ZA200505151B

ZA200505151B - Method for manufacturing recombinant polyclonal proteins

Info

Publication number: ZA200505151B
Application number: ZA200505151A
Authority: ZA
Inventors: John S Haurum; Vincent W Coljee; Chiou-Ying Yang; Finn C Wiberg; Jacqueline Sharon
Original assignee: Symphogen As
Priority date: 2003-01-07
Filing date: 2006-01-19
Publication date: 2006-03-29
Also published as: CN1723283A; CN100383244C

Description

METHOD FOR MANUFACTURING RECOMBINANT POLYCLONAL PROTEINS

. FIELD OF THE.INVENTION . The present invention forms the basis of a technology platform for producing recombinant polyclonal proteins, such as proteins from the immunoglobulin superfamily, e.g. soluble or membrane-bound forms of B or T cell receptors, to be used as a new class of therapeutic in the treatment, amelioration or prevention of various infections, inflammatory diseases, transplantation rejection, cancer, and allergies.

BACKGROUND OF THE INVENTION

A number of infectious diseases and cancers lacks efficient therapies. Monoclonal antibodies have generally not been successful against these targets, partly due to variability of the com- plex targets and adaptive mutations of target proteins causing immune escape from mono- clonal antibody recognition. Polyclonal antibodies on the other hand are able to target a plu- rality of dynamic targets, e.g., on viruses or cancer cells. Also, polyclonal antibodies have the highest probability of retaining activity In the event of antigenic mutation.

Different commercially available polyclonal antibody therapeutics exist including: 1) normal human immunoglobulin isolated from the blood of normal human donors; 2) human hyper- immune immunoglobulin derived from the blood of individual human donors carrying anti- bodies against a particular disease target, e.qg., a virus, which they previously have encoun- tered either through infection or vaccination; and 3) animal hyperimmune immunoglobulin derived from the blood of immunized animals.

Immunoglobulin purified from human blood has proved effective against infections with hepatitis B virus, respiratory syncytial virus, cytomegalovirus and other herpes viruses, ra- bies virus, botulinum toxin, etc, as well as in the neonatal rhesus D prophylaxis. Immuno- globulin purified from the blood of rabbits immunized with human T cells is used to afford T cellimmunosuppression in the treatment or prevention of transplant rejection (e.qg., Thymo- globulin). Normal human immunoglobulin has been utilized to boost the immune system of « immunodeficient patients, as well as in the therapy of various autoimmune disorders.

Nevertheless, widespread immunoglobulin use has been limited due to the constrained supply of donor blood raw material, problems with batch-to-batch variations, and variable safety.

Animal-derived immunoglobulins in particular are faced with the same problems of immuno- genicity as was observed for animal-derived monoclonal antibodies in the 1980s and 1990s,

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Finally, as with other blood products, the risk of transmission of infectious agents such as

HIV, herpes or hepatitis viruses or prions remains. Accordingly, while clinicians acknowledge ‘ that polyclonal antibodies are a preferred therapeutic in some situations, their use has been very limited.

New approaches to generate human immunoglobulins arose with the transgenic animal tech- niques. Transgenic mice carrying human immunoglobulin loci have been created (U.S. Patent

No. 6,111,166). These mice produce fully human immunoglobulins, and antibodies against a specific target can be raised by usual immunization techniques. However, larger antibody yields are limited because of the relatively small size of mice. Larger animals have also been made transgenic for the human immunoglobulin genes, e.g., cows, sheep, rabbits, and chick- ens (Kuroiwa, Y. et al. Nature Biotechnology; 2002; 20: 889-893). However, producing poly- clonal antibodies for therapy from the blood of such animals is not without complications.

First, the immunophysiology of the animal and humans may display considerable differences, causing a difference in the resulting immune repertoire, functional rearrangement, and diver- sity of the antibody response. Second, mitotic instability of the introduced immunoglobulin loci might influence the long-term production of antibodies. Third, it is technically challen- ging to delete the animal's own immunoglobulin loci so that e.g., the animal antibody pro- duction will not exceed the production of human antibody. Fourth, the risk of transmission of infectious agents such as viruses, prions or other pathogens accompanies the administration of human antibodies produced in animals.

Accordingly, there is a need for manufacturing technologies for producing recombinant poly- clonal proteins, such as antibodies, in sufficiently large amounts and with minimal batch-to- batch variations for safe clinical uses. Efficient methods for manufacturing homogenous re- combinant proteins using eukaryotic (in particular mammalian) expression cell lines have been developed: for the production of a variety of proteins including monoclonal antibodies, interleukins, interferons, tumor necrosis factor, coagulation factors VII, VIII and IX. Many of these techniques are based on transfection and random integration of the gene of interest

Into the genome of the expression cell line followed by selection, amplification, and charac- terization of a high-producer expression clone and propagation of this clone as a master ex- pression cell line.

The expression of an inserted foreign gene may be influenced by “position effects” from syr- rounding genomic DNA. In many cases, the gene Is inserted into sites where the position effects are strong enough to inhibit the synthesis of the product of the introduced gene. Fur- thermore, the expression is often unstable due to silencing mechanisms (i.e. methylation) imposed by the surrounding chromosomal host DNA.

Systems allowing Integration and expression of a gene of interest in mammalian cells at a specific genomic location have been developed for the expression of a homogenous recombi- A . nant protein composition (U.S. Patent Nos. 4,959,317 and 5,654,182; WO 98/41645; WO 01/07572). wo 98/41645 describes the site-specific integration for production of a mam-

R 5 malian cell line that secretes, for example, antibody. However, this expression is monoclonal and there is no indication that transfections could be done with a library of vectors. Nor are there any suggestions how to maintain the original diversity generated by specific Vy4-V, com- binations in a library.

DISCLOSURE OF CONTRIBUTION

The present invention provides solutions for generating a manufacturing cell line for expres- sion and production of a recombinant polyclonal protein, avoiding significant bias among the individual members constituting the polyclonal protein.

Further, the present invention does not utilize animals in the polyclonal protein production, thereby obviating the ethical and clinical difficulties associated with such approaches,

SUMMARY OF THE INVENTION

The present invention provides methods for producing a recombinant polyclonal manufactu- ring cell line for the production of a recombinant polyclonal protein, often selected from the immunoglobulin superfamily. Especially the production of polyclonal antibodies, polyclonal T cell receptors or polyclonal fragments thereof are of interest. The present invention allows for the commercial production of a recombinant polyclonal protein for use in pharmaceutical compositions. One important feature of the invention Is that during the manufacturing pro- cess biased expression of the individual molecules constituting the polyclonal protein is kept to a non-significant level, minimizing unwanted batch-to-batch variation.

One aspect of the present invention relates to a method for manufacturing a recombinant polyclonal protein of interest, wherein said polyclonal protein comprises (or consists of) dis- ’ tinct members that bind a particular antigen, said method comprising: a) providing a collec- tion of cells comprising a library of variant nucleic acid sequences, where each of said nucleic ) acid sequences encodes a distinct member of said polyclonal protein and where each of said nucleic acid sequences are integrated at the same, single site of the genome of each indivj- dual cell in said collection of cells; b) culturing sald collection of cells under conditions faciii- tating expression of said polyclonal protein; and C) recovering said expressed polyclonal protein from the cell culture, cell fraction or cell culture medium.

A further aspect of the present invention relates to a method for generating a collection of cells suitable as a recombinant polyclonal manufacturing cell line, said method comprising: a) . providing a library of vectors comprising a population of variant nucleic acid sequences, wherein each of said vectors comprises 1) one single copy of a distinct nucleic acid sequence . 5 encoding a distinct member of a polyclonal protein comprising (or consisting of) distinct members that bind a particular antigen and 2) one or more recombinase recognition sequen- ces; b) introducing said library of vectors into a host cell line, wherein the genome of each individual cell of said host cell line comprises recombinase recognition sequences, matching those of the vector, at a single specific site in its genome; ¢) ensuring the presence in said cells of one or more recombinases so that the variant nucleic acid sequences of step (a) are integrated site-specifically in the cells of the host cell line, where said one or more recombi- nases is/are either i) expressed by said cells into which said nucleic acid sequence is intro- duced; ii) operatively encoded by the vectors of step a; Iii) provided through expression from a second vector; or iv) provided to the cell as a protein; and d) selecting cells comprising an ' integrated copy from said library of variant nucleic acid sequences.

In both methods of the invention, it will be understood that the polyclonal protein normally is one that is not naturally associated with the cells wherein expression is effected.

The present invention describes several methods by which a library of variant nucleic acid

Sequences can be introduced into a host cell fine in order to generate a collection of cells suitable as polyclonal manufacturing celi line. These methods include bulk transfection of a coliection of cells with the library, semi-bulk transfection of aliquots of cells with fractions of the library or individual transfection where host cells are transfected with individual members of the library followed by pooling of the clones generated upon selection. Preferably the pre- sent invention utilizes mammalian cells (cell lines or cell types) as host cell line.

In one aspect of the invention, the individual members of a polyclonal protein are encoded from pairs of independent gene segments. Polyclonal proteins, where the individual members are comprised of two polypeptide chains, include soluble or membrane-bound forms of anti- bodies and T cell receptors. In further embodiments of the present invention a pair of gene segments encode an antibody heavy chain and light chain variable region, or a T cell receptor ' 30 alpha chain and beta chain variable region or a T cell receptor gamma chain and delta chain variable region. :

The present invention further provides a recombinant polyclonal manufacturing cell line comprising a collection of cells transfected with a library of variant nucleic acid sequences, wherein each cell in the collection is transfected with and capable of expressing one member of the library, which encodes a distinct member of a polyclonal protein that binds a particular antigen and which is located at the same single site in the genome of individual cells in said collection, wherein said nucleic acid sequence is not naturally associated with said cell in the : collection. The cell line preferably originates from a mammalian cell line such as Chinese hamster ovary (CHO) cells, COS cells, BHK cells, myeloma cells (e.g., 5p2/0 cells, NSO), . 5 YB2/0, NIH 3T3, fibroblast or immortalized human cells such as Hela cells, HEK 293 cells, or

PER.C6. However, non-mammalian eukaryotic or prokaryotic cells, such as plant cells, insect cells, yeast cells, bacteria, fungi etc., can also be used.

Also embraced by the present invention is a library of vectors for site-specific integration comprising a population of naturally occurring variant nucleic acid sequences, wherein each of said vectors comprises 1) one copy of a distinct nucleic acid sequence encoding a distinct member of a polyclonal protein that binds a particular antigen and 2) one or more recombi- nase recognition sequences.

In another aspect, the invention provides a pharmaceutical composition comprising, as an active ingredient, a recombinant polyclonal antibody (or fragment thereof) or polyclonal T cell receptor (or fragment thereof), preferably obtained by the methods of the invention. The recombinant polyclonal protein of the composition Is specific for or reactive against a prede- termined disease target. Such pharmaceutical compositions can be used for the treatment, amelioration or prevention of diseases such as cancer, infections, inflammatory diseases, allergy, asthma and other respiratory diseases, autoimmune diseases, immunological mal- functions, cardiovascular diseases, diseases in the central nervous system, metabolic and endocrine diseases, transplant rejection, or undesired pregnancy,. in a mammal such as a human, a domestic animal, or a pet.

Definitions

By “protein” or “polypeptide” is meant any chain of amino acids, regardless of length or post- translational modification. Proteins can exist as monomers or multimers, comprising two or more assembled polypeptide chains, fragments of proteins, polypeptides, oligopeptides, or peptides.

As used herein, the term “polyclonal protein” or “polyclonality” refers to a protein composi- tion comprising different, but homologous protein molecules, preferably selected from the ‘ 30 immunoglobulin superfamily. Thus, each protein molecule is homologous to the other mole- cules of the composition, but also contains one or more stretches of variable polypeptide sequence, which is/are characterized by differences in the amino acid sequence between the individual members of the polyclonal protein. Known examples of such polyclonal proteins

Include antibody or immunoglobulin molecules, T cell receptors and B cell receptors. A poly-

clonal protein may consist of a defined subset of protein molecules, which has been defined by a common feature such as the shared binding activity towards a desired target, e.g., in : the case of a polyclonal antibody against the desired target antigen. . The term “polyclonal protein of interest” covers a defined polyclonal protein subset, which shares a common feature, such as binding activity towards a desired target, e.g., in the case of polyclonal antibodies described by the binding activity or specificity against the target an- tigen, said antigen being one or more of €.g., separate proteins, microorganisms, parasites, cell types, allergens, or carbohydrate molecules, or any other structures, molecules, or sub- stances, which may be the target of specific antibody binding, or mixtures of said antigens.

The terms “one member of a recombinant polyclonal protein composition” or “one member of a recombinant polyclonal protein” denote one protein molecule of a protein composition com- prising different, but homologous protein molecules, where each protein molecule is homolo- gous to the other molecules of the composition, but also contains one or more stretches of variable polypeptide sequence, which is/are characterized by differences in the amino acid sequence between the individual members of the polyclonal protein.

The terms “variable polypeptide sequence” and “variable region” are used interchangeably.

The terms “a distinct member of a recombinant polyclonal protein” denotes one protein mole- cule of a protein composition comprising different, but homologous protein molecules, where each protein molecule is homologous to the other molecules of the composition, but also contains one or more stretches of variable polypeptide sequence, which is/are characterized by differences in the amino acid sequence between the individual members of the polyclonal protein.

The term “antibody” describes a functional component of serum and Is often referred to ej- ther as a collection of molecules (antibodies or immunoglobulin) or as one molecule (the an- tibody molecule or immunoglobulin molecule). An antibody molecule is capable of binding to or reacting with a specific antigenic determinant (the antigen or the antigenic epitope), which in turn may lead to induction of immunological effector mechanisms. An individual antibody ' molecule is usually regarded as monospecific, and a composition of antibody molecules may be monoclonal (i.e., consisting of identical antibody molecules) or polyclonal (i.e., consisting ‘ 30 of different antibody molecules reacting with the same or different epitopes on the same an- tigen or even on distinct, different antigens). Each antibody molecule has a unique structure that enables it to bind specifically to its corresponding antigen, and all natural antibody mole- cules have the same overall basic structure of two identical light chains and two identical heavy chains. Antibodies are also known collectively as immunoglobulins. The terms antibody i 7 or antibodies as used herein are also intended to include chimeric and single chain antibo- dies, as well as binding fragments of antibodies, such as Fab, Fv fragments or scFv frag- ' ments, as well as multimeric forms such as dimeric IgA molecules or pentavalent IgM. ) The term “polyclonal antibody” describes a composition of different antibody molecules which is capable of binding to or reacting with several different specific antigenic determinants on the same or on different antigens. Usually, the variability of a polyclonal antibody is thought to be located in the so-called variable regions of the polyclonal antibody. However, in the context of the present invention, polyclonality can also be understood to describe differences between the individual antibody molecules residing in so-called constant regions, e.g., as in the case of mixtures of antibodies containing two or more antibody isotypes such as the hu- man isotypes IgG1, 1gG2, 1gG3, 1gG4, IgAl, and IgA2, or the murine isotypes I1gG1, 1gG2a,

IgG2b, IgG3, and IgA.

A “recombinant polyclonal antibody of interest” describes a defined recombinant polyclonal antibody subset, which is characterized by the ability to bind to a desired target or desired set of targets, said targets being e.g., a separate protein, a microorganism, a parasite, a cell, an allergen, or a carbohydrate molecule, or another structure, molecule, or substance which may be the target of specific antibody binding, or mixtures thereof.

The term “immunoglobulin” commonly is used as a collective designation of the mixture of antibodies found in blood or serum, but may also be used to designate a mixture of antibo- dies derived from other sources.

The term “immunoglobulin molecule” denotes an individual antibody molecule, e.g., as being a part of immunoglobulin, or part of any polyclonal or monoclonal antibody composition.

When stating that a member of a polyclonal protein binds to an antigen, It is herein meant a binding having binding constant that is below 1 mM, preferably below 100 nM, even more preferred below 10 nM. . . The term “a library of variant nucleic acid molecules of interest” is used to describe the col- lection of nucleic acid molecules, which collectively encode a “recombinant polyclonal protein of interest”. When used for transfection, the library of variant nucleic acid molecules of inter- est is contained in a library of expression vectors. Such a library typically have at least 3,5, 10, 20, 50, 1000, 10% 10° or 10° distinct members.

As used herein the terms “one copy of a distinct nucleic acid sequence of interest” are not to be taken literally as a single stretch of nucleic acids corresponding to a single gene segment,

but rather as one copy of all the gene segments required to produce all the subunits of one molecule of the protein of interest, and assembled into one nucleic acid molecule such as e.g. ’ a vector, Some examples, where more than one gene segment usually is required to give rise to a complete molecule of a protein of interest include B cell receptors, antibodies and frag- : 5S ments of antibodies such as Fab's and variable domains, or T cell receptors, When introduced into the cell, the gene segments, which together encode the fully assembled protein of inter- est, reside in the same vector, thus being linked together in one nucleic acid sequence, pos- sibly as separate transcriptional elements under control of different promoters.

The term “a gene of interest” as used herein, refer to a nucleic acid sequence composed of one or more gene segments (genomic or cDNA) that encode one member of a protein of in- terest. The plural form “genes of interest” refers to a library of nucleic acid sequences en- coding a polyclonal protein of interest. The term “GOI” is used as an abbreviation of (a) gene(s) of interest.

As used herein, the term “vector” refers to a nucleic acid molecule into which a nucleic acid sequence can be inserted for transport between different genetic environments and/or for expression in a host cell. A vector capable of integrating into the genome of a host cell at a pre-determined, specific locus in the genome is herein named “a vector for site-specific inte- gration”. If the vector carries regulatory elements for transcription of the nucleic acid se- quence inserted in the vector (at least a suitable promoter), the vector is herein called “an expression vector”, If the expression vector is capable of integrating at a pre-determined, specific locus in the genome of the host cell, the expression vector may be called “an expres- sion vector for site-specific integration”. If the nucleic acid sequence inserted into the above identified vectors encodes a protein of interest as herein defined, the following terms are used “vector of interest”, “vector of interest for site-specific integration”, “expression vector of interest” and “expression vector of interest for site-specific integration”. The term “an iso- type-encoding vector” refers to a vector carrying nucleic acid sequences encoding an anti- body isotype. In the present specification, “phagemid vector” and “phage vector” are used interchangeably. The terms “plasmid” and “vector” are used interchangeably. The invention is intended to include such other forms of vectors, which serve equivalent functions for example plasmids, phagemids and virus genomes or any nucleic acid molecules capable of directing the production of a desired protein in a proper host.

The term “each member of the library of vectors of interest” is used to describe individual vector molecules with a distinct nucleic acid sequence derived from a library of vectors of interest, where the nucleic acid sequence encodes for one member of the recombinant poly- clonal protein of interest.

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The term “mass transfer” is used to describe the transfer of nucleic acid-sequences of interest from one population of vectors to another population of vectors and doing so for each DNA ’ simultaneously without resorting to isolation of the individual DNA'’s of interest.. Such popula- tions of vectors can be libraries containing for example variable regions, promoters, leaders : 5 or enhancing elements of interest. These sequences can then be moved without prior isola- tion from for example a phage vector to a mammalian expression vector. Especially for anti- body sequences this technique ensures that the linkage between V,, and Vv, diversity Is not lost while moving libraries from, for example, a selection vector (e.g., a phage display vec- tor) to a mammalian expression vector. Hereby the original pairing of V,, and V. is retained.

The term “transfection” is herein used as a broad term for introducing foreign DNA into a cell.

The term is also meant to cover other functional equivalent methods for introducing foreign

DNA into a cell, such as e.g., transformation, infection, transduction or fusion of a donor cell and an acceptor cell.

The term “selection” is used to describe a method where cells have acquired a certain char- acteristic that enable the isolation from cells that have not acquired that characteristic. Such characteristics can be resistance to a Cytotoxic agent or production of an essential nutrient, enzyme, or color,

The terms “selectable marker gene”, “selection marker gene”, “selection gene” and “marker gene” are used to describe a gene encoding a selectable marker (e.g., a gene conferring re- sistance against some cytotoxic drug such as certain antibiotics, a gene capable of producing an essential nutrient which can be depleted from the growth medium, a gene encoding an enzyme producing analyzable metabolites or a gene encoding a colored protein which for example can be sorted by FACS ) which is co-introduced into the cells together with the gene(s) of interest.

The term “recombinant protein” is used to describe a protein that is expressed from a cell line transfected with an expression vector comprising the coding sequence of the protein, . As used herein, the term “operably linked” refers to a segment being linked to another seg- ment when placed into a functional relationship with the other segment. For example, DNA encoding a signal sequence is operably linked to DNA encoding a polypeptide if it is ex- pressed as a leader that participates in the transfer of the polypeptide to the endoplasmic reticulum. Also, a promoter or enhancer is operably linked to a coding sequence if it stimu- lates the transcription of the sequence.

The term “a majority of the individual cells” refers to a percentage of the cells such as more than 80%, preferably more than 85%, more preferably 90%, 95%, or even 99% or higher.

As used herein, the term “genome” is not to be taken literally as the normal complement of : chromosomes present in a cell, but also extra-chromosomal elements that can be introduced into and maintained in a cell. Such extra-chromosomal elements can include, but are not limited to, mini-chromosomes, YACs (yeast artificial chromosomes), MACs (mouse artificial chromosomes), or HACs (human artificial chromosomes).

The term “promoter” refers to a region of DNA involved in binding the RNA polymerase to initiate transcription.

The term “head-to-head promoters” refers to a promoter pair being placed in close proximity so that transcription of two gene fragments driven by the promoters occurs in opposite direc- tions. A head-to-head promoter can also be constructed with a stuffer composed of irrelevant nucleic acids between the two promoters. Such a stuffer fragment can easily contain more than 500 nucleotides.

An “antibiotic resistance gene” Is a gene encoding a protein that can overcome the Inhibitory or toxic effect that an antibiotic has on a cell ensuring the survival and continued proliferation of cells in the presence of the antibiotic,

The term “internal ribosome entry site” or “IRES” describes a structure different from the normal 5’ cap-structure on an mRNA. Both structures can be recognized by a ribosome to initiate scanning for an AUG codon to initiate translation. By using one promoter sequence and two initiating AUG’s, a first and a second polypeptide sequence can be translated from a single mRNA. Thus, to enable co-translation of a first and a second polynucleotide sequence from a single bi-cistronic mRNA, the first and second polynucleotide sequence can be tran- scriptionally fused via a linker sequence Including an IRES sequence that enables translation of the polynucleotide sequence downstream of the IRES sequence. In this case, a transcribed bi-cistronic RNA molecule will be translated from both the capped 5’ end and from the inter- nal IRES sequence of the bi-cistronic RNA molecule to thereby produce both the first and the second polypeptide.

The term “inducible expression” is used to describe expression that requires interaction of an inducer molecule or the release of a co-repressor molecule and a regulatory protein for ex- pression to take place.

The term “constitutive expression” refers to expression which is not usually inducible.

Claims

So PCT/DK2004/000001 CLAIMS

1. A method for generating a collection of cells suitable as a recombinant polyclonal manu- facturing cell line, said method comprising: a) providing a library of vectors comprising a population of variant nucleic acid 5S sequences, wherein each of said vectors comprises 1) one single copy of a distinct nucleic acid sequence encoding a distinct member of a polyclonal protein comprising distinct members that bind a particular antigen and 2) one or more recombinase recognition sequences; b) Introducing said library of vectors into a host cell line, wherein the genome of each individual cell of said host ceil line comprises recombinase recognition sequences, matching those of the vector, at a single specific site in its genome; c) ensuring the presence in said cells of one or more recombinases so that the variant nucleic acid sequences of step (a) are integrated site-specifically in the celis of the host cell line, where said one or more recombinases is/are either I) expressed by sald cells into which said nucleic acid sequence Is introduced; il) operatively encoded by the vectors of step a; iii) provided through expression from a.second vector; or iv) provided to the cell as a protein; and d) selecting cells comprising an integrated copy from sald library of variant nucleic acid sequences.

2. The method according to claim 1, wherein the polyclonal protein is not naturally associ- ateu with said collection of cells. :

3. The method according to claim 1 or 2, wherein said polyclonal protein is a polyclonal antibody or antibody fragment.

4. The method according to claim 1 or 2, wherein said polyclonal protein Is a polyclonal T ) 25 cell receptor or T cell receptor fragment.

5. The method according to any one of the preceding claims, wherein sald iibrary of vectors Is introduced into said host cell line by bulk transfection of a collection of said host cells with said library of vectors.

6. The method according to any one of claims 1-4, wherein said library of vectors is intro- duced into said host cell line by semi-bulk transfection of aliquots of said host cells with frac- tions comprising S to 50 individual vectors of said library of vectors, and said cells are pooled to form a collection of cells suitable as a recombinant polyclonal manufacturing cell line prior or subsequent to the selection of step (d). CLEAN COPY

PCT/DK2004/000001

7. The method according to any one of claims 1-4, wherein said library of vectors for site- specific integration is intraduced into said host cell line by transfecting said host cells sepa- rately with individual members of said library of vectors, and said cells are pooled to form a collection of cells suitable as a recombinant polyclonal manufacturing cell line prior or subse- quent to the selection of step (d).

8. The method according to any one of the preceding claims, wherein the population of vari- ant nucleic acids in step (a) are isolated or identifled by the aid of a screening procedure that enables identification and/or isolation of nucleic acids that encode protein which bind said particular antigen.

9. The method according to claim 8, wherein the screening procedure includes a biopanning step and/or an immunodetection assay.

10. The method according to claim 8 or 9, wherein said screening procedure is selected from the group consisting of phage display, ribosome display, DNA display, RNA-peptide display, covalent display, bacterial surface display, yeast surface display, eukaryotic virus display, ELISA and ELISPOT.

11. The method according to any one of the preceding claims, wherein said library of variant nucleic acid sequences comprises at least 3 variant nucleic acid sequences.

12. The method according to any one of the preceding daims, wherein individual members of said library of variant nucleic acid sequences are integrated in a single predefined genomic locus of individual cells in said collection of cells, said locus being capable of mediating high- level expression of each member of said recombinant polyclonal protein.

13. The method according to any cne of the preceding claims, wherein each distinct nucleic acid sequence comprises a pair of gene segments that encode a member of a polyclonal protein comprised of two different polypeptide chains.

14. The method according to claim 13, wherein said pair of gene segments comprise an anti- body heavy chain variable region encoding sequence and an antibody light chain variable region encoding sequence.

15. The method according to claim 13, wherein said pair of gene segments comprise a T cell receptor alpha chain variable region encading sequence and a T cell receptor beta chain vari- able region encoding sequence. CLEAN COPY

PCT/DK2004/000001 i 16. The method according to claim 13, wherein said pair of gene segments comprise a T cell receptor gamma chain variable region encoding sequence and a T cell receptor delta chain variable region encoding sequence.

17. The method according to any one of the preceding claims, wherein said library of variant S nudeic acid sequences comprises a naturally occurring diversity located within the variant nucleic acid sequences.

18. The method according to claim 17, wherein the naturally occurring diversity is located in CDR regions present in said variant nucleic acid sequences.

19. The method according to any one of the preceding daims, wherein said collection of cells is derived from a mammalian cell line or cell type.

20. The method according to claim 19, wherein said mammalian cell line is selected from the group consisting of Chinese hamster ovary (CHO) cells, COS cells, BHK cells, YB2/0, NIH 3T3, myeloma cells, fibroblasts, Hela, HEK 293, PER.C6, and cell lines derived thereof.

21. A method for the manufacture of a polyclonal protein, wherein said polyclonal protein comprises distinct members. that bind a particular antigen, said method comprising: a) providing a collection of cells comprising a library or variant nucleic acid sequences, where each of said nucleic acid sequences encode a distinct member of said polyclonal protein and where each of sald nucleic acid sequences are integrated at the same, single site of the genome of each individual cell In said collection of cells; b) culturing said collection of cells under conditions facilitating expression of said polyclonal protein; and c) recovering said expressed polyclonal protein from the cell culture cells or cell

. culture supernatant,

22. The method according to claims 21, wherein the collection of cells in step (a) Is gene- rated according to the method of any one of claims 1-20.

23. The methad according to claim 21 or 22, wherein the polyclonal protein is not naturally associated with said collection of cells.

24. The method according to any one of claims 21-23, wherein the library of variant nucleic acids in step (a) are isolated or identified in an eariler step by the aid of a screening proce- : 30 dure that enables identification and/or isolation of nucleic acids that encode protein which bind said particular antigen. CLEAN COPY

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25. The method according to claim 24, wherein the screening procedure includes a biopan- ning step and/or an immunodetection assay.

26. The method according to claim 24 or 25, wherein said screening procedure is selected from the group consisting of phage display, ribosome display, DNA display, RNA-peptide dlis- S play, covalent display, bacterial surface display, yeast surface display, eukaryotic virus dis- play, ELISA, and ELISPOT.

27. The method according to any one of claims 21-26, wherein said polyclonal protein is a polyclonal antibody or antibody fragment.

28. The method according to any one of claims 21-26, wherein said polyclonal protein is a polyclonal! T cell receptor or T cell receptor fragment.

29. The method according to any one of claims 21-28, wherein the relative expression levels of the variant nucleic acid sequences are monitored.

30. The method according to claim 29, wherein said expression levels are monitored at mRNA level and/or protein level. 1s

31. The method according to claim 29 or 30, wherein the culturing in step (b) is terminated at the latest when the relative expression levels are outside a predetermined range.

32. A recombinant polyclonal manufacturing cell line comprising a collection of cells trans- fected with a library of variant nucleic acid sequences, wherein each cell in the collection Is transfected with and capable of expressing one member of the library, which encodes a dis- tinct member of a polyclonal protein that binds a particular antigen and which is located at Co the same single site in the genome of individual cells In said collection, wherein said nucleic acid sequence is not naturally associated with said cell in the collection.

33. The recombinant polyctonal manufacturing cell line according to claim 32, wherein said library of variant nucleic acid sequences encodes a polyclonal antibody or antibody fragment having a naturally occurring diversity among the individual members of said polyclonal anti- body or antibody fragments.

34. The recombinant polyclonal manufacturing cell line according to claim 32, wherein said library of variant nucleic acid sequences encodes a polyclonal T cell receptor or T cell receptor CLEAN COPY

PCT/DK2004/000001 fragment having a naturally occurring diversity among the individual members of said polyclonal T cell receptor or T-cell receptor fragment.

35. The recombinant polyclonal manufacturing cell line according to any one of claims 32-34, wherein said collection of cells is derived from a mammalian cell fine or cell type.

36. The recombinant polyclonal manufacturing cell line according to claim 35, wherein said mammalian cell line is selected from the group consisting of Chinese hamster ovary (CHO) cells, COS cells, BHK cells, YB2/0, NIH 3T3, myeloma cells, fibroblasts, Hela, HEK 293, PER.CS6, and derivative cell lines thereof.

37. Alibrary of vectors for site-specific integration comprising a population of naturally occurring variant nucleic acid sequences, wherein each of said vectors comprises 1) one copy of a distinct nucleic acid sequence encoding a district member of a polyclonal protein that binds a particular antigen and 2) one or more recombinase recognition sequences.

38. The library according claim 37, wherein said population of naturally occurring variant nucleic acid sequences encode a polycional antibody or antibody fragment.

39. The library according claim 37, wherein said population of naturally occurring variant nucleic acid sequences encode a polyclonal T cell receptor T cell receptor fragment.

40. The library according to any one of claims 37-39, wherein each member of said library of vectors further comprises a recombinase encoding nucleic acid sequence.

41. A collection of cells comprising a library of variant nucleic acid sequences, where each of said nucleic acid sequences encodes a distinct member of a polyclonal protein comprising distinct members that bind a particular antigen and wherein each of said nucleic acid sequences is integrated at the same single site of the genome of each individual cell in said coliection of cells.

42. The collection of cells according to claim 41, wherein the library of variant nucleic acid sequences is a library according to any one of the claims 37-40.

43. The collection of cells according to claim 41 or 42, wherein at least 50% of the encoding sequences originally present in the library can be identified as different individual members of the final polyclonal! protein expressed from said collection of cells. AMENDED SHEET

. PCT/DK2004/000001

44. A polyclonal antibody expressing cell line transfected with a library of pairs of Vy and Vi gene segments, wherein each cell in the cell line is transfected with and capable of expressing one Vy and V, gene pair of the library, which encodes a distinct member of a polyclonal antibody that binds a particular antigen and which is located at the same single site in the genome of individual cells in said cell line, wherein said nucleic acid sequence is not naturally associated with said cell in the collection.

45. The cell line according to claim 44, wherein the library of pairs of Vy and V_gene segments is a library according to claim 38.

46. A method according to claim 1, or claim 21, substantially as herein described and illustrated.

47. A cell line according to claim 32, or claim 44, substantially as herein described and illustrated.

48. A library of vectors according to claim 37, substantially as herein described and illustrated.

49. A collection of cells according to claim 41, substantially as herein described and illustrated.

50. A new method for generating a collection of cells, a new method for manufacturing a protein, a new cell line, a new library of vectors, or a new collection of cells, substantially as herein described. AMENDED SHEET