expressed from polynucleotidas encoding antibody fusion proteins lacking a signal sequence. Methods for screening, purifying and analyzing phage antibodies are described in the above patents. Another example of such display technologies is yeast display (examples are disclosed in U.S. Patent No. 6,300,065, which is incorporated herein by reference in its entirety).
[0080] Phage-display technology has generally made use of the filamentous
bacteriophage Ml3 or the closely related phage fd. Thesephages are composed of circular, single-stranded DNA surrounded by a cylinder of coat proteins. Most of the viral capsid consists of the major protein pVHI, of which there are approximately 2,700 copies per plmge. At one end of the phage particle., there are five copies each of pill and pVI that are involved in host-cell binding and in the termination of the assembly process. At the other end, there are five copies each of pVII and pIX, hydrophobic peptides of 33 and 32 amino acids, respectively, required for the initiation of assembly and for maintenance of virion stability.
[0081] Embodiments of the invention include chiraeric antibodies and synthetic
antibodies. The early monoclonal technologies described above produced non-human antibodies. These antibodies ore potentially immunogemc in humans and this immunogenicity has severely hampered the development of therapeutic antibodies. The
production of BO called "chimeric antibodies," e,g,, variable regions fiom one species jorauu to constant regions from another species, has bean somewhat sucoess&l, but does not overcome the immunogenloity problem in many cases. Exemplary methods for chimBrizing antibodies are disclosed in, for example, U.S. Patent No. 4,816,567, which is incorporated herein by reference hi its entirety,
(0082] Recombinant DNA technology has been utilized to produce hiummoglobulins
which have human framework regions from one species combined with complementarity determining regions (CDR's) from another species' imrnunoglobulin (see, e.g., EPO Publication No. 0239400). Those new proteins are called "reshaped" or "humanized" (when the framework regions are human) immunoglobulins and the process by which the donor imrnunoglobulin is converted tato a human-lilce imrnunoglobulin by combining its CDR's with a human framework is called "humanization". Exemplary methods for humanization of antibodies are disclosed in, for example, U.S. Patent No, 6,180,370, which is incorporated herein by reference in its entirety.
[0083] Artificial antibodies and fragments thereof can be constructed baaed on known
antibody sequences, which reflect the structural properties of a whole group of homologous antibody genes. Therefore it is possible to reduce the number of different genes without any loss in the structural repertoire. This approach leads to a limited set of artificial genes, which can be synthesized de novo, thereby allowing introduction of cleavage sites and removing unwanted cleavages sites, Furthermore, Ms approach enables (i), adapting the codon usage of the genes to mat of highly expressed genes in any desired host cell and (ii), analyzing all possible pairs of antibody light (L) and heavy (H) chains in terms of interaction preference, antigen preference or recombinant expression titer, which is virtually impossible using the complete collection of antibody genes of an organism and all combinations thereof.
[0084] The use of a limited set of completely synthetic genes makes it possible to
create cleavage sites at the boundaries of encoded structural sub-elements. Therefore, each gene is built up from modules which represent structural sub-elements on the proteiny(poly)peptide level. In the case of antibodies, the modules consist of "framework" and "CDR" modules. By creating separate fimnework and CDR modules, different combinatorial assembly possibilities are enabled, Moreover, if two or more artificial genes carry identical pairs of cleavage sites at the boundaries of each of the genetic sub-elements, pre-built libraries of sub-elements can be inserted in these genes siniulta-neously, without any
additional information related to any particular gene sequence, This strategy enables rapid optimization of, for example, antibody affinity, since DNA cassettes encoding libraries of genetic sub-elements can be (i), pie-built, stored and reused and (ii), inserted in any of these sequences at the right position without knowing the actual sequence or having to determine the sequence of the individual library member, Exemplary methods for generating synthetic libraries of antibodies are disclosed in, for example, U.S. Patent No. 5,885,793 and 6,300,064, which are incorporated herein by reference in their entirety.
[0085] In one embodiment the antibodies are epitope-focused human antibodies
created by methods for engineering antibodies where the resulting antibodies retain epitope binding specificity and affinity while at the same lime having most of the non-human sequences replaced with human sequences, as described in patent application U.S. Patent Application Sei. No. 11/040,159, filed January 2.0, 2005, which ia incorporated herein by reference in its entirety. This is accomplished by transferring a BSD pair from the refeience antibody, e.g., & protein of a CDR3 pair (CDRSj). In antibodies that are affinity-matured, e.g,, the reference antibody, the heavy chain and light chain BSDs are in close contact with one another and are optimized for mutual stabilization of the combined antigen-binding conformation, hence, they form a unit, i.e., a BSD pair. The antigen-binding conformation is, of course, dependent on the support of the underlying frameworks of the V-iegions. When an affinity-matured BSD, e.g., that of the reference antibody, is combined with the structural diversity and stability of the complete human repertoire of heavy chain or light chain V-segment pairs, scaffolds that fully support the optimal antigen-binding conformation of the BSD are readily identified with the aid of selection systems including, but not limited to, phage display, cell viability, colony lift binding assays (CLBA),, or a variety of imnwnoassays, e.g., ELISA assays.
[0086] Further, transfer of a BSD pair to diverse germline V-segments often result in
selection of V-regions that that have affinities of greater than 50 nM. These selected V-regians can also be incorporated into the affinity maturation process of any antibody, V-aegment libraries are relatively small without CDR3 repertoires, thua selection of human V-regions can also be combined with limited mutagenic diversification of one or both BSDs in libraries of searchable size for many conventional selection systems.
[0087] The V-segment repertoire used in generating libraries to replace the heavy
and/or light chain V-segmdnt of the reference antibody can be from any source. The human
repertoires can be generated, e.g.s by polymeraae chain reaction (PCR.) amplification using primers appropriate for the desired segments from cDNA obtained from peripheral blood or spleen, in which case the repertoires are expected to contain clones with somatic mutations. Alternatively, the repertoires can be obtained by amplification of genomic DNA from non-immune system cells in order to obtain germline-encoded sequences,
10088] The human germline V- segment repertoire consists of 51 heavy chain V-
regions, 40 K light chain V-segroeuts, and 31 "k light chain V-segments, maldng a total of 3,621 germline V-region pairs. In addition, there are stable allelic variants for most of these V-segments, but the contribution of these variants to the structural diversity of the germline repertoire is limited. The sequences of all human germ-line V-segment genes are Icnown and can be accessed in me V-base database,- provided by the MRC Centre for Protein Engineering, Cambridge, United Kingdom (see, Chothia et alt 1992, J Mol Biot 227:776-798; Toralinson et [0089] Human V-region repertoires, both germline and affinity-matured, can bo
recovered, e.g.t from peripheral blood lymphocytes (PBL), often pooled from multiple (e,g., at least 10) healthy individuals, using conventional cDNA cloning methods (Sambrook and Russell, eda, Molecular Cloning: A Laboratory Manwt, 3rd Ed, vols. 1-3, Cold Spring Harbor Laboratory Press, 2001). Insofar as the germline frequency distribution is not uniform in expressed sequences, it is prudent to capture at least 103 independent clones for each of the three V-region isotypes (VH, Vic, and VX) to ensure optimal diversity of the repertoires. The PCR can be used to amplify V-region sequences during the cloning process. However, exponential amplification mechanisms are prone to random biases, and this may be compounded by the use of degenerate primers, which have variable priming efficiencies, resulting in a loss of diversity, Thus, when amplification is desired, it may be desirable,
where possible, to use a primer-independent linear amplification method, such as in vitro transcription (Sambiook awl Russell, eels, Molecular Cloning: A Laborcttoiy Manual, 3rd Ed, vols, 1-3, Cold Spring Harbor Laboratory Press, 2001),
[00901 • BSDs from the reference antibody are transferred to a library of V-segment sequences generated as described above. The BSDs can be incorporated into the expression vector before or after the population of V-segments is cloned into the expression vector. The BSD that is transferred can be a CDR3-FR4, a CDR3, a D segment (where the BSD is from the heavy chain), a MEBSD, or any other fragment of CDR3-FR4 that has binding specificity in combination with the complementing BSD from the other chain of the reference antibody. It ia understood that when transferring a BSD from a reference antibody to a different V-region, ths structure of the heavy or light chain V region is maintained in the resulting V-ragion. Thus, if the BSD from the reference antibody is a subregion of CDR3-FR4, the complete CDR3-FR4 structural length is maintained, i.e.t the remainder of the CDR3-FR4 residues that are not from the reference antibody are made up of other residues, typically human gennline residues,
[0091] As noted, the BSD can include Framework 4 regions, e.g,, from the reference
antibody, which are part of the J-segraents, but which are highly conserved among mammals, and are important for CDR3 structure. These sequences can, for example, be amplified by PCR willi primers containing restriction sites for in-frame ligation, to Framework 3, and other unique restriction sites downstream from the carboxyl terminus of Framework 4, e.g., for ligation to the C-region. Each CDR3-PR4 is then transferred into the appropriate sites of the V-region library construct. Alternatively, the desired sequence or mix of sequences for the CDR3-FR4 region can be synthesized as one continuous oligonucleotida or mix of oligonucelolides and can be joined to the V segment repertoire by primer extension using in vitro transcribed cRNA synthesized from the repertoire as a template for first-strand cDNA synthesis. Diversity can be introduced into & region, e.g., CDR3 and^or FR4.
[0092] The BSD can also be a sequence that is leas than the complete CDR3, e.g, the
D segment of a heavy chain CDR.3 or a MEBSD. As appreciated by one of skill in the art, when the reference antibody BSD is less than a complete CDR3, a complete CDR3 still results in the antibody expression library, as the remaining CDR3 residues are incorporated into the construct. For example, appropriate oligonucleotides can be designed to incorporate
human sequences, e.g., germUne J segments, to replace the CDR3 residues that are not part of the MEBSD.
[0093] The MEBSD is tha region within a GDR3 sequence or a pair of CDJUfl that is
required to retain the binding specificity of the reference antibody when combined with human sequences that re-constitute me remainder of CDR3 and the rest of the V^region. Tha MEBSD can be defined empirically or con be predicted from structural considerations.
[0094] The antibody library can be a library where the antibody is an IgG, an Fv, ft
Fab, a Fab', a F(ab')2, a single chain Fv, an IgG with, a deletion of one more domains, or any other antibody fragment that includes Hie V-region.
[0095] The antibodies can be displayed on l}ie surface of a virus, cell, spore or viros-
like particle, For this purpose, one or both chains of the antibody fragment are typically expressed as a fusion protein, for example as a fuaion to a phftge coat protein for display on tha surface of filamentous phage. Alternatively, the antibodies of the antibody library can be secreted from a host cell.
[0096] The following provides an exemplary description using secretion systems to
express the antibodies as Fab or Fab' fragments. It is readily apparent to those in the art, however, mat the axpression systems can be adapted for any library format For this general example, a library of complete V-regions is constructed by ligation of oligonucleotides encoding CDR3-FR4 segments to the V-segment repertoire as described above, The amplified sequences encoding complete V-regions are cloned into a suitable expression vector and can be fused to constant region sequences at this stage for expression of Fab or Fab' molecules. The antibody fragments can be secreted from prokaryotic or eukaryotic cells including bacteria, yeast, plant cells andmamnmlian ceils.
[0097] Filter screening methodologies have been described for detection of secreted
antibodies specific for a particular antigen. In one format, the secreted antibody fragments are trapped on a membrane which is probed with soluble antigen (Skerra et al., 1991, Anal Jitochem, 196:151-5). In this case, bacteria Harboring plasmid vectors that direct the secretion of Fab iragmonts into the bacterial peripkun are grown on a membrane 01 filter. The secreted fragments are allowed to diffuse to a second "capture" membrane coated with antibody which can bind the antibody fragments (e.g., witHmmunoglobulm antisentm) and the capture filter is probed with specific antigen. Antibody - enzyras conjugates can be used
to detect antigen-bindmg antibody fragments on the capture membrane as a colored spot, The colonies are re-grown on the first membrane and the clone expressing the desired antibody fragment recovered.
[0098] Colony lift binding assays have also been described in which the antibodies
are allowed to diffuse directly onto an antigen-coated membrane, Giovannoni et al have described auch a protocol for the screening of single-chain antibody libraries (Giovannoni et al, 2001 Nucleic Acids Research, Vol. 29, No, 5 e27).
[00991 Libraries of secreted antibody fragments can also be screened by ELISA,
either using pools of multiple clones or screening of individual clones each secreting a unique antibody sequence. One such method for screening individual clones is described by Watkiris et at, 1997, Anal Biochem. 253: 37-45. In this case, tnicrotiter wells were coated with anti-Fab antibody to capture Fab fragments secreted directly in the wells, The Fab samples were than probed with soluble biotinylated antigen followed by detection with streptavidin-alkalme phosphatase conjugates.
Fusion Antibodies
[00100] In. one embodiment, the polypeptides may be fusion antibodies. In one
embodiment, the antibody fragment may comprise one or more C-termiiwl peptide tags to facilitate detection and purification, m another embodiment the antibody may be fused to a peptide or polypeptide for display on the surface of a cell, spore or virus. For example one chain of the antibody fragment may be displayed as a fusion protein on the surface of a bacteriophage such as a filamentous phage. Methods for display of antibodies on phage ara well Imown in the art and include fusion to pHI and pVHI proteins of a filamentous phage. In a preferred embodiment at least one of the peptides comprising an antibody - ph'age protein fusion is expressed without a signal peptide in a prl strain of E. coli and is presented on the surface of a phage.
[00101] The secreted chains may retain the N-termmal methionine (or N-fornjyl-methiontne), Alternatively, and in some cases, depending on the sequence of the antibody, the initial methionine may be removed by proteolytic processing by the host cell.
100102) Embodiments also include other display technology, such as yeast cell display, bacterial cell display, ribosome display, and mammalian cell display, In one embodiment, screening is performed by screening pools of library members.
[00103] Fragment and subunit complementation systems can be used in me invention
to select/screen for antibodies having desired properties ("complementation system"). In general, fragment complementation systems are comprised of a responder that is fragmented or separated into two (or more) parts that must reassociate to mate a functional teeponder. The rragvnents/subumts of the responder are fused individually to members of a binding ensemble, and the reassembly of the reeponder is then driven by the direct or indirect interaction of the two binding ensemble members. In a preferred embodiment the binding ensemble is comprised of an antibody(s) and an antigen(s), Examples of fragmerd/subunit complementation systems mat may be used in the invention are disclosed in U.S. Patent Nos, 6,342,345, 6,270,964, 6,294,330, 5,503,977, 5,585,245, which are incorporated herein by reference in their entirety, PCX patent application WO 00/71702, and Fields et at., 1989, Nature 340:245-247; Bai et al,, 1996, Meth. EnxymoL 273:331-347, Luo et al, 1997, Biatechniques 22:350-352, which are hereby incorporated by reference in their entirety.
[00104] Reactivation-based molecular interaction systems (e,g., RAIR.TM.) can be used in the invention to select/screen for antibodies having desired properties ("reactivation system")- In general, reactivation-based molecular interaction systems are comprised of responders, inhibitors, reactivators, and binding ensembles of two or more members. The system has two complexes, one containing the responder, the inhibitor, and a binding ensemble member (the responder complex), and the other containing the raactivator and a binding ensemble member (the reactivator complex). The responder is inhibited in its complex, and docking of the reactivator complex to the responder complex by direct or indirect interaction of the binding ensemble members allows the reactivatoi to "reactivate" the responder by displacing Ihe inhibitor. Typically, a responder complex comprisea a responder molecule, an inhibitor of the reapondev, and a first binding ensemble member. The components of Ihe icsponder complex may be arranged in various configurations by covalent or non-covalent linkages. In a preferred embodiment the binding ensemble is comprised of an antibody(s) and an antigen(a),
100105] In a. preferred reactivation system, molecular interactions can be detected by a
process termed "reactivation of an auto-inhibited responder," or "RAIR." The RAJR systems
•comprise the following components; a tesponder complex and a reactivator complex. By avito-inlnbited, we mesa, that the responder is directly United to the responder so that the base state is automatically inhibited until the inhibitor is displaced and the responder activated by a reactivator complex. "Where this linkage is by a covalent bond, the covalant linkages may farther comprise a linker. A reactivate! complex comprises a reactivate molecule to displace the inhibitor and a second binding ensemble member. Like the components of the reapondei' complex, the raactivator and binding ensemble member may be linked either oovalently or non-covalently.
[003061 Molecular interaction between the fitst and the second ensemble members caii be detected by the following mechanism; the signal or activity of the raspondor in the responder complex is sequestered by the inhibitor present in me complex, i.e., the reaponder is auto-inhibited; when a reactivator complex is introduced, if the second ensemble member in the reactivator complex binds with sufficient affinity to the first ensemble member in the responder complex, the reactivate will be able to displace the inhibitor in the responder complex and lead to the so-called "reactivation of an auto4nhtbited tesponder," The detection of responder activity or signal indicates an interaction between the first and the second ensemble members.
[00107] Variations of the RAIR systems can be used for interaction mapping, improving the affinity of a first binding pair member, and isotropic selection of a plurality of binding molecules. In some variations, a third ensemble member may be used.
100108] Examples of reactivation systems are disclosed in U.S, Patent Application Ser. No. 10/208,730, which is incorporated herein by reference in its entirety.
(001091 Systems using molecular sensors activated by competition can also be used in the invention to select/screen for antibodies having desired properties. These systems are designated COMPACT.TM. In general, competitive activation systems are comprised of a binding ensemble, a vesponder, and an inhibitor. The responder is complexed with one binding ensemble member and the inhibitor is complexed to another binding ensemble member. The binding ensemble members, upon binding to one another, bring the Tesponder and inhibitor together so that the responder is inhibited. Antibodies of the invention that disrupt the binding ensemble or inhibit binding ensemble formation and thereby activate the responder can than be selected. In a preferred embodiment, the binding ensemble is an
antibody(s) and an antigan(s), and the "competitive activator" is an antibody. For example, the binding ensemble antibody might be a reference antibody, and the competitive activator may comprise a library of antibodies which compete with the reference for binding to the antigen. Examples of competitive activation systems that may be used in the invention are disclosed in U,S. patent application Ser, No, 10/076,845, which is incorporated herein by reference in its entirety.
[00110] Such a system may further employ a "mask" to control the sensitivity of the system. These systems are described, e.g., in co-pending U.S. application Ser, No. 10/076,845, filed February 14, 2002. A "mask", in the context of a competitive activation system, refers to a molecule that has low affinity for a reporter 01 inhibitor, such that the mask does not bind appreciably at working concentrations unless it is tethered covalently to the reporter or inhibitor. The mask does not affect reporter activity only the binding of the inhibitor and vice versa. Control of the system with Masks permits a high-affinity inhibitor to be uaed without fear of increasing the background inhibition because its association rate constant is greatly reduced by the Mask without affecting the dissociation rate constant of the reporter-inhibitor complex, thereby reducing the overall affinity while retaining the stability of the high-affinity reporter-inhibitor complex.
Libraries
[00111] In another embodiment of the invention, the secreted polypeptides may be a
diverse library of antibodies, antibody fragments, or antibody-related polypepticles with different binding characteristics expressed fiom a prokaryolic host cell such as a strain of E. coli expressing a prl mutation, wherein one or more antibody chain IB expressed without a signal peptide. In another embodiment, the secreted polypeptides are expressed in a signal-independent maianer. The libraries according to this aspect of the invention show broader representation of VH and VL subclasses,
[00112] It is known in the art liiat different antibodies are secreted at different levels
into the periplaam and that certain sub-classes of antibody are only poorly secreted in soluble correctly-folded form. In many cases the V-region sequences of the antibody can affect the ability of the antibody to be secreted from E. coli. Murine V-regions, for example, fold poorly in the periplasm and may lead to the accumulation of aggregated and inactive antibodies (Skerra and Pluckthun, 1991, Prot Eng, 4:971; Bcthmann and Pluckthun, 1998,
Nature Biotech 18; 376; Helle rf a/., 1995, Proc, Natt Acad. Sci. USA 92: 11907). Cbaperone proteins may improve the folding and expression of some antibody fragments (Bothmann and Pluckthun, 1998, Nature Biotech. \ 8:376; Bolhrnann and Pluclcthun, 2000, J. Bid, Chem 275:17100). Bias in secretion of some sub-classes of antibody from bacteria can lead to bias in the sequences of the antibodies which may be screened from ft library, An embodiment of the present invention addresses allows for higher yields of functional correctly folded antibodies and antibody fragments from bacterial secretion systems by secretion of antibody chains in a signal-independent manner.
[00113] Embodiments of this invention include naKve libraries and immunized
libraries. Naive libraries are made from the B-lympliocyteB of a suitable host which has not been challenged with any immunogea, nor which ie exhibiting symptoms of infection or inflammation. Immunized libraries are made from a mixture of B-cells and plasnaa cells obtained from a suitably "immunized" host, i.e., a host that lias been challenged with an hnmunogen. In one embodiment^ the mRNA from these cells is translated into cDNA using methods well known in the art (e.g., oligo-dT primers and reverse transcriptase). In an alternative embodiment, nucleic acids encoding antibodies from the host cells (mRNA or genomic DNA) are amplified by PCR with suitable primers. Primers for such antibody gene amplifications are well known in the art (e.g., U.S. Patent No. 6,096,551, which is incorporated herein by reference in its entirety, and PCT Patent Application WO 00/70023A1 disclose uuch primers). In a hybrid embodiment, the ,mRNA from the host cells is synthesized into cDNA and these cDNAs are then amplified in a PCR reaction with antibody specific primers (e,g., U.S. Patent No, 6,319,690, which is incorporated herein by reference in
I
its entirety, discloses such a hybrid method), Alternatively, the repertoires may be cloned by conventional cDNFA cloning technology (Sambrook and Russell, eds, Molecular Cloning,- A Laboratory Manual, 3rd Ed, vols. 1-3, Cold Spring Harbor Laboratory Press, 2001), without using PCR.
(00114) In one embodiment of the invention, a database of published antibody sequences of human origin is established where the antibody sequences are aligned to each other. The database is used to define subgroups of antibody sequences which show a high degree of similarity in both the sequence and the canonical fold of CDR bops (as determined by analysis of antibody structures). For each of the subgroups B consensus sequence is deduced which represents the members of mis subgroup; the complete collection of
consensus sequences represent therefore the complete structural repertoire of human antibodies.
[001J5] These artificial genee are then constructed, eg,, by total gene synthesis or by the use of synthetic genetic subunits, These genetic aubunits correspond to structural sub-elements on the (polyjpeptide level. On the DNA level, these genetic subunits are defined by cleavage sites at the start and the end of each of the sub-elements, which are unique in the vector system. All genes which are members of the collection of consensus sequences ara constructed such that they contain a similar pattern of corresponding genetic sub-sequences. Moat preferably, said (poly)peptides are or are derived from the HuCAL consensus genes: Vkl, Vk2, Vk3, Vk4. Vll, V12, V13, VH1A, VH1B, VH2, VH3, VH4S VH5, VH6, Ck, Cl, CHI cr any combination of said HwCAL consensus genes,
[00116J This collection of DNA molecules can then be used to create "synthetic libraries" of antibodies, preferably Fv, disulphide-linked Fv, single-chain Fv (scFv), Fab fragments, or .Fab' fragments which may be used as sources of specificities against new target antigens. U,S. Patent No. 6,300,064, which is incorporated herein by reference in its entirety, diacloaes methods for making synthetic libraries containing more than 10* transformants,
[00117] lu atiother embodiment, synthetic human antibodies have now been made by synthesis from defined V-gene elements. "Winter (EP 0368 684 Bl) lias provided a method for amplifying (by PCR), cloning, and expressing antibody variable region genes, Starting with these ganes he was able to create libraries of iunctianal antibody fragments by randomizing the CDR3 of the heavy and/or the light chain. This process is AinctionaUy equivalent to the natural process of VJ end VDJ recotnbination which occurs during the development of B-cellg in the immune system. For example, repertoires of human germ Ime VH gene segments can be rearranged in vitro by joining to synthetic "D-segments" of five random ammo acid residues and a J-segment, to create a synthetic third complementarity determining region (CDR) of eight residues. U.S. Patent No. 5,885,793, which is incorporated herein by reference in its entirety, discloses methods of making such antibody libraries such as these that create libraries containing 107 plmge clones.
[00118] The antibody fragments according to this aspect of the invention may be
soluble secreted antibody fragment's or may be presented as a fusion protein on the surface of a cell, spore or virus. Thus, for example, the library of antibodies may be a phage-display
library in which one or more chains of the antibody fragment ate expressed as a fusion protein with a phage protein in which at least one of the peptides comprising the fusion protein is expressed without a signal peptide. If tbe antibody fragment is comprised of a heavy and a light chain, it is preferred that both chains ate expressed without a signal peptide, In this aspect of tho invention the host strain is chosen to he suitahle for expression of the antibody library and may be a mutant strain such as prlA4 or may be a strain chosen for another purpose, for example a strain with high transformation frequency, in which the mutant prl protein is expressed from a plasmid expression vector,
[00119] Both pill and pVIII have been used to display peptide and antibody libraries, Display of nonimmune or "narve" antibody-phase libraries on pIII has been used to isolate human, antibodies against a variety of target antigens. Antibodies can be isolated in either scFv or Fab formats, the scFv or one of the Fab chains being fused at the N-terminus of the phage protein. In all cases described previously, a signal peptide is fused at the N-terminus of the antibody chain in order to direct secretion of the antibody-phage fusion protein. The other proteins of the phage coat have also been used to display antibody chains. pVTC and pDC have been used to display the antibody variable heavy-chain legion (VH) and variable light-chain region (VL), respectively. pIX display has also been used to construct a naive human antibody library based on the fusion of scFv to the N terminus of pIX using a PelB signal peptide for secretion (Gao et al, 2002, Proc. Natl Acad. Sci 99: 12612), Because of the high efficiency of phage transducticm, phage-displayed antibody libraries can be large, with diversities in excess of 109 antibody molecules or sometimes in excess of 10)0 or even 10U antibodies per library.
[00120] In some embodiments, the library can he a library of epitope-focuaed human antibodies as described in U.S. Patent Application Serial No. 11/040,159, filed January 20, 2Q05, which is incorporated herein by reference in its entirety. For example, such a library can comprise a plurality of nucleic acids that encode a diverse popvilation of heavy chain V segments, wherein the V segments are not linked to a CDR3, The invention also provides a library comprising nucleic acids that encode a diverse population of light chain V segments, wherein the V segments are not linked to a CDR3, The V segments of either OT both libraries can be, e.fi-,, human gennline. Libraries of epitope-focused hutnaii antibodies can range in size from 103 to 10s antibodies per library,

(001Z1] Antibody libraries may also be focused libraries comprising predominantly members of one or more sub-class of VH or VL gene segments. Thus, for example, in humans there are 1 recognized VH sub-classes (VH1 - VH7) and 16 VL sub-classes (Vkcappal - Vkappa6 and Vlambdal-VlambdalO) and a focused library may be constructed comprising members of one or more VH sub-class in combination with a diverse library of Vkappa chains or Vlambda light chains. Alternatively, a focused library of one or more VL sub-classes may be combined with a. diverse library of heavy chains. As a farther alternative, a. focused library maybe constructed comprising predominantly members of a single VL subclass and a single VH sub-class. Antibody fragments of the VH3 sub-class are typically expressed efficiently in E. colt when expressed in a signal-dependent fashion. Antibodies of other VH sub-classes are not efficiently secreted using signal peptides. Similarly, antibody fragments with murine V-regions are poorly secreted using signal peptides. The present invention allows improved representation of secreted antibodies of different sub-classes and allows efficient secretion of antibody libraries comprising murine V-regious.
[00122] In another embodiment, the invention provides a library comprising a
plurality of human antibody V-iegion pairs where a V-region pair comprises: i) an unselected heavy chain V-region comprising a human V segment and a heavy chain CDR.3 'from a reference antibody, and ii) an unselected light chain V-region comprising a human V segment and a light chain CDR3 from the reference antibody.
[001235 In other embodiments, the library is a library comprising nucleic acids encoding human antibody V-region pairs, where the VH and VL V segments are each linked to a MEBSD from a reference antibody of interest.
[00124] A library of the invention can also comprise nucleic acids encoding a'plurality of VH. or VL regions, wherein the VH or VL regions comprise V segments from one VH or VL subclass, wherein the V regions lack D and/or I segments. In one embodiment, the V segments of the VH regions are germline and/or the V segments of the VL regions are germline,
[00125] The invention also provides a library comprising & plurality of antibody V
region pairs, wherein a pair comprises: i) n. heavy-chain V region comprising a binding specificity determinant BSD from a heavy chain CDR3 from a reference antibody joined to a diversity of V segments, and ii) a light chain V region comprising a BSD from a light chain
CDB.3 from the reference antibody joined to a diversity of V segments, wherein at least one of the BSDs comprises lees than the reference antibody CDR3,
MI JLT1MERIC PROTEINS
[00126] Proper assembly of polypeptide Bxvbumts of a multiraedc protein to form a stable complex is required to ensure the biological function of the rnultimeric protein. An embodiment of the present invention enables expression, secretion and assembly of selected raoncitnsric polypeptidas to effect efficient production of heteroinultimers outside of the cytoplasm. One or more of the monomer polypeptides of the multimeric protein can be made without a signal sequence in the methods of the invention, and the other monomer polypeptides can be expressed with or without signal Bequence(s). Assembled multimeric proteins that may be produced by the present invention include antibodies, antibody fragments or antibody-related polypeptides,
NUCLEIC ACIDS
[00127] The rmcleic acid sequences that are useful in the methods of this invention, i.e., those that encode at least in part the individual peptides, polypeptides and proteins secreted in the method of the invention, or those expressed in or comprising the libraries of this invention, may be native, synthetic 01 a combination thereof. They may be mKNA, DMA or cDNA. In the preferred embodiment, the nucleic acids encode antibodies.
[00128] Recombinant DNA methodologies may be used to create antibody fragments that cannot be made by enzymatic digestion. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whale antibodies, or i those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al.} 1990, Nature 348:552-554), Nucleic acids encoding the polypeptides of the invention can be obtained using routine techniques in the field of recombinant genetics (see, e.g., Sambrook and Russell, eds, 2001, Molecular Cloning: A Laboratory Marnial, 3rd Ed, vols. 1-3, Cold Spring Harbor Laboratory Press; and Ausubel, ed,, 1997, Cwrent Protocols in Molecular Biology, John Wiley & Sons, Inc. New York).
[00129] Often, the nucleic acid sequences encoding the polypeptides of the invention
are cloned from cDNA or genomic DNA libraries by hybridization with probes, or isolated
using amplification techniques with oligonucleotide primers. Amplification techniques can bus vised to amplify and isolate sequences from DNA or UNA (see, e.g., Dieffbnbach & Dvekaler, 1995, PCR Primers: A Laboratory Manual). Alternatively, overlapping oligonucleotidea can be produced synthetically and joined to produce one or more of the domains. Nucleic acids encoding the component domains can also be isolated from expression libraries using antibodies as probes.
[00130] In an example of obtaining a nucleic acid encoding a polypeptide of the invention using PCR> the nucleic acid sequence or subsequence is PCK. amplified, using a sense primer containing one restriction site and an anu'sense primer containing another restriction site, This will produce a nucleic acid encoding the deaired polypeptide and having terminal restriction sites, This nucleic acid can then be easily Ugated into a vector having the appropriate corresponding restriction aitea, If the desired polypeptide is a fusion protein, (he domains can be directly joined or may be separated by a Inter, or other, protein sequence. Suitable PCR primers can be determined by one of skill in the art using the sequence information provided in GenBank or other sources. Appropriate restriction sites can also be added to the nucleic acid encoding the protein or protein subsequence by site-directed mutagenesis. The plasmid containing the polypeptide encoding sequence of the invention is cleaved with the appropriate restriction endonuoleaae and taen ligated into an appropriate vector for amplification and/or expression according to standard methods.
[00131] Examples of techniques sufficient to direct persons of sldll through in vitro amplification methods are found in Berger, Sarahrook, and Ausubel, as well as U.S. Patent No. 4,683,202, which is incorporated herein by reference in its entivaty; Innis et al,t eds, 1990, PCR Protocols A Guide to Methods and Application, Academic Press Inc. San Diego, CA; Amheim £ Levmson, 1990, C&EN 36-47, The Journal Of NIH Research 3: 81-04; Kwoh et al.t 1989, Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al, 1989, J. Clin. Chem., 35; 1826; Landegren et al., 1988, Science 241: 1077-1080; Van Brunt, 1990, Biotechnology 8: 291-294; Wu and Wallace, 1989, Gene*; 560; andBarringeref al, 1990, Gene 89:117*
100132] In some embodiments, it may be desirable to modify the polypeptides of the
invention. One of skill will recognize many ways of generating alterations in a given nucleic acid construct. Such welHcnown methods include site-directed mutagenesis, PCR amplification using degenerate oligonucleotides, exposure of cells containing the nucleic acid
to mutagenlo agents or radiation, chemical synthesis of a desired oligonucleovide (e.g., in conjunction with ligatton and/or cloning to generate large nucleic acids) and other well-known techniques. See, e.g, Giliman and Smith, 1979, Gene 8;81-97, Roberts et al, 1987, Nature 328; 731-734.
[00133] In some embodiments, the recombinant nucleic acids encoding the polypeptidee of the invention are modified to provide preferred codons which enhance translation of the nucleic acid in a selected organism (e.g, yeast preferred codons are substituted into a coding nucleic acid for expression in yeast),
[00134] The polynucleotidos of the invention also include polymicteotides including
rmcleotide sequences that are substantially equivalent to the polynucleotides of the invention. Polymicleotides according to the invention can have at least about 80%, more typically at least about 90%, and even more typically at least about 95%, sequence identity to a polynucleotide of the invention. The invention also provides the complement of the polynucleotides including anucleotide sequence that has at least about 80%, more typically at least about 90%, and even vnoie typically at least about 95%, sequence identity to a polynucleotide encoding a polypeptide recited above. The polynucleotide can be; DNA (genomic, cDNA, amplified, or synthetic) or RNA. Methods and algorithms for obtaining suchpolymicleotides are well known to those of alcill in 1ihe art and can include,' for example, methods for determining hybridization conditions which can routinely isolate polynucleotides of the desired sequence identities.
[00135] The nucleic acids useful in this invention may be naturally diverse, synthetic diversity may be introduced into those naturally diverse members, or the diversity may be entirely synthetic. For example, synthetic diversity can be introduced into one or more CDRs of antibody genes. Preferably, it is introduced into CDR1 and CDR2 of imunoglobulins. Preferably, natural diversity is captured in the CDR3 regions of the imnrnnoglobulin genes of this invention from B cells, Most preferably, the nucleic acids of this invention comprise a population of iromunoglobulin genes that comprise synthetic diversity in at least one, and wore preferably both of the CDR1 and CDR2 and diversity in CDR3 captured from B cells.
[00136] Nucleic acids which encode piotein analogs in accordance with this invention
(i.e., wherein one or more amino acids are designed to differ from the wild type polypeptide) may bo produced using site directed mutagenesis or PCR amplification in which the primer(s)
have the desired point mutations. For a detailed description of suitable rnutageuesis techniques, see gambrook et al, 1989, Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and/or Ausubel et al,, editors, 1994, Current Protocols in Molecular Biology, Green Publishers Inc, and Wiley and Sons, N.Y. Chemical synthesis using methods described by Engels et al, 1989, in Angew. Chem, Intl. Ed., Volume 28, pages 716-734, may also be used to prepare such nucleic acids.
[00137] "Recombinant variant" refers to any polypeptide differing from natirally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using recombinant D1S1A techniques. Guidance in determining which amino acid residues may be replaced, added, or deleted without abolishing activities of interest, such as enzymatic or binding activities, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology.
[00138] Preferably, amino acid "substitutions" are the result of replacing one amino
acid with another amino acid having similar structural and/or chemical properties, i.e, conservative amino acid replacements. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydiophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanme, tryptophan, and meuiionrae; polar neutral amino acids include glycine, serine, threonine, cysteme, tyrosine, asparagine, and glutatnine; positively charged (basic) amino acids include argmiue, lysine, and hisn'dine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
[00139] "Insertions" or "deletions" are typically in the range of about 1 to 5 amino acids. The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.
[00140] Alternatively, where alteration of function is desired, insertions, deletions or non-conservative alterations can be engineered to produce altered polypeptides or chimeric polypeptides. Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention. For example, such
alterations may change polypeptide characteristics such, as ligand-bmdmg affinities, interchain affinities, ot degradation/turnover rate. Further, such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells chosen for expression. For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate diaulfide bridges.
[00141] Alternatively, teoambinant variants encoding these same or similar polypeptides may be synthesized or selected by making vise of the "redundancy" in the genetic code. Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system. Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/tumover rate.
[00142J The nucleic acid sequences of the invention are further directed to sequences which encode variants of the described nucleic acids, These amino acid sequence variants may be prepared by methods known in the art by introducing appropriate micleotide changes into a native or variant polynucleotide. There are two variables in the construction of amino acid sequence variants; the location of the mutation and the nature of the mutation, The ammo acid sequence variants of the nucleic acids are preferably constructed by mutating the polynucleotide to give an amino acid sequence that does not occur in nature, These amino acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions .(constant regions). Sites at such locations will typically be modified in series, e.g., by substituting first with conservative choices (e,g., hydrophobic amino acid to a different hydrophobic ainino acid) and then with more distant choices (e.g., hydrophobic amino acid to a charged amino acid), and then deletions or insertions may be made at the target site.
(00143] Amino acid sequence deletions generally range from about ] to 30 residues, preferably about 1 to 10 residues, and are typically contiguous. Ainino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one to one hundred or more residues, as well as mtrasequence insertions of single or multiple ammo acid residues. Intrasequenca insertions may range generally from about 1 to 10 amino residues,
preferably from 1 to 5 residues. Examples of terminal insertions include the haterologous signal sequences necessary for intracellular targeting in different host cells.
[00144] In a preferred method, polynucleotides encoding the novel nucleic acids are changed via site-directed mutageneais. This method uses oligonuoleotide sequences that encode tha polynucleotide sequence of the desired amino acid variant, us well as a sufficient adjacent nucleotide on both sides of the changed arnino acid to form a stable duplex on either side of the site of being changed, In general, the tectoiques of site-directed rautagenesis are well Icnown to those of skill in the art and this technique is exemplified by publications such as, Edelraan et al,, 1983, DNA 2:183. A versatile and efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller and Smith, 1982, NudeicAcidsK.es. 10:6487-6500.
[00145] PCR may also be used to create amino acid sequence variants of the novel
nucleic acids, When small amounts of template DNA are used as starting material, primer(s) that differs slightly in sequence from the corresponding region in me template DNA can generate the desired amino acid variant. PCR amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the collagen at the position specified by the primer, The product DNA fragments replace the corresponding region in the plasraid and this gives the desired amino acid variant.
[00146] A further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells et ol, 1985, Gene 34:315; and other mutagenesis techniques well known in the art, such as, for example, the techniques in Sawbrook et «/., supra, and Current Protocols in Molecular Biology, Ausubele? al
[00147] Due to the inherent degeneracy of the genetic code, other DNA sequences
which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the invention for the cloning and expression of these novel nucleic acids. Such DNA sequences include those which are capable of hybridizing to the appropriate novel nucleic acid sequence under stringent conditions. In addition, nxicletc acids encoding the same amino acid sequence as that of the polypeptide of the invention but having very different nucleic acid sequences d.ue to the degeneracy of the genetic code are also encompassed within tha invention.
HOST CELLS
[00148] Another aspect of the invention provides a prokaryotio host cell allowing for
secretion of antibodies or antigen-binding antibody fragments or raultimeric proteins without the need for a signal peptide. Strains suitable for use in the invention do not have a generally increased permeability but selectively secrete proteins naturally destined for seoietion including the antibodies and antibody fragments of the invention. In a preferred embodiment, the prokEiryote is preferably a grain-negative bacterium, and most preferably is the bacterium E. coli.
[00149] Multiple pathways have been described in Gram-uegative bacteria for the
secretion of proteins from the cytoplasm either to the periplasim or through both the inner and outer membranes, traditionally grouped into four different systems, The Type III and Type IV systems are generally used for direct transfer of bacterial proteins to adjacent eukaryotic host cells. The Type I system forma a "tunnel" that links the outer and inner membranes such that proteins exported by this pathway are secreted directly in,to the extracellular medium. The Type II secretion system, also known as the general secretory pathway or Sec pathway, is responsible for the secretion of the majority of proteins through the inner membrane into tha periplasm. An additional secretion system, which also makes use of specific N-terramal signal peptidea 'to direct secretion of proteins via the periplasm, is the twin-arginine translocBtion (TAT) pathway. In contrast to the Sec system, which secretes loosely folded proteins to the periplaam where protein assembly takes place, the TAT pathway is used for the secretion of already folded enzymes (reviewed by Berks et. al., 2005, Current Opinion 8; 174-181).
[00150] . The Type II secretion system, has been widely used for the secretion of recombinant proteins from £. coli. In an embodiment of the invention, the host cells comprise a mutant gene(s) in the Type E, or Ssc, pathway. The addition of a short N-tenninal signal sequence to the recombinaivt protein serves to direct the recombinant protein to this secretion pathway and the signal peptide ia removed during the secretion process. The pathway has been used to express antibody fragments such as Fab fragments directed by signal sequences from bacterial proteins naturally secreted using this pathway, including OrnpA, PelB and PboA. The antibody heavy and light chains have been shown to assemble in the periplasm to form antigen-binding Fab fragments (e,g., Skerra and Pluckthun, 1991, Protein Eng. 4:971-979).
[00151] The translocation machinery of the Sec pathway is a well-studied enzyme complex, the ttanslocase, which consists of several integral membrane proteins and an associated ATPase to provide the energy for traualocation (reviewed in Fekkes and Driesseu, 1999, Microbiology and Molecular Biology Reviews, 63:161-173; van der Wollc et al., 1998, EMBO J. 17: 3631-3639). The core of this enzyme complex consists of the membrane-embedded hetarotrirner consisting of the SecY, SecE and SecG proteins (the SecYEG complex), and the peripheral homodimeric ATPase SecA, The SecD, SecF and YajC proteins form a separate heterotrimeric complex that associates with the SecYEG complex to form the complete tranalocase.
[00152] Newly synthesized precursor proteins are bound by the chaperone SecB which stabilizes the pro-protein in a loosely folded conformation competent for translocation, SecB and the signal sequence target the pre-protein to the membrane, and both associate with SecA which is bound with high affinity to the SecY siibunit of the SecYEG complex. As a result of the SecB-SecA interaction, the pre-proteiti is transferred to SecA, which binds both its signal
)
sequence and its mature domain. The release of SecB from the membrane requires the binding of ATP at one of the two ATP-binding sites of SacA. At this stage, a loop of the signal sequence and the N-teiminal region of the pre-protein are presented to the periplasmic face of the membrane, allowing cleavage of the signal sequence by leader peptidase.
[00153] The N-terminal signal sequence of pre-proteins has been regarded as important
for the initial targeting event, the recognition of toe pre-protein by SecA. Aberrant signal sequences are not efficiently recognized by the translocase resulting in. a deficiency in translocation.
[00154] The Type I secretion system has also been used to direct the secretion of
heterologous proteins from E. coli, In an embodiment of the invention, the hoat cell comprises a mutant gene in the Type I secretion system. The secretion signals recognized by the components of the Type I pathway are located at the carboxy terminus of the secreted protein and, in most cases, the secretion signals are not claaved off either during or after secretion. A well-characterized E. coli protein secreted via. the Type I secretion pathway is alpha-hemolysin. The signal sequence from this protein has been used to direct the secretion of a number of heterologous proteins including a scFv antibody fragment by fusion of the C-terminal region of hemolysin to the C-termirma of the scFv coding region (Fernandez and
Lorenzo, 2001, Mol Microbiology 40: 332-346)- In this case the C-termwml hemolyain signal peptide was retained in the secreted product.
[00155] Several mutations in the Sec system components are known which allow
efficient secretion of normally secreted E, coli proteins with defective or absent signal sequences. Such protein localization (prl) mutations have been identified in multiple components of the Sec system, including for example, SecY (prIA), SecE (p/'/G), SecG (prlH) and SecA (prlD) (Boat.and Belin, 1997, J. Biol Cheat. 272:4087-93, which ia incorporated herein by reference in its entirety).
[00156] It has also been found that prl mutations can rescue secretion of some
naturally secreted E. coli proteins in the complete absence of a signal peptide, Thus E. coli maltose binding protein and alkaline phosplmtase lacking a signal peptide have been secreted from prlA mutants (Derman el al., 1993, EMBO J. 12; 879). The bacteriophaga protein LamB can also be secreted without a signal sequence inprlA mutants (Flower at al, 1994, / Bacterlol 176; 18). However, heterologoiiB proteins such as eulcaryotic proteins have not bean previously shown, to be secreted without a signal sequence from prl strains, Wong et aL, 1988, Gene 68:193) achieved successful secretion of insulin-like growth factor-1 from^E. coli using the signal sequences from the LamB or OmpF genes and showed that the processing efficiency of LamB-IGF-1 and OmpF-IGF-1 was enhanced in a strain bearing the prlA4-mutation. Human CD4 iused to OmpA, PhoA, or OmpF signal peptides has also been shown to express efficiently in prlA mutants (Rockeribach et al, 1991, Appl Microbiol Biotechnal 35:32-7), Overexpresaion of SecY mutant proteins on a plasmid vector has also been explored for enhancing expression of a human protein from E. coli. Thus overexpression of the prlA4 mutant SecY protein together with secE increased secretion of human TL-6 fused to the OmpA signal peptide (Perez-Perez et at,,, 1994, BioTechnology 12: 178).
[00157] PrlA strains have also been used to improve diversity of peptides displayed on surface of filamentous phage fd using the pIll signal peptide (Peters et al, 1994, J. Bacterial. 176:4296) and to express bovine pancreatic trypsiu inhibitor as a phage fusion protein from a PhoA signal peptide (US Patent No. 5,223,409, which is incorporated herein by reference in its entirety; Ladner et al). Thus it appears that prlA mutant proteins can facilitate the expression of certain eukaiyotic proteins expressed in a signal-peptide-dependent manner,
comprise a mutant SecY or SecE gene expressed on a plasraid, Such a plasmid may be constructed using a constitutive 01 an inducible promoter allowing for induction of secretion of polypeptides without signal sequences only at desired times, Methods for regulating expression in E. coll are well known in the art and include the use of inducible promoters such as the lac, trc, or tac promoters which are inducible by IPTG, and arabinose-inducible promoters. The mutant Sec gene may be a mutant Sec gana from E. coli or ftom another gram-negative bacterium. Thus, for example, the prlA4 mutant form of SacY may be expressed from a plasmid in an E. coli host cell in order to permit secretion of B Fab fragment expressed without a signal peptide.
[00161] The host cell may be a wild type & coli strain such as W3110 or may be
another strain of E. coli. Suitable host strains include TOPIC, DH5, DHSalpha, Origami and HB101. The host cell may be chosen to provide mutations in other chaperones and genea which affect the folding, assembly and secretion of heterologous proteins. It has been demonstrated that a combination of molecular chaperones such as bacterial DnalC and GroE systems, can augment refolding df proteins that interact with the chaperoning yet fail to fold properly (Buchberger, A., Schroder, H,, Hesterkampj T., Sohonfeld, H. J., and Bulcau, B,, 1996, /, Mol Biol 261, 328-233, Petit, M. A., Bedale, W., Osipiuk, J., Lu, C., Rajagopalan, M., Mclnemey, P., Goodman, M. P., Echols, H,, 1994, J. Biol. Chen. 269, 23824-23829). DnaK also cooperates with Trigger Factor in folding of newly synthesized proteins.
100162] Library embodiments of mis invention may be expressed inprl mutant strains
in order to allow secretion for screening of antibody fragments in various functional assays. The identical vectors may also bo expressed in alternative strains of E. coli for expression within the cytoplasm without me need to re-engineer the antibody molecules, Intracellular expression may be used for efficient production of antibody fragments, for example using trxB gor mutants to provide an oxidizing cytoplasm to allow dimxlphide-bond formation. High level expression of correctly folded and assembled Fab fragments can be achieved in the cytoplasm of E. coli carrying mutations in the glutathione oxjdoreductase (gar) and the thioredaxin reductase (trxB1) genes (Venturi et al, 2002, Mol Biol. 315:1-8). Expression and assembly of correctly folded antibody fragments can be further enhanced using coexpression of molecular chaperones (Levy et al, 2001 Protein Expr Purif. 23: 338-47; Jurado et «/., 2002 J. Moimol 28: 320:1-10).
[00158] Proteins that ate normally secreted from calls often form insoluble inclusion bodies when reoombinantly expressed in E. coli without a signal sequence. For example, genes encoding antibody heavy and light chains or antibody fragments have been expressed without signal sequences in E. coli and ths proteins produced typically accumulate as insoluble prodiictG in inclusion bodies inaida the cell (Boss et al, 1984, A/we/. Acids Res 12: 3791; Cabilly et al,, 1984 Proc. Natl, Acad. Sci. USA 81:3273). Such proteins ars not available for transport across the oytoplasrnic membrane and do not form functional protein. Methods for re-folding antibody fragments from inclusion bodies are provided in US Patents 6,331,415 and 4,816,567- However, such methods for generating antibodies are inefficient and provide poor yields of functional antibody and cannot be used for library screening purposes.
[001S9] In a preferred embodiment of the invention, the prokaryotic host cell contains
a prl mutation in one or more components of the secretory apparatus which allows secretion of the antibody in the absence of an encoded signal peptide. The prl mutant is a mutation which permits secredon of signal-less monoraoric proteins and may comprise a mutation affecting the activity of a SecY, SecA, SecE or SecG gene or another gene, Prl mutants also permit secretion of proteins encoded with a signal peptide in a signal-independent fashion, One embodiment of the invention comprises secreting an antibody polypeptide with a signal sequence in a host strain with aprt mutation and achieving assembly of the antibody into a functional multimeric protein. This is an unexpected result since secretion and chain assembly aro, thought to be closely co-ordinated. Another embodiment carnpri6e.s secreting a polypeptide lacking a signal sequence in a host strain with uprl mutation. Preferably the prl mutation comprises a mutation in SecY (prtA) or SecE (prlG). Most preferably the prl mutant comprises one or more mutations in SecY such as the prlA4 mutant strain (Erar and Bassford, 1982, J. Biol Chem 257: 5852-5860), The prlA4 allele contains two missenge mutations in the seoY gene, resulting in the amino-acid substitutions F286Y and 140 8N. Most preferably, tlieprlA mutant comprises at least an 140 8N mutation in SecY. The sequence of faeprlAJ mutant SecY protein is shown in Figure 1,
(00160] The prl mutant may contain a prl mutation in the chromosome of the host
attain, such as the prlAA mutant strain of E. coli. Alternatively, the prl mutant may be derived by over-expression of an additional copy of a mutant Sec gene, for example, by expression in a plasmid-based expression vector. Thus, for instance, the prl mutant may
[00163] Another embodiment of the invention includes mutants in a second secretory pathway, the twin arginine tamslocation or TAT pathway. It is intended that all tat-dependent signal peptides are to be encompassed by the presant invention, Specific examples include but are not limited to the pnoD and the lipA sequences.
[00164] Another embodiment of the. invention includes mutants in a third secretory pathway, referred to as lie Type III secretion system. Type 01 secretion machinery is present in numerous gram-negative bacteria (including members of the species Shigella, Salmonella, Yersitita, Escherichta, Pseiidomonas, Xcaithomonas, ftalstonia, and Erwinia) that are pathogenic for man, animals, and plants, For example, the Sec-independent type HI secretion pathway is involved in secretion of Yersima anti-host proteins. In Salmonella and Shigella species, it is involved in the process of entry into epithelial cells. It is also implicated in EPEC signal transducing proteins, Pseudomonas aeruginosa toxins, and virulence factors of many plant pathogens, as well as in flagelhun assembly of bacteria such as S. typhimvrium and Bacillvn subtilis.
[00165] Features of this secretion pathway can include activation of secretion by
contact of the bacterium with host colls (Menard et a/., 1994, The secretion of me Shigella flexneri Ipa invasins is activated by epithelial cells and controlled by IpaB and IpaD, EMBO J., 13:5293-5302; Watarai et alt 1995, Contact of Shigella with host cells triggers release of Ipa invasine and is an essential function of invafiiveneBB, EMBO J,, 14:2461-2470; Zierler and Galan» 1995, Contact with cultures epithelial cells stimulates secretion of Salmonella typhimurium invasion proteins IrrvJ, Infect, Immun,, 63:4024-4028); that some of the secreted proteins are delivered into Hie cytoplasm of host cells (Rosqvist et al, 1994, Target cell contact triggers expression and polarized transfer of Yersinia YopE cytotoxin into mammalian cells, EMBO J., 13:964-972; Sory and Cornells, 1994, Translocatum of an hybrid YopE-adenylate-cyclase from Yerainia enterocoliu'Da into HeLa cells, Mol. Microbiol., 14:583-594; Wood et aL, 1996, SopE, a secreted protein of Salmonella dublin, is translocated into the target eukaryotio cell via a sip-dependent mechanism and promotes bacterial entry, Mol Microbiol, 22:327-338; Collazo and Galaii, 1997, The invasion-associated type HI system of Salmonella typhirourium directs the tranalocation of Sip proteins into the host cell, Mol. Microbiol, 24:747-756); and that transcription of genas encoding secreted proteins is controlled by secretion of regulatory proteins (Hughes et al., 1993, Sensing structural intermediates in bacterial flagellar assembly by export of a negative regulator, Science,
262:1277-1280; Pettersson et al., 1996, Modulation of virulence factor expression by pathogen target cell contact, Science, 273:1231-1233).
[00166] In another embodiment of this aspect of the invention, the host strain may be selected for other mutations impacting secretion. For this purposa, a secreted selectable marker protein is expressed without a signal peptide in the host call and mutants are selected which permit secretion of the marker protein, The host strain may be treated with a mutogen to increase the number of mutations or another method to introduce mutations may be used such as transposon mutagenesis. A suitable marker protein is bata-lactamaae, which confers resistance to beta-lactam antibiotics such as ampicillin, Beta-lactamase is expressed without a signal peptide and ampicilliu-resistant mutants are selected. These mutants are screened for the ability to secrete other proteins such as antibody fragments in the absence of signal peptides in order to identify prl mutations. By this means, a mutation allowing secretion without a signal sequence can he introduced into any desired strain of E. coti such as a wild-type W3110 strain or a strain with a high transformation frequency or a strain with mutations in other chaperone proteins.
[00167) Some embodiments of the invention use singly or multiply protease-deficiont
mutant hosts. Different proteins will be more or less sensitive to different proteases normally produced by the microorganisms. Strains may be used which are deficient in proteases such as ompT and degp, Protease HI, La Protease, ClpYQ, ClpXP and ClpAP.
[00168] This invention will be better understood from the Experimental Details which
follow. However, one skilled in flie art will readily appreciate that the specific methods and
results discussed are merely illustrative of the invention as described more folly in the claims
whi ch follow there after. ,
EXAMPLES
Example 1, Expression and secretion of human anti-PorV Fab fragment without sigjial
peptidas
[00169] The Fab fragment of human antibody 1A8 was expressed and secreted from E,
coli mutant prlA4 without signal peptides. Fab 1A8 is an engineered human antibody fragment which binds specifically to an epitope on the PcrV protein of Pseudomonas aeruginosa with high affinity. It competes for binding with a mouse antibody Mabl66
identified to the same epitope (Frank et al, 2002, J Infect Dis. 186:64-73). The light chain of 1A8 consists of a Vkl -kappa light chain and the Fd chain is a VH3 sub-class V-region fused to an IgGl CHI domain,
[00170] A signal-less expression vector for the expression of Fab 1A8 was derived from pGEX-4T-l (GE Healthcare) as follows. The Ampidllin-resistance gene in pGEX-4T-l was deleted by digestion with Aatn and AlwNI, and was replaced with a Chloraraphenicol resistance gene obtained by PCR amplification from plasmid pACYCDuet (Novagen) to form pGEX-CAT. A point mutation of I to A at position 256 was generated by PCR-mutagenesis to mtrodwce a unique Bstll07l restriction site in pGEX-CAT just before the translation initiation codoti downstream of the pTac promoter.
[00171] The pTac promoter of pGEX-CAT was used to express the light chain which was cloned between the Bstll07I and EcoRI sites by PCR using following primers to give vector KB-L, A T7 terminator sequence was incorporated in the Primer 2 before the EcoRI
site.
Primer 1: GGAAACAGTATACATGGACATCCAGTTGACCCAGTC (SEQ ID N0;4)
Primer 2: GCCAGTGAATTCAAACCCCTCAAGACCCGTTTAGAGGCCCCAA
GGGGTTATOCTAGTTAATCGATTTAACACTCTCCCCTGTTGAAGC TC(SEQIDNO:5)
This primer pair amplifies the mature light-chain coding sequence of 1A8 and adds a translation-initiation codon and an upstream sequence to provide an appropriate distance between the Shine-Dalgarno ribosome-binding sequence (AGGA (SEQ ID N0;6)) and the initiation codou of 9 nucleotidos. The predicted amino acid sequence of the N-terminus of the light chain (in single-letter amino-acid code) is:
MDIQLTQ (SEQ ID NO;7)
[00172] The heavy chain (Fd chain) of Fab 1A8 was cloned similarly by PCR, using primers 3 and 4, and introduced between the Bstll07I and NotI sites of pGEX-CAT to give vector KB-H.
Primer 3: GGAMCAGTATACATCGAGGTGCAGCTGGTGGAQTC (SEQ ID NO: 8)
Primer 4; CACGATGCGGCCOCTTAACAAGATTTGGGCTCAACTTTC (SEQ1D NO: 9)
This primer pair amplifies the mature Fd chain coding sequence and adds a translarion-initiation codon and sequences to provide a Shine-Dalgarno - ATG distance of 9 nucleotides. The predicted amino acid sequence of the N-tarminus of the heavy chain is:
MEVQLVE (SEQ ID NO: 10)
[00173] The pTac expression cassette of KB-H was then amplified by PCR. using
Primer 4 and 5 and cloned into KB-L between EcoRI and Notl sites to give vector KB-LH,
Primer 5: CGATGCGAATTCGACTCTAGCGCTGTGGTATGGCT GTGCAGGTCG (SEQ ID NO; 11)
[00174] The final signal-less expression vector for expression of Fab 1AS was constructed by cloning the EcoRI and Fspl fragrnant (138 nucleotides) of pUC19 (Fermentas) into KB-LH between EcoRI and Afel sites to provide a spacer between 2 pTac expression cassettes. A map of the plasim'd. KB5246 is shown in Figure 2.
[00175] E, coli strain SE6004, containing theprlA4 mutation (Emr et al, 1982, J. Biol.
Chem 257: 5852; Wong et a/., 1988, Gote 68: 193), was obtained from the Netherlands Culture Collection of Bacteria (NCCB catalog number 2976).
[00176] Plasmid KB5246 was introduced into SE6004 by electroporation. Electro-
competent cells were prepared using standard techniques as described in Short Protocols in Molecular Biology (3rd edition), Auaubel et at., (John Wilay and Sons foe). Electropration was carried out using a Biorad E. coli Pulser elactroporation apparatus according to the manufacturer's man-actions with a 1.8 kV pulse and a 5 ms time constant. Electroporation cuvettes were from BTX. Transfonnants selected on 34 µg/ml chloramphenicol were cultured in 2xYT medium and expression of the heavy and light chains of Fab 1 AS was induced using isopropyl-beta-D-thiogalactopyranoside (JPTG) at concentrations up to 1 mM. Induction was carried out. for 3 hours for analysis of Fab expression in the periplasm, or cultured for 16 hours for analysis of Fab released into the medium.
[00177] For analysis of Fab secreted across the cytoplasmic membrane into the periplasm, cells were fractionated as follows. The bacterial cell pallet from a 1 liter culture was resuspeuded in I Oral of TES buffer (0.2M Tria pH 8.0, 17.12% sucrose trad O.5mM EDTA) and incubated at 4"C for 15 minutes. After the addition of 12.Sral of TES / H2O at a ratio of 1 / 4, the cell mixture was incubated at 4"C for a further 15 minutes, The cells were pelleted by centrifugation at 7000 rpm in a Sorvall banch-top centrifuge for 15 minutes and the superaatant was kept. The pellet was then resuspended in 10ml TES supplemented with 15mM Mg2S04 and incubated at 4"C for 10 minutes followed by ropefletwg at 7000 rpm and retention of the supernatant.
[00178] 10 µl of periplasmic extract was run on an SDS-PAGE gel under naiwrediidng
conditions, transferred to PVDF membrane and western blotted using an anti-Human Kappa specific antibody conjugated .to Horseradish peroxidase (Zymed labs). The Peroxidase substrate ECL plus (GE Healthcare) was use to produce luminescent signal which was then detected on radiographic film to detect Fab secretion. Figure 3 shows a representative Western blot demonstrating secretion of assembled 1A8 Fab detected in the periplasm. A small amount of immuiioglobulm-related protein of lower molecular weight is also detectable. These bands are consistent with the secretion of light-chain dimera and moncroeric light chain as typically found on secretion of Fab fragments from E. coli in other, signal-dependent secretion systems.
[00179] Fab 1A8 secreted either into lie periplasin or into the medium was analyzed
for antigen-binding activity using a specific antigen-based enzyme-linked immunasarbent assay (ELISA). For this purpose, recombinant PcrV antigen, closed as a fusion protein in frame with an amino terminal glutathione S-transferase (GST) purification tag, was used as described previously (Frank et at., 2002, J. Infectious Diseases 186: 64-73). The PcrV coding sequence is cloned in the expression vector pGEX 2TK (GE Healthcare) to generate the GST-PcrV fusion protein.
[00180] For production of antigen for use in ELISA for the detection of functional anti-PcrV Fabs, GST-PcrV fusion protein was expressed from JS. coli (BL21) transformed witli pGEX 2TK-PcrV and purified as follows. 4 liter liquid culture batches of JS. coli expressing GST-PcrV were grown in 2xYT medium to an optical density of 0.6 at 60Qmn before induction of protein expression with 0.5 mM IPTG and a farther 3 hours growth. The bacterial cells were pelleted by centrifiigation and lysed in a solution of Bug Buster
(Novagen) supplemented with lU/ml iLysozyrae (Novagen) and a protease inlribitor cocktail (Sigma-Aldrich) diluted to the mamifecturer's insn-uctions, After clearing the lyaate by centrifugation and filtration, it was passed over a glutathione sepharose column (GSTrap FF, GE Healthcare), washed and the pure GST-PcrV was eluted in lOmM Glutathione. Tlie antigen was desalted back into PBS.
[00181] Antigen-binding ELISAs for detection of anli-PcrV Fab in periplftsm fractions
i
or in medium samples were carried out as follows. ELISA plates (Costar ElA / R1A) were coated with 100 ng/well GST-PcrV in PBS (see above) by incubating at 4°C for 16 hours and blocking for 1 hour with a 5% solution of non-fat dry railk in PBS 0.1% Tween 20 (PBST). periplasmic fraction samples were diluted in a 1 fold series and applied to the ELISA plate for Ihour at 33DC. After washing with PBST, antibody fragments binding to the antigen were detected with goat anti-human. kappa-ERP. conjugate (US Biological) at a dilution of 1/1000 hi PBST. Antibody binding was revealed using the peroxidase substrate Tetramethyl benzidine (TMB) (lOOjjl / well), and the reaction was stopped with the addition of lOOul 2N HaSO* and read by a standard plate-reader.
[00182] Antigen-binding ELISA confirmed the presence of functional Fab 1 AS in the periplasm (see Figure 4) and released into the medium of SE6QQ4 transfarmants containing plasmid ICB5246. Figure 4 demonstrates secretion of significant amounts of Fab fragment capable of binding to PcrV in. comparison with a standard preparation of Fab fragment in the periplaam of cells expressing Fab 1A8 in a signal-dependent manner (preparation 1150 in Figure 4),
i
[00183] Thus the heavy and light chains of Fab 1A8 are secreted form prlA4 mutant E. coli without the need for a signal peptide on either chain. The two chains assemble to form Fab fragment which can be detected in the periplasm and released into the culture medium as functional antigen-binding molecules.
Example 2. Detection of antigen-binding Fabs by Colony-Lift Binding Assay (CLBA)
[00184] Libraries of antibody Fab fragments cloned in plasmid KB5246 and
transformed into SE6004 are plated onto 2YT agar (Becton, Dickinson Difco™ 2xYT yeast extract tryptone medium) containing -the appropriate antibiotic (chlorampheniool at 34µg/ml). The plating efficiency is adjusted so the resulting bacterial colonies are discreet but dense enough to maximize the area of the plate. Various sizes of plate are used depending on the
number of clonal colonies to be screened, Thus, at optimal density a 10cm diameter plate contains 4000 colonies, a 15cm diameter plate contains 10000 colonies and a 25cm square plate contains 50,000 colonies,
[00185] Nitrocellulose filters (Schlmcher & Schuell BA85) of diameter 8,2cm, 13.2cm or 20cm square are pre-coated with antigen in. Phosphate Buffered Saline (PBS) at an empirically determined concentration (usually between 0.5 and 20µg/ml). Tha volume of coating solution depends upon the filter size, 4ml, 8ml or 20ml can be used for the various filter sizes listed above. Filters are placed face down in a pool of the antigen and capillary action evenly distributes the antigen. The filters are coated for 2-3 hours at 33°C with occasional agitation. Hie filters.are then rinsed once with excess PBS and blocked with a 5% solution of non-fat dry milk in PBS for an additional 2 hours at 25°C with agitation. The filters are then drained and rinsed once in PBS supplemented with 0.1% Tweeii 20 (PBST) and twice in excess 2YT liquid media supplemented with antibiotic selection (34 µg/ml chlommphenicol) and transcriptional tnducer (IPTG). The IPTG concentration can be optimized for each library but is typically in the range 0.01 - 0.1 mM. After allowing tha filters to drain, they are placed on a 2YT-agar plate supplemented with the same concentration of antibiotic and inducer (the expression plate).
[00186] Un-coatedj dry nitrocellulose membrane is placed face-down on the plates of colonies containing the antibody-fragment library. Once the filters are visibly wet (~20sec) ' and in one movement, the filters are lifted and placed colony side up onto the coated filter which is already on the expression platfl. A sterile needle is used pierce Ilia filters in a pattern which will allow alignment.
[00187] The expression plate with the nitrocellulose filter sandwich is placed at 33DC for 12-16 hours. During this time, tha antibody fragments are secreted and diffuse through the first nitrocellulose membrane to the second, antigen-coated membrane. If the antibody fragment ftom a given bacterial colony has antigen binding activity, it is retained on the antigen filter and is subsequently detected,
[00188] After the 12-16 hour expression period, the colony filter is removed from the
expression, plate and stored at 4°C on a 2YT-agar plate with antibiotic selection but no trgnscriptional inducer.
[00189] The antigen-coated filter is removed and washed throe times (5 minute washes) in excess PBST followed by blocking with a 5% solution of non-fat dry milk in PBST for 1.5 hours at 25°C. The antibody fragments retained on the antigen filter are then detected by first incubating with one of the following alternative primary antibodies: Goat anti-human Kappa-HRP conjugate (US Biological) is used to reveal binding. After four 10-mhiuta washes, the filters are incubated in peroxidase substrate solution (EGL plus, GE Healthcare) and used to expose light-sensitive photographic film. Alternatively, antibodies conjugated with fluorescent labels may be used. In this case a flatbed excitation scanner such as the Typhoon (GE Healthcare), FX-Pro (Biorad) or Odyssey (Licor) can be used to visualize the positive spots,
[00190] Using a light box for back illumination, the pattern of spots on the photographic film or digital image is aligned with the colony filter (the filter can be removed from the 2YT-agur plate and placed on a plastic transparency for this process). The identified positive colonies are picked and used to inoculate ft 2YT liquid mini-culture. Bacteria from the primary screen are then re-plated at a lower density and picked for subsequent analysis to ensure that a clonal population is expanded.
Example 3. Detection of anti-PcrV Fab secreted froro prlA4 cells without signal peirtides using CLBA
[00191] For Fab fragments expressed without signal peptides in plasmid KB 5246, transformed cells were plated on 2YT expression plates containing chloramphemcol (34|J-g/ml) and 10µM IPTG. Cells were induced for 16 hours and antibody fragments binding to GST-PcrV on the antigen-coated filter were detected as described in Example 2, using a goat ant-Miwnan kappa antibody - Horseradish peroxidase conjugate (US Biological) at a dilution of 1/5000 in PBST. After four 15-mmute washes and the application of ECL Plus (GE Healthcare), the filters were weed to expose autoradiographic film (Hyperfihn from GE Healthcare),
[00192] Plasmid KB5246, expressing FablAB, was transformed into SE6004 cells,
which have a mutant SecY gene (containing the prlA44 mutation), and into TOPIC cells which contain a wild type SecY gene. Positive colonies secreting FablA8 were detected in the PcrV antigen-CLBA only from SE6004 tranaformants; the TOP 10 tamsformants did not secrete detectable amounts of FablA8. This result indicates that the prlA4 mutant strain is
able to secrete Fab fragment without the need for a signal peptide on either the heavy or light chain, The heavy and light chains assemble and are capable of forming Mly ftinetional Fab fragment capable of binding the cognate antigen coated onto a nitrocellulose filter.
Example 4, Screening for binders to specific antigens of Fabs eecretad without signal peptidea
[00193] A second human Fab FB42-8, specific for a human cytakine, was expressed without signal peptides in SE6004 by cloning the appropriate V-regtons sequences into KB5246 in place of the FablA8 V-regions, Cells expressing the two Fabs (FB42-8 and FablAS) were mixed in a 50/50 ratio and plated on 2xYT agar, A CLBA was performed as described in Example 2, with the coated, antigen being either PCRV or the cytoldne antigen specific for FB42-8. Duplicate CLBA, detection and alignment showed that Fabs specific for each antigen could ba picked from, a mixture of the two tranafonnants.
(00194] A library of diverse Fabs can be screened for binders to a specific antigen in
the same manner.
Example 5. Efficiency of secretion of Fabs without signal peptidas
[00195] A Fab was cloned into either a typical bacterial expression plaatnid with
bacterial secretion leader-peptides (KB 1150) or into KB5246 hi place of the Fab 1A8 coding sequences, These two constructs were compared for efficiency of expression and secretion at various IPTG induction conditions. Cultures were grown until OD0.6 at 600nm and then induced, Growth was continued for lolirs, Fab secreted into the culture medium was detected on a western blot by an aflti-kappa-HRP polyclonal, as in Example 1 (see figure 5).
[00196] Fabs containing murins V-regions are known to be difficult to express to high yields in bacteria. In this experiment the Fabs secreted, more efficiently without signal peptides from SE6004 than when expressed with signal peptides in the wild-type TOP 10 straia Indeed, Fab secretion was undetectable using signal-peptide mediated secretion in TOP10F' cells and was readily detectable in the medium when the SE6004 strain was used for secretion of signal-less Fab. Thus Fabs and other antibodies which are poorly expressed in E. coli may advantageously be produced by secretion in the absence of signal peptides from appropriate mutant strains such as the prlA4 mutant SE60Q4,
Example 6. Construction of an expression vector for a orlA4 mutant SacY gene
[00197] An expression vector, plSA, for expression of genes in bacterial cells under the control of a strong bacterial promoter, Ifte trc promoter, was constructed as follows.
[00198] Plasmid pACYC!77 (Fermantas) was digested with BanI and partially digested with Stul. The 2386 bp DNA fragment was then blunt-ended using Klenow fragment of DNA polymerase I. The pTrc promoter was PCR amplified from the plasmid pfixHis-GFP (Clontech) with the following primara:
Primer 1: TCTTCCAGGCCTGAGCTCGAGCTGTTGACAATTAATCA (SEQ ID NO: 12)
Primer 2: CAGTTACAGGCCTGGTACCTCACCGGCCGTTAAACCCCCCAT GGriTATTCC (SEQ ID NO: 13)
The PCR product was then digested with Still, and ligatod with the 2386 DNA fragment of pACYC!77 to give vector plSA, which has Ncol and Kpnl sitee after the pTrc promoter.
[00199] The prlA4 mutant SecY gene was cloned from SE6004 cells by PCR
amplification using the following primers:
Prirner3; ACGGAATTCACCATGGCTAAACAACCGGGATTAGATTTTC (SEQ ID NO: 14)
Primer4: CAGTTACGGTACCrfATCGGCCGTAGCCTTTCAGGTTC (SEQ ID NO: 15)
[00200] The PCR product was then digested with Ncol and Kpnl and cloned into vector pi 5A between the same two sites to give KB5282 which expresses the mutant SecY gene under the control of the bacterial trc promoter (pTrc; see Figure 6). Transformation of E. coli strains with KB5282 confers theprW phenotypa on the host cell and allows secretion via Hie periplasm of heterologous proteins such as antibodies from coding sequences which do not encode signal peptides.
[00201] Electro-competent DHS-alplia cells were transformed with plasmid KB5282
by elesclroporation and transformants were selected uauig 35 |ig/ml kanamycin in 2xYT medium.
[00202] The expression of FablA8 in DH5 cells in the presence of oveiexpressed mutant SocY was assessed as follows. DH5 cells were co-transformed, by electropor&tion with plasmids KB5246 (expressing the Fab without signal sequences) and KB5282 (expressing mutant SecY). Transformants selected on ohloratnphenicol and Kanamycm were cultured in 2xYT medium and expression of the heavy and light chains of Fab 1 AS was induced using isopropyl-bata--thiogalactopyratioside (IPTG) at a concentration of 20^iM or 200uM. Expression was continued for 16 houra at 33°C with shaldng. The levels of expressed and secreted intact Fab fragments from DH5 cells with the co-transformation of mutant SecY was compared with expression of Fab from tiffiprlAJ strain SE6004 using the same concentrations of IPTG, as described in Example 1. Western blots using a detection antibody specific for human Kappa chains were carried out on expression madia ran on SDS-PAGE under non-reducing conditions (Figure 7). High levels of secreted Fab ware detected in the media of DH5α cells expressing mutant SecY. Indeed these cells secreted higher levels of Fab when induced using 20 µM IPTG than SE6004 cells. In contrast, no detectable secretion of Fab was observed when 1CB5246 was transformed into TOPIC F' cells, a strain which expresses wild-type SecY, (see Figure 7).
Bxarnple 7. Expression of antibody fragments with signal sequences in prl mutant K call strains
[00203] Expression vectors which encode antibody polypeptides including signal
peptides can also be expressed in a prl mutant E. coli strain as follows, A signal peptide is introduced at the N-terminus of the heavy chain coding sequence, the light chain coding sequence or both in order to secrete assembled and fanctional Fab or Fab' fragments from the prl mutant strain.
[00204] To generate a convenient prl mutant strain for the expression of antibody
fragments containing signal peptides, plasmid ICB5282 (Example 6) is used to transform DH5-alpha cells. The kananrychvvesistant transformants have the prlA phenotype and can secrete Fab fragments lacking signal peptides as described in Example 6. In this case the KB5282 DH5-alpha Irarisformants ate subsequently transformed by electroporatiou with an expression vector expressing antibody heavy and light chains in which one or both of the chains is expressed with a signal peptide. Electrocorapetent cells are prepared according to standard techniques as described in Short Protocols in Molecular Biohgy (3rd edition),
Auaubel et al (John Wiley and Sana Inc.) and elactroporation is carried out as described in Example 1.
[00205) Functional Fab or Fab' fragments are identified and may be isolated from the peripksmic fraction or the culture medium as desoribod in Examples 1,2 and 3 above.







WE CLAIM:
1. A method for producing an antibody, antibody-fragment, or antibody-related polypeptide
in a prokaryotic host cell without the need for a signal peptide, comprising the steps of:
(a) Obtaining a prokaryotic host cell, characterized in that said host cell comprises a prl mutation-and at least one expression vector comprising a polynucleotide molecule encoding a heavy chain polypeptide of the antibody and a polynucleotide molecule encoding a light chain polypeptide of the antibody, further characterized in that at least one of the polynucleotide molecules does not encode a signal peptide,
(b) Expressing the prl mutant protein and the polynucleotide encoding the heavy chain and the polynucleotide encoding the light chain polypeptide of the antibody, characterized in that at least one of the polypeptides lacks a signal peptide, and,
(c) Forming at least one antibody by allowing the host cells containing expressed prl mutant protein and expressed heavy and light chain polypeptides to secrete the heavy chain polypeptide and the light chain polypeptide across a cytoplasmic membrane.
2. The method as claimed in claim 1, wherein the antibody is selected from the group consisting
of a human antibody, a mouse antibody, a rat antibody, a rabbit antibody, a camel antibody, a
sheep antibody, a chimeric antibody, a humanized antibody, an engineered an antibody and an
epitope-focused antibody.

3. The method as claimed in claim 1, wherein the polynucleotides encoding the heavy chain polypeptide and the light chain polypeptide are present in a plurality of different vectors.
4. The method as claimed in claim 1, wherein the polynucleotides encoding the heavy chain polypeptide and the light chain polypeptide are, both present on the same vector.
5. The method as claimed in claim 1, wherein the polynucleotide encodes the heavy chain polypeptide lacking a signal peptide.
6. The method of claim 1, wherein the polynucleotide encodes the light chain polypeptide lacking a signal peptide.
7. The method as claimed in claim 1, wherein the polynucleotides encode the heavy and light chain polypeptides lacking signal peptides.

8. The method as claimed in claim 1, wherein the host cell is a gram negative bacterium.
9. The method as claimed in claim 8, wherein the gram negative bacteria is an E.coli.
10. The method as claimed in claim 9, wherein the E. coli cell comprises a mutant gene in a Sec pathway.

11. The method as claimed in claim 10, wherein the mutant gene comprises a mutant in a SecY
gene.
12. The method as claimed in claim 11, wherein the host cell comprises a SecY gene encoding a
protein having substantially wild-type function and a SecY gene encoding a protein substantially
lacking wild-type function.
13. The method as claimed in claim 11, wherein the host cell comprises a mutant SecY gene carried on a vector.

Documents:

518-delnp-2008-1-Correspondence-Others-(27-02-2013).pdf

518-DELNP-2008-Abstract-(02-02-2012).pdf

518-delnp-2008-abstract.pdf

518-DELNP-2008-Claims-(02-02-2012).pdf

518-delnp-2008-Claims-(05-03-2013).pdf

518-delnp-2008-claims.pdf

518-delnp-2008-correspondenc others (10-03-2010).pdf

518-delnp-2008-correspondenc others (14-02-2008).pdf

518-delnp-2008-correspondenc others (23-06-2009).pdf

518-DELNP-2008-Correspondence Others-(02-02-2012).pdf

518-delnp-2008-Correspondence Others-(05-03-2013).pdf

518-DELNP-2008-Correspondence Others-(07-02-2012).pdf

518-DELNP-2008-Correspondence-Others-(10-03-2010).pdf

518-DELNP-2008-Correspondence-Others-(27-02-2013).pdf

518-delnp-2008-correspondence-others.pdf

518-DELNP-2008-Description (Complete)-(10-03-2010).pdf

518-delnp-2008-description (complete).pdf

518-DELNP-2008-Drawings-(02-02-2012).pdf

518-delnp-2008-drawings.pdf

518-delnp-2008-form-1.pdf

518-delnp-2008-Form-13 (10-03-2010).pdf

518-delnp-2008-form-18 (14-02-2008).pdf

518-delnp-2008-form-2.pdf

518-delnp-2008-form-3 (23-06-2009).pdf

518-delnp-2008-Form-3-(02-02-2012).pdf

518-DELNP-2008-Form-3-(27-02-2013).pdf

518-delnp-2008-form-3.pdf

518-delnp-2008-form-5.pdf

518-DELNP-2008-GPA-(02-02-2012).pdf

518-DELNP-2008-GPA-(27-02-2013).pdf

518-delnp-2008-pct-101.pdf

518-delnp-2008-pct-301.pdf

518-delnp-2008-pct-304.pdf

518-delnp-2008-pct-311.pdf

518-DELNP-2008-Petition-137-(02-02-2012).pdf


Patent Number 257016
Indian Patent Application Number 518/DELNP/2008
PG Journal Number 35/2013
Publication Date 30-Aug-2013
Grant Date 27-Aug-2013
Date of Filing 18-Jan-2008
Name of Patentee KALOBIOS, INC.
Applicant Address 3427 HILLVIEW AVENUE, SUITE 200, PALO ALTO, CA 94304, UNITED STATES OF AMERICA.
Inventors:
# Inventor's Name Inventor's Address
1 CHRISTOPHER ROBERT BEBBINGTON 132 AVILA ROAD, SAN MATEO, CALIFORNIA 94402, USA.
2 GEOFFREY YARRANTON 1148 BALBOA AVENUE, BURLINGAME, CA 94010, USA.
PCT International Classification Number C40B 30/06
PCT International Application Number PCT/US2006/026334
PCT International Filing date 2006-07-07
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/701,902 2005-07-22 U.S.A.
2 11/273,906 2005-11-14 U.S.A.