WO2006106341A2 - Amorces ovines - Google Patents

Amorces ovines Download PDF

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Publication number
WO2006106341A2
WO2006106341A2 PCT/GB2006/001263 GB2006001263W WO2006106341A2 WO 2006106341 A2 WO2006106341 A2 WO 2006106341A2 GB 2006001263 W GB2006001263 W GB 2006001263W WO 2006106341 A2 WO2006106341 A2 WO 2006106341A2
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seq
primer set
antibody
pcr
isolation
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WO2006106341A3 (fr
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Karen Dorothy Cromie
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UCB SA
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UCB SA
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/005—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production

Definitions

  • the present invention relates primer sets and their use in the isolation of sheep antibody variable regions which are useful as reagents for the development of therapeutic, prognostic and diagnostic agents.
  • these antibodies may be humanised.
  • Hybridoma technology for the production of monoclonal antibodies is well- known and historically used mice as the immunised species.
  • Production of monoclonal antibodies requires immortalization of splenocytes by somatic fusion to a myeloma cell line partner to produce a hybridoma.
  • One disadvantage of hybridomas is that, although they can be immortal, their maintenance may depend on a feeder cell layer and they may be genetically unstable.
  • a second disadvantage is that only a fraction of B cells from a spleen population can be recovered as stable lines after the fusion and screening process. Since the inception of hybridoma technology (Kohler, G.
  • lymphocyte antibody method (Babcook et al, 1996, Proc. Natl. Acad. Sci, 93, 7843-7848; WO 92/02551; de Wildt et al., 1997, J. Immunol. Methods, 207:61-67 and in Lagerkvist, et al., 1995, BioTechniques 18(5):862-869) which enables cells producing high affinity antibodies generated during in vivo immune responses to be isolated from any species.
  • a major difficulty after that isolation then lies in the ability to obtain the antibody sequence, in particular the variable region sequences.
  • mice and rats due to the large number of possible variable region sequences, a considerable problem arises in that in order to ensure that all possible expressed variable region sequences will be represented, the utilisation of large numbers of primers, i.e. a high complexity primer set, is required.
  • the usual method for obtaining antibody sequence is to use primers designed to anneal to the mature nucleic sequence encoding the antibody, for example within framework 1; often large numbers of possible variable region sequences exist necessitating a large primer set to ensure coverage.
  • primer design is influenced by the mechanism by which antibody diversity is generated, hi particular, there is a need for sets of primers that will efficiently recover a large majority of possible expressed variable regions, ensuring cloning of said regions from a single, or small numbers of, B cells or other antibody producing cells without alteration to variable region sequences.
  • Sets of suitable primers that permit coverage of most or all of the possible expressed variable region sequences in the sheep have not been previously described. Indeed, a limited number of sheep antibody sequences are publicly available (see, for example, Charlton et ah, 2000, J. Immunol. 164:6221-6229, who use a bacteriophage library approach).
  • the invention described herein includes novel primer sets which provide coverage of a large majority of possible expressed variable regions in the sheep, permitting the isolation of a nucleic acid comprising a sheep antibody variable region sequence expressed by a single or low numbers of antibody producing cells, for example an antibody of interest from B cells, B cell lines, or other antibody producing cells, e.g. a hybridoma cell.
  • a nucleic acid comprising a sheep antibody variable region sequence expressed by a single or low numbers of antibody producing cells, for example an antibody of interest from B cells, B cell lines, or other antibody producing cells, e.g. a hybridoma cell.
  • oligonucleotide primer set comprising or consisting of:
  • the primer set comprises SEQ ID NO:1 (Set A).
  • the primer set comprises or consists of SEQ ID NOS: 1, 2 and 3 (Set B), and use of said primer set in a PCR enables the isolation of a nucleic acid comprising the variable heavy chain region sequence a sheep antibody heavy chain; and hence the corresponding polypeptide sequence of those regions.
  • Such nucleic acid sequences are of use in cloning the full length sequence of a sheep antibody heavy chain. They are also of use in designing a humanised antibody or fragment thereof.
  • Primers of SEQ ID NOS: 1, 4-6, 9-11, 13, 16-18 and 20-22 are forward primers; SEQ ID NOS:2, 3, 7, 8, 12, 14, 15, 19 and 22 are reverse primers.
  • a primer set as used herein includes a primer set comprising at least one primer.
  • the primer sets described herein are useful for the isolation of sheep antibody variable regions.
  • the amount of template will influence the selection of primer sets.
  • the template DNA is obtained from a single or low numbers of antibody producing cells, for example but not limited to, using reverse transcription of mRNA into cDNA, one may screen for the presence of template before committing the use of expensive reagents. Alternatively, no screen is performed.
  • a DNA template may be prepared using standard means known in the art, for example by cloning rearranged genomic DNA, or mRNA via cDNA (reviewed by e.g. Mountain and Adair, 1992, Biotech. Gen. Eng. Rev. 10:1-142; Ehlich and Kuppers 1995, Curr. Opin. Immunol. 7:281-284).
  • RNA obtained from a single antibody producing cell by lysis is used to prepare a DNA template. It may be preferable to culture the antibody producing cells to increase the cell number.
  • a single antibody producing cell e.g.
  • a B cell or a hybridoma cell is cultured to increase the cell number before preparation of cDNA from mRNA for use as a template for cloning in order to determine the sequence of, in particular, the variable heavy and light chain regions of a sheep antibody of interest, hi another embodiment, multiple antibody producing cells are used as a source for deriving template DNA, which cells may or may not be cultured to increase cell number, hi one embodiment, the multiple antibody producing cells are clonal. Alternatively, said cells are not clonal.
  • the invention also provides an oligonucleotide primer set comprising or consisting of:
  • the primer set comprises SEQ ID NOS:4 to 6 (Set C).
  • the oligonucleotide primer set comprises or consists of SEQ ID NOS :4 to 8 (Set D) and use of said primer set in a PCR enables the isolation of a nucleic acid comprising the variable region sequence of a sheep antibody kappa light chain; and hence the corresponding polypeptide sequence.
  • nucleic acid sequences are of use in cloning the full length sequence of a sheep antibody kappa light chain. They are also of use in designing humanised antibodies or fragments thereof.
  • the invention further provides primer sets designed for the isolation of nucleic acids comprising the variable regions of sheep antibody lambda chain sequences. Accordingly, provided is an oligonucleotide primer set comprising or consisting of: . (e) at least one primer selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11;
  • the oligonucleotide primer set comprises SEQ ID NO:9 to 11 (Set E).
  • the primer set comprises or consists of SEQ ID NOS:9 to 12 (Set F) and enables the isolation of a nucleic acid comprising the variable region sequence of a sheep antibody lambda light chain; and hence the corresponding polypeptide sequence.
  • Such nucleic acid sequences are of use in cloning the full length sequence of a sheep antibody lambda light chain sequence. They are also of use in designing humanised antibodies or fragments thereof.
  • the invention further provides oligonucleotide primer sets for performing nested PCR. These primer sets can alternatively be used to perform a first round PCR.
  • an oligonucleotide primer set comprising or consisting of:
  • any of the primer sets defined in (h), above, in a PCR enables the isolation of a nucleic acid comprising the variable region sequence of a sheep antibody heavy chain.
  • These primer sets have the following restriction sites incorporated: 5 '-Hind III (underlined), 5'-Apa I (bold and underlined) and 3'-Xho I (bold and italic). It will be understood by one skilled in the art that any restriction site desired may be incorporated into the primers, for example by replacing the restriction site encoded with coding for the desired restriction site. In another embodiment the upstream restriction site and bases for annealing to the vector may be omitted.
  • the primer set comprises SEQ ID NO: 13 (Set G).
  • the primer set comprises or consists of SEQ ID NO:13, SEQ ID NO: 14 and SEQ ID NO: 15 (Set H) and use of said primer set in a PCR enables the isolation of a nucleic acid comprising the variable region sequence of a sheep antibody heavy chain; and hence the corresponding polypeptide sequence.
  • nucleic acid sequences are of use in cloning full length sheep antibody heavy chain sequences. They are also of use in designing humanised antibodies or fragments thereof.
  • the invention also provides an oligonucleotide primer set comprising or consisting of: (i) at least one primer selected from the group consisting of SEQ ID NOS : 16,
  • primer sets defined in (i) to (k), above, in a PCR enables the isolation of a nucleic acid comprising the variable region sequence of a sheep antibody kappa light chain.
  • These primer sets have the following restriction sites incorporated: 5'-SfU I (italic), 5'-Hind III (underlined) and 3'-Bsi WI (underlined and italic). It will be understood by one skilled in the art that any restriction site desired may be incorporated into the primers, for example by replacing the restriction site encoded with coding for the desired restriction site.
  • the oligonucleotide primer set comprises SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 (Set I). Most preferably, the primer set comprises or consists of SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19 (Set J) and enables the isolation of a nucleic acid comprising the variable region sequence of a sheep antibody kappa light chain; and hence the corresponding polypeptide sequence. Such nucleic acid sequences are of use in cloning full length sheep antibody kappa light chain sequences. They are also of use in designing humanised antibodies or fragments thereof.
  • the invention further provides an oligonucleotide primer set comprising or consisting of:
  • any one of the primers defined in (1) to (n), above, in a PCR enables the isolation of a nucleic acid comprising the variable region sequence of a sheep antibody lambda light chain.
  • These primer sets have the following restriction sites incorporated: 5'-Sfu I (italic), 5'-Hind III (underlined) and 3'-Bsi WI (italic and underlined). It will be understood by one skilled in the art that any restriction site desired may be incorporated into the primers, for example by replacing the restriction site encoded with coding for the desired restriction site.
  • the oligonucleotide primer set comprises SEQ ID NO:20, SEQ ID NO.21 and SEQ ID NO:22 (Set K).
  • the primer set comprises or consists of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23 (Set L) enabling the isolation of a nucleic acid comprising the variable region sequence of a sheep antibody lambda light chain; and hence the corresponding polypeptide sequence of that region.
  • Such antibody regions isolated are useful for the cloning of the full sequence of a sheep antibody lambda light chain. They are also of use in designing humanised antibodies or fragments thereof.
  • primer sets Preferably, one primer set is used to isolate a nucleic acid comprising the variable heavy chain region sequence, and one primer set is used to isolate a nucleic acid comprising the variable light chain region sequence of a sheep antibody. Alternatively, two or more primer sets may be used.
  • any one of the sets defined in (a) and (h), above may be used in conjunction with any one of the sets defined in (b)-(d), or (i)-(k), above, enabling the isolation of a nucleic acid comprising the variable region sequences of a sheep antibody heavy chain and kappa light chain; and (2) any one of the sets defined in (a) and (h), above, may be used in conjunction with any one of the sets defined in (e)-(g) or (l)-(n), above, enabling the isolation of a nucleic acid comprising the variable region sequences of a sheep antibody heavy chain and lambda light chain.
  • primer set B or primer set H is used in conjunction with primer set D or J (or primer set F or L) 3 above, and even more preferably, primer set B is used in conjunction with primer set D (or primer set F) and primer set H is used in conjunction with primer set J (or primer set L) enables the isolation of nucleic acids comprising the variable region sequence of the heavy and light chain region sequences of a sheep antibody. Such nucleic acid sequences are of use in cloning full length sheep antibody heavy and light chain sequences.
  • the invention described herein also provides methods for the isolation of a nucleic acid comprising the variable region sequence of a sheep antibody heavy chain comprising use of an oligonucleotide primer set in a PCR, wherein the primer set comprises or consists of:
  • any one of the primer sets defined in (I), above is used in a PCR in conjunction with template derived from at least one antibody producing cell of interest, for example by reverse transcription of mRNA into cDNA.
  • the method comprises use of the oligonucleotide primer set consisting of Set B, defined above, in a PCR, preferably a first PCR, and enables the isolation of a nucleic acid comprising the variable region sequence of a sheep antibody heavy chain.
  • any of said primer sets defined in (I), above may be used in a second PCR, using template derived from a first PCR.
  • any one of the primer sets defined in (I), above can be used in a first and a second PCR.
  • the invention further provides a method for the isolation of a nucleic acid comprising the variable region sequence of a sheep antibody heavy chain comprising use of an oligonucleotide primer set in a PCR, wherein the primer set comprises or consists of:
  • any one of the primer sets defined in (II), above is used in a PCR.
  • the method comprises use of the primer set of Set H, defined above, in a PCR, most preferably a second PCR, using as template nucleic acid derived from a first PCR, said method enabling the isolation of a nucleic acid comprising the variable region sequence of a sheep antibody heavy chain.
  • any of the primer sets defined in (II), above is used in a first PCR in conjunction with template derived from at least one antibody producing cell of interest, for example by reverse transcription of mRNA into cDNA.
  • the primers set defined in (II), above can be used in a first and a second PCR.
  • the method comprises two PCRs; a first PCR and a second PCR using as template, DNA from the first PCR.
  • a method for the isolation of the variable region of a sheep antibody heavy chain comprising use of at least one primer set in a first PCR and at least one primer set in a second PCR, said first primer set comprising or consisting of any one of the primer sets defined in (I), above, and said second primer set comprising or consisting of any one of the primer sets defined in (II), above.
  • the method comprises use of primer Set B, defined above, in a first PCR and primer Set H, defined above, in a second PCR using as template, DNA from the first PCR.
  • Said method enables the isolation of a nucleic acid comprising the variable region sequence of a sheep antibody heavy chain.
  • said PCRs are performed individually.
  • said PCRs are performed jointly.
  • Such antibody regions are useful for the cloning of the full sequence of a sheep antibody heavy chain.
  • more than two PCRs may be used, for example, but not limited to three, four, five or more PCRs.
  • fewer numbers of cycles are used then one or more additional PCRs may be performed to produce the amplification required.
  • the invention described herein also provides methods for the isolation of a nucleic acid comprising the variable region sequence of a sheep antibody kappa light chain, said method comprising use of an oligonucleotide primer set in a PCR wherein the primer set comprises or consists of:
  • any one of the primer set defined in, (M)-(V), above is used in a PCR in conjunction with template derived from at least one antibody producing cell of interest, for example by reverse transcription of mRNA into cDNA.
  • the method comprises use of the oligonucleotide primer Set D, defined above, in a PCR, preferably a first PCR enabling the isolation of a nucleic acid comprising the variable region sequence of a sheep antibody kappa light chain.
  • any one of the primer sets defined in (HI)-(V), above may be used in a second PCR in conjunction with template derived from a first PCR.
  • any one of the primer sets defined in (HI)-(V), above can be used in a first and a second PCR.
  • a nucleic acid comprising the variable region sequence of a sheep antibody kappa light chain
  • said method comprising use of an oligonucleotide primer set in a PCR, wherein the primer set comprises or consists of:
  • any one of the primer sets defined in (VI)-(VIII), above is used in a PCR.
  • the method comprises use of the oligonucleotide primer Set I, defined above, in a PCR, preferably a second PCR and enables the isolation of a nucleic acid comprising the variable region sequence of a sheep antibody kappa light chain.
  • any of the primer sets defined in (VI)-(VIII), above is used in a first PCR in conjunction with template derived from at least one antibody producing cell of interest, for example by reverse transcription of mRNA into cDNA.
  • any one of said primer sets can be used in a first and a second PCR.
  • the method comprises two PCRs, a first PCR and a second PCR using as template, DNA from the first PCR. Accordingly, provided is a method for the isolation of a nucleic acid comprising the variable region sequence of a sheep antibody kappa light chain comprising use of at least one primer set in a first PCR and at least one primer set in a second PCR, said first primer set comprising or consisting of any one of the primer sets defined in (HI)-(V), above, and said second primer set comprising or consisting of any one of the primer sets defined in (VI)-(VIII), above.
  • the method comprises use of primer Set D, defined above, in a first PCR and primer Set J, defined above, in a second PCR, using as template, DNA from the first PCR enabling the isolation of a nucleic acid comprising the variable region sequence of a sheep antibody kappa light chain.
  • the PCRs are performed individually. Alternatively, said PCRs are performed jointly. Such antibody regions isolated are useful for the cloning of the full sequence of a sheep antibody kappa light chain.
  • more than two PCRs may be used, for example, but not limited to three, four, five or more PCRS. Thus, one skilled in the art will appreciate that if, for example, fewer numbers of cycles are used then one or more additional PCRs may be performed to produce the amplification required.
  • the invention described herein further provides a method for the isolation of a nucleic acid comprising the variable region sequence of a sheep antibody lambda light chain comprising use of an oligonucleotide primer set in a PCR, wherein the primer set comprises or consists of:
  • (IX) at least one primer selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11;
  • any one of the primer sets defined in (IX)-(XI), above, is used in a first PCR in conjunction with template derived from at least one antibody producing cell of interest, for example by reverse transcription of mRNA into cDNA.
  • the method comprises use of the oligonucleotide primer Set F, defined above, in a PCR, preferably a first PCR enabling the isolation of a nucleic acid comprising the variable region sequence of a sheep antibody lambda light chain.
  • any one of the primer sets defined in (DC)-(XI), above may be used in a second PCR in conjunction with template derived from a first PCR.
  • any one of said primer sets can be used in a first and a second PCR.
  • a nucleic acid comprising the variable region sequence of a sheep antibody lambda light chain
  • said method comprising use of an oligonucleotide primer set in a PCR, wherein the primer set comprises:
  • any one of the primer sets defined in (XII)-(XIV), above is used in a PCR.
  • the method comprises use of the oligonucleotide primer set L, defined above, in a PCR, most preferably a second PCR, using as template nucleic acid derived from a first PCR, said method enabling the isolation of a nucleic acid comprising the variable region sequence of a sheep antibody lambda light chain.
  • any one of the primer sets defined in (X ⁇ I)-(XrV), above is used in a first PCR in conjunction with template derived from at least one antibody producing cell of interest, for example by reverse transcription of mRNA into cDNA.
  • any one of said primer sets can be used in a first and a second PCR.
  • the method comprises two PCRs, a first PCR and a second PCR using as template, DNA from the first PCR. Accordingly, provided is a method for the isolation of a nucleic acid comprising the variable region sequence of a sheep lambda light chain comprising use of at least one primer set in a first PCR and at least one primer set in a second PCR, said first primer set comprising or consisting of the primer set defined in (IX)-(XI), above, and said second primer set comprising or consisting of any one of the primer sets defined in (XH)-(XIV), above.
  • the method comprises use of primer Set F, defined above, in a first PCR and primer Set L, defined above, in a second PCR, using as template, DNA from the first PCR enabling the isolation of a nucleic acid comprising the variable region sequence of a sheep antibody lambda light chain.
  • said PCRs are performed individually.
  • said PCRs are performed jointly.
  • Such antibody regions isolated are useful for the cloning of the full sequence of a sheep antibody lambda light chain.
  • more than two PCRs may be used, for example, but not limited to three, four, five or more PCRs.
  • fewer numbers of cycles are used then one or more additional PCRs may be performed to produce the amplification required.
  • the sequence of at least the variable regions of both the heavy and light chains of a sheep antibody can be determined by use of the primer sets and methods described above.
  • All methods described herein for the isolation of at least the variable region of a sheep antibody sequence comprise the performance of at least one PCR and provision of: 1. at least one primer set as provided herein;
  • the additional primer set as required can be a forward or a reverse primer set depending upon whether the primer set provided herein is a forward or a reverse primer set.
  • Such additional primer sets may be designed to anneal to any desired region of an antibody, particularly a sheep antibody. It will also be understood that suitable use of primer sets requires that forward and reverse primers are used.
  • Reverse transcription and PCR performance reagents include reagents as known in the art, such as without limitation, DNA polymerase, dNTPs, buffers such as but not limited toTris buffers, cations such as Mg2+, detergents, for example but not limited, to Nonidet NP-40, reducing agents such as dithiothreitol or mercaptoethanol and RNAasin.
  • any one of the primer Sets B, D, F, H, J and L provided herein are utilised in a single PCR.
  • suitable use means that one or more additional primers will be required.
  • one or more PCRs may be performed utilising a portion of the primers present within a primer set, for example but without limitation, one or two or more primers from any one of Sets A to L, plus additional primers where required, may be utilised in a single PCR.
  • one or more of the primer sets A, C, E, G, I or K (which are designed to complement the variable regions of an antibody, in particular a sheep antibody), or one or more primers comprising any of the sets, may be used in combination with primers designed to anneal to downstream regions of a nucleic acid encoding an antibody of interest.
  • one or more of the primer sets A, C, E, G, I or K, or one or more primers comprising any of the sets may be used for RACE, where the source cDNA can be homopolynucleotide tailed at the 3 '-end using terminal transferase enzyme, providing a location for strand synthesis using a complementary primer.
  • primers described herein can be made as desired.
  • sequence of one or more primers may be shortened or lengthened, hi particular, the restriction sites may be altered or removed altogether.
  • Suitable annealing temperatures for using such primers may be selected as known in the art, for example but without limitation, lowering the annealing temperature may allow less stringent priming and result in a PCR product where previously, at higher temperatures, no product was isolated.
  • antigens of interest can be used for immunisation of an animal, particularly a sheep.
  • antigens include any substance that can be recognised by an antibody, including proteins, glycoproteins and carbohydrates.
  • Antigens also include biologically active proteins, such as hormones, cytokines, and their cell surface receptors, bacterial or parasitic cell membranes or purified components thereof, and viral antigens.
  • Antigens may also include cells. Such cells may express endogenous proteins as antigens, for example, presented on the cell surface. Such cells may also transiently or stably express antigens.
  • Antigens of particular interest are those involved in diseases and which show aberrant expression or aberrant activity associated with a disease. Such diseases include cancers, inflammatory disorders and immune disorders.
  • Antibody sequences provided using the methods of the invention described herein include sequences of functionally active fragments, derivatives or analogues and may be, but are not limited to, polyclonal, monoclonal, bi-, tri- or tetra-valent antibodies, humanised or chimeric antibodies, single chain antibodies, Fab fragments, Fab' and Fab' 2 fragments, fragments produced by a Fab expression library, anti- idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
  • Humanised antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule (see e.g., US 5,585,089).
  • Antibodies include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules that contain an antigen binding site that specifically binds an antigen.
  • the immunoglobulin molecules of the invention can be of any class (e.g. IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulin molecule.
  • an antibody can be used therapeutically alone or in combination with a cytotoxic factor(s) and/or cytokine(s).
  • antibodies provided using the primer sets and methods of the invention can be conjugated to a therapeutic agent, such as a cytotoxic agent, a radionuclide or drug moiety to modify a given biological response.
  • a therapeutic agent such as a cytotoxic agent, a radionuclide or drug moiety to modify a given biological response.
  • the therapeutic agent is not to be construed as limited to classical chemical therapeutic agents.
  • the therapeutic agent may be a drug moiety which may be a protein or polypeptide possessing a desired biological activity.
  • Such moieties may include, for example and without limitation, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin, maytansinoid (DMl), a protein such as tumour necrosis factor, ⁇ -interferon, ⁇ - interferon, nerve growth factor, platelet derived growth factor or tissue plasminogen activator, a thrombotic agent or an anti-angiogenic agent, e.g.
  • a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin
  • a protein such as tumour necrosis factor, ⁇ -interferon, ⁇ - interferon, nerve growth factor, platelet derived growth factor or tissue plasminogen activator, a thrombotic agent or an anti-angiogenic agent, e.g.
  • angiostatin or endostatin angiostatin or endostatin; angiogenin, gelonin, dolstatins, minor groove-binders, bis-iodo-phenol mustard, or, a biological response modifier such as a lymphokine, interleukin-1 (IL- 1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF) or other growth factor.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • GM-CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • NGF nerve growth factor
  • Therapeutic agents also include cytotoxins or cytotoxic agents including any agent that is detrimental to (e.g. kills) cells.
  • cytotoxins or cytotoxic agents including any agent that is detrimental to (e.g. kills) cells.
  • examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vinca alkaloids, e.g.
  • Therapeutic agents also include, but are not limited to, anti-folates (e.g. aminopterin and methotrexate), antimetabolites (e.g.
  • methotrexate 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine, 5-fluoro-2'-deoxyuridine
  • alkylating agents e.g. mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU)
  • cyclothosphamide busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin
  • anthracyclines e.g.
  • daunorubicin (formerly daunomycin) and doxorubicin, adriamycin, idarubicin, morpholinodoxorubicin, epirubicin, doxorubicin hydrazides), antibiotics (e.g. dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC), calicheamicins or duocarmycins, CC- 1065, enediyenes, neocarzinostatin), and anti-mitotic agents (e.g. vincristine and vinblastine). See Garnett, 2001, Advanced drug Delivery Reviews 53:171-216 for further details.
  • antibiotics e.g. dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC), calicheamicins or duocarmycins, CC- 1065, enediyenes, neocarzin
  • therapeutic moieties may include radionuclides such as 131 1, 111 In and 90 Y, Lu 177 , Bismuth 213 , Bismuth 212 , Californium 252 , Iridium 192 and
  • the antibody sequences provided using the primer sets and methods of the invention can be modified, for example but without limitation, by the covalent attachment of any type of molecule. Preferably, said attachment does not impair immunospecific binding.
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate (see US 4,676,980).
  • the antibody sequences provided using the primer sets and methods of the invention are provided as fusion proteins of the antibodies (or functionally active fragments thereof), for example but without limitation, where the antibody or fragment thereof is fused via a covalent bond (e.g. a peptide bond), at optionally the N-terminus or the C-terminus, to an amino acid sequence of another protein (or portion thereof; preferably at least a 10, 20 or 50 amino acid portion of the protein).
  • a covalent bond e.g. a peptide bond
  • the antibody, or fragment thereof is linked to the other protein at the N-terminus of the constant domain of the antibody.
  • an antibody fusion protein may facilitate depletion or purification of a polypeptide as described herein, increase half-life in vivo, and enhance the delivery of an antigen across an epithelial barrier to the immune system.
  • the fusion protein is an antibody fragment linked to an effector or reporter molecule, this may be prepared by standard chemical or recombinant DNA procedures.
  • a preferred effector group is a polymer molecule, which may be attached to the modified Fab fragment to increase its half-life in vivo.
  • the polymer molecule may, in general, be a synthetic or a naturally occurring polymer, for example an optionally substituted straight or branched chain polyalkylene, polyalkenylene or polyoxyalkylene polymer or a branched or unbranched polysaccharide, e.g. a homo- or hetero- polysaccharide.
  • Particular optional substituents which may be present on the above-mentioned synthetic polymers include one or more hydroxy, methyl or methoxy groups.
  • Particular examples of synthetic polymers include optionally substituted straight or branched chain poly(ethyleneglycol), poly(propyleneglycol) poly(vinylalcohol) or derivatives thereof, especially optionally substituted poly(ethyleneglycol) such as methoxypoly(ethyleneglycol) or derivatives thereof.
  • Particular naturally occurring polymers include lactose, amylose, dextran, glycogen or derivatives thereof.
  • Derivatives as used above is intended to include reactive derivatives, for example thiol-selective reactive groups such as maleimides and the like.
  • the reactive group may be linked directly or through a linker segment to the polymer. It will be appreciated that the residue of such a group will in some instances form part of the product as the linking group between the antibody fragment and the polymer.
  • the size of the polymer maybe varied as desired, but will generally be in an average molecular weight range from 500Da to 50000Da, preferably from 5000 to 40000Da and more preferably from 25000 to 40000Da.
  • the polymer size may in particular be selected on the basis of the intended use of the product.
  • a small molecular weight polymer for example with a molecular weight of around 5000Da.
  • a higher molecular weight polymer for example having a molecular weight in the range from 25000Da to 40000Da.
  • Particularly preferred polymers include a polyalkylene polymer, such as a poly(ethyleneglycol) or, especially, a methoxypoly(ethyleneglycol) or a derivative thereof, and especially with a molecular weight in the range from about 25000Da to about 40000Da.
  • a polyalkylene polymer such as a poly(ethyleneglycol) or, especially, a methoxypoly(ethyleneglycol) or a derivative thereof, and especially with a molecular weight in the range from about 25000Da to about 40000Da.
  • Each polymer molecule attached to the modified antibody fragment may be covalently linked to the sulphur atom of a cysteine residue located in the fragment.
  • the covalent linkage will generally be a disulphide bond or, in particular, a sulphur- carbon bond.
  • the antibody fragment may have one or more effector or reporter molecules attached to it.
  • the effector or reporter molecules may be attached to the antibody fragment through any available amino acid side-chain or terminal amino acid functional group located in the fragment, for example any free amino, imino, hydroxyl or carboxyl group.
  • An activated polymer may be used as the starting material in the preparation of polymer-modified antibody fragments as described above.
  • the activated polymer may be any polymer containing a thiol reactive group such as an ⁇ -halocarboxylic acid or ester, e.g. iodoacetamide, an imide, e.g. maleimide, a vinyl sulphone or a disulphide.
  • a thiol reactive group such as an ⁇ -halocarboxylic acid or ester, e.g. iodoacetamide, an imide, e.g. maleimide, a vinyl sulphone or a disulphide.
  • Such starting materials may be obtained commercially, for example from Nektar Therapeutics, Inc (Huntsville, AL), or may be prepared from commercially available starting materials using conventional chemical procedures.
  • Standard chemical or recombinant DNA procedures in which the antibody fragment is linked either directly or via a coupling agent to the effector or reporter molecule either before or after reaction with the activated polymer as appropriate may be used.
  • Particular chemical procedures include, for example, those described in WO 93/06231, WO 92/22583, WO 90/09195, WO 89/01476, WO 99/15549 and
  • the linkage may be achieved using recombinant DNA procedures, for example as described in WO 86/01533 and EP 0392745.
  • Fab fragment is PEGylated, i.e. has PEG
  • a PEG modified Fab fragment has a maleimide group covalently linked to a single thiol group in a modified hinge region.
  • a lysine residue may be covalently linked to the maleimide group.
  • To each of the amine groups on the lysine residue may be attached a methoxypoly(ethyleneglycol) polymer having a molecular weight of approximately 20,000 Da. The total molecular weight of the entire effector molecule may therefore be approximately 40,000 Da.
  • the invention includes an antibody or fragment thereof isolated according to any one of the methods described, above. Repertoires of such antibodies, or fragments thereof, are also useful in bacteriophage libraries. Such antibodies are particularly useful as therapeutic agents, hi one embodiment, antibodies isolated using the methods of the invention are humanised' (see, for example, Adair et al, 1992, Immunol Rev. 130:5-40 and WO91/09967). The antibodies of the invention are also of use as diagnostic and prognostic reagents. Thus, further provided is the use of an antibody for the manufacture of a medicament for the treatment and/or prophylaxis of a disease involved in aberrant expression or activity of an antigen recognised by said antibody.
  • Antibodies isolated according to the methods of the invention are also useful in diagnosis.
  • a method of screening for and/or diagnosis or prognosis of a disease in a subject, and/or monitoring the effectiveness of therapy for said disease which comprises the step of detecting and/or quantifying in a biological sample obtained from said subject, the expression of an antigen recognised by an antibody isolated according to the methods of the invention.
  • the step of detecting comprises contacting the sample with the antibody and detecting whether binding has occurred between the antibody and the antigen in the sample.
  • Preferred features of each embodiment of the invention are as for each of the other embodiments mutatis mutandis. All publications, including but not limited to patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.
  • a sheep was immunised with an antigen, X.
  • B cells were collected and screened for production of antibody recognising antigen X. A single B cell was selected.
  • cDNA was prepared by reverse transcription using a SuperscriptTM III Reverse Transcriptase kit (Invitrogen cat.# 18080-044) and RNasin, an RNAase Inhibitor (Promega cat. # N2511). The following master mix (20 ⁇ l) was added directly to the selected single sheep B cell:
  • Oligo dTVx primers were as follows: tttttttttttttttttttttttttttttttva (SEQ ID NO:24), tttttttttttttttttttttttvg (SEQ ID NO:25), tttttttttttttttttttttvc (SEQ ID NO:26), and ttttttttttttttttttttttttttttvt (SEQ ID NO:27), where v is a, g, or t. RT-PCR was performed at 50°C for 60min followed by 70°C for 15min.
  • Primary PCR fragments were prepared by adding 2 ⁇ l of the cDNA and the appropriate primary primers in conjunction with a TaqPlus Precision PCR system (Stratagene Cat. No. # 600211) mix in a total volume of 50 ⁇ l as described below: dH 2 O 37.5 ⁇ l 1OX Precision PCR buffer 5 ⁇ l
  • Secondary PCR fragments were prepared by adding 2 ⁇ l of the primary PCR product and the appropriate primary primers in conjunction with a TaqPlus Precision PCR system (Stratagene cat No. # 600211) mix in a total volume of 50 ⁇ l as described below: dH 2 O ⁇ 33.5 ⁇ l
  • V H (Set G) or V L (Set K) at 0.25 ⁇ M 4 ⁇ l
  • V H SEQ ID NOS: 14 & 15
  • V L SEQ ID NO: 19
  • variable region (V H and Vu ⁇ pa ) of the antibody recognising antigen X was confirmed by running a 5 ⁇ l sample of the product on a 2%w/v agarose gel.
  • V H and V L The DNA product present in each case (V H and V L ) was approximately 450bp and was cloned into an expression vector using the Hind III/Xho I/Bsi WI/Sfu I/Apa I restriction sites.

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Abstract

L'invention concerne des ensembles d'amorces destinés à isoler des séquences régionales variables de l'anticorps ovin. L'invention concerne également des anticorps comprenant ces régions qui sont utiles dans le développement de thérapeutiques et, plus précisément, d'anticorps humanisés.
PCT/GB2006/001263 2005-04-06 2006-04-05 Amorces ovines Ceased WO2006106341A2 (fr)

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GB0507042A GB0507042D0 (en) 2005-04-06 2005-04-06 Primers
GB0507042.0 2005-04-06

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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GONTIER E ET AL: "Developmental progression of immunoglobulin heavy chain diversity in sheep" VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY, AMSTERDAM, NL, vol. 103, no. 1-2, 10 January 2005 (2005-01-10), pages 31-51, XP004697475 ISSN: 0165-2427 *
JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 15 JUN 2000, vol. 164, no. 12, 15 June 2000 (2000-06-15), pages 6221-6229, XP002387522 ISSN: 0022-1767 *
LI Y ET AL: "Sheep monoclonal antibody fragments generated using a phage display system" JOURNAL OF IMMUNOLOGICAL METHODS, ELSEVIER SCIENCE PUBLISHERS B.V.,AMSTERDAM, NL, vol. 236, no. 1-2, March 2000 (2000-03), pages 133-146, XP004190753 ISSN: 0022-1759 *

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