HK1088359A

HK1088359A - Modified antibodies stably produced in milk and methods of producing same

Info

Publication number: HK1088359A
Application number: HK06108514.4A
Authority: HK
Inventors: H.M.米德; E．伯克-威尔森; D．普洛克
Original assignee: Gtc生物治疗学公司
Priority date: 2002-11-27
Filing date: 2003-11-26
Publication date: 2006-11-03

Description

Modified antibody stably produced in milk and preparation method thereof

Technical Field

The invention provides methods for producing antibodies in the milk of transgenic mammals. The method comprises providing a transgenic mammal whose somatic and germ cells have coding sequences encoding at least one heavy and one light chain and at least one hinge region, wherein the hinge region has been altered from the hinge region normally associated with the heavy chain constant region to improve the stability and folding properties of the resulting recombinant antibody.

Background

IgG is the most abundant isotype antibody in adult serum, and constitutes about 80% of total serum immunoglobulin. IgG is a monomeric molecule with a quaternary structure, consisting of two immunoglobulins^Pu heavy chain and two immunoglobulin light chains: (^P2 or^SE) And (4) forming. The light and heavy immunoglobulin chains are typically interconnected by disulfide bonds. The antibody additionally includes a proline-rich hinge region, which confers flexibility to the fragment of the molecule. IgG demonstrates multiple biological functions including antigen agglutination, opsonization, antibody-dependent cell-mediated cytotoxicity, crossing the placenta, complement activation, neutralizing toxins, immobilizing bacteria, and neutralizing viruses.

Due to the lack of effector functions, IgG4 may be useful as a therapeutic agent. Unfortunately, the IgG4 antibody has labile properties under acidic treatment or non-reducing polyacrylamide gel electrophoresis (PAGE), and can produce an 80kDa protein (also known as a "half molecule"). A half molecule would be produced if there were no disulfide bonds linking the two heavy chains together.

The production of IgG4 in tissue culture has been variously successful. Depending on the cell line, the percentage change of "half molecule" IgG4 ranged from 5-25%. One of the difficulties in generating IgG4 molecules is the lack of convenient methods for separating half-molecules from the intact IgG4 molecule. Many production units simply believe that different levels of impurity "half-molecules" will be produced during processing.

Summary of The Invention

The present invention is based, in part, on the discovery that production of antibodies in the milk of transgenic animals results in up to 50% of the resulting antibody being in a semi-molecular form, while by modifying the hinge region of the antibody, increased levels of assembled antibody can be obtained in the milk of the animal. While not intending to be bound by theory, the increased levels of the half molecule found in the milk of transgenic animals may be due in part to the inability of the mammary gland to fold the antibody properly and/or to form disulfide bonds between the heavy chains, but still provide efficient secretion. By modifying the hinge region of such antibodies, the half-molecule level is reduced.

Accordingly, one aspect of the invention features a method of producing an antibody in the milk of a transgenic mammal. The method comprises providing a transgenic mammal whose somatic and germ cells have coding sequences encoding an exogenous heavy chain variable region or antigen-binding fragment thereof, at least one heavy chain constant region or fragment thereof, and a hinge region operably linked to a promoter that directs expression in mammary epithelial cells, wherein the hinge region has been altered from the hinge region normally associated with the heavy chain constant region.

In one embodiment, at least 70%, 75%, 80%, 90% or 95% of the antibodies present in the milk are in assembled form. In another embodiment, the somatic and germ cells of the transgenic mammal additionally comprise sequences encoding a light chain variable region, or antigen-binding fragment thereof, and a light chain constant region, or functional fragment thereof, operably linked to a promoter that directs expression in mammary epithelial cells.

In other embodiments, the method comprises the step of obtaining milk from the transgenic mammal to provide the antibody composition. In addition, the method may include the step of purifying the exogenous antibody from the milk.

The promoter used may be any promoter known in the art to direct expression of mammary epithelial cells, such as a casein promoter, a whey protein promoter, a beta lactoglobulin promoter, or a whey acidic protein promoter. In a preferred embodiment, the transgenic animal may be, for example, cattle, goats, mice, rats, sheep, pigs and rabbits.

The antibody may be any antibody from any antibody class, such as IgA, IgD, IgM, IgE or IgG, or fragments thereof. In a preferred embodiment, the antibody is an IgG antibody, such as an IgG1, IgG2, IgG3, or IgG4 antibody. In another preferred embodiment, the antibody is an IgG4 antibody.

The invention encompasses various alterations of the hinge region of an antibody. For example, in one embodiment, all or part of the hinge region of the antibody is modified. In another embodiment, all or part of the hinge region of the antibody is replaced, for example by a hinge region or part thereof which is different from the hinge region normally associated with the heavy chain constant and/or variable region. In a preferred embodiment, the hinge region of an antibody having an IgG antibody heavy chain constant region or portion thereof may be replaced with a hinge region or portion thereof of an antibody other than an IgG antibody. For example, the hinge region or portion thereof of an IgG antibody (e.g., an IgG1, IgG2, IgG3, or IgG4 antibody) can be replaced with a hinge region or portion thereof derived from an IgA, IgD, IgM, IgE antibody. In another embodiment, the hinge region or portion thereof of an antibody having an IgG antibody (e.g., an IgG1, IgG2, IgG3, or IgG4 antibody) heavy chain constant region or portion thereof can be replaced with a hinge region or portion thereof derived from another IgG antibody, e.g., the hinge region of an IgG1, IgG2, IgG3, or IgG4 antibody can be replaced with a hinge region derived from another IgG subclass. In yet another preferred embodiment, the hinge region of an antibody having the constant region of the heavy chain of the IgG4 antibody may be replaced with a hinge region derived from IgG1, IgG2, or IgG 3.

In yet another embodiment, the hinge region has been modified such that at least one nucleic acid residue of the nucleic acid sequence encoding the hinge region of the antibody differs from the naturally occurring nucleic acid sequence of the hinge region normally associated with the constant region of a heavy chain of the antibody. In another embodiment, the amino acid sequence of the hinge region of the antibody differs from the amino acid sequence of the hinge region naturally occurring with the constant region of a heavy chain of the antibody by at least one amino acid residue.

In a preferred embodiment, the hinge region has been modified such that one or more amino acids of the hinge region naturally associated with the heavy chain constant region are substituted with amino acids at positions corresponding to the hinge region associated with the heavy chain constant region of a different class or subclass of antibody. Preferably, the heavy chain constant region of the antibody produced is from an IgG antibody and the hinge region is substituted with one or more amino acids of the IgA, IgD, IgM or IgE antibody hinge region. In another preferred embodiment, the heavy chain constant region of the antibody produced is from an IgG antibody, e.g., an IgG4 antibody, and the hinge region is substituted with one or more amino acids of the hinge region of a different subclass of antibody (e.g., IgG1, IgG2, and IgG3 antibodies).

In another embodiment, at least one amino acid of the hinge region other than a cysteine residue may be replaced with a cysteine residue. The modification may comprise altering at least one glycosylation site of the antibody, for example in the heavy or light chain, or heavy chain hinge region of the antibody.

In another embodiment, the heavy chain constant region of the antibody prepared is from an IgG4 antibody, and the serine residues of the hinge region may be replaced with proline residues. For example, the serine residue at amino acid number 241 of the hinge region can be replaced with a proline residue.

The antibody may be, for example, a chimeric, human or humanized antibody or fragment thereof.

In another embodiment, the milk of the transgenic mammal is substantially free of a half-molecule form of the exogenous antibody. Preferably, the ratio of assembled exogenous antibody to half-molecular form antibody present in the milk of the transgenic mammal is at least 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1 or higher (e.g., 20: 1).

In another aspect, the invention features a method of making a transgenic mammal whose somatic cell-colonizing cells include a modified antibody coding sequence, wherein the modified antibody coding sequence encodes an antibody molecule or portion thereof having an altered hinge region. The method comprises the step of introducing into the mammal a construct comprising sequences encoding an exogenous heavy chain variable region or antigen-binding fragment thereof, at least one heavy chain constant region or fragment thereof, and a hinge region, operably linked to a promoter that directs expression in mammary epithelial cells, wherein the hinge region has been altered from the hinge region normally associated with the heavy chain constant region of the antibody produced. In one embodiment, the hinge region has been altered such that at least 70%, 75%, 80%, 85%, 90%, 95% of the exogenous antibody present in the milk of the transgenic mammal is in assembled form. In another embodiment, the construct comprises sequences encoding a light chain variable region, or antigen binding fragment thereof, and a light chain constant region, or functional fragment thereof, operably linked to a promoter that directs expression in mammary epithelial cells.

The invention encompasses various alterations of the hinge region of an antibody. For example, in one embodiment, all or part of the hinge region of the antibody is modified. In another embodiment, all or part of the hinge region of the antibody is replaced, for example by a hinge region or part thereof which is different from the hinge region normally associated with the heavy chain constant and/or variable region. In a preferred embodiment, the heavy chain constant region or part thereof is from an IgG and the hinge region of the antibody may be replaced with the hinge region or part thereof of an antibody other than an IgG antibody. For example, the hinge region or portion thereof of an IgG antibody (e.g., an IgG1, IgG2, IgG3, or IgG4 antibody) can be replaced with a hinge region or portion thereof derived from an IgA, IgD, IgM, IgE antibody. In another embodiment, the hinge region or portion thereof of an antibody having an IgG antibody (e.g., an IgG1, IgG2, IgG3, or IgG4 antibody) heavy chain constant region or portion thereof can be replaced with a hinge region or portion thereof derived from another IgG antibody, e.g., the hinge region of an IgG1, IgG2, IgG3, or IgG4 antibody can be replaced with a hinge region derived from another IgG subclass. In yet another preferred embodiment, the hinge region of an antibody having the constant region of the heavy chain of the IgG4 antibody may be replaced with a hinge region derived from IgG1, IgG2, or IgG 3.

In a preferred embodiment, the hinge region has been modified such that one or more amino acids of the hinge region naturally associated with the heavy chain constant region are substituted with amino acids at positions corresponding to the hinge region associated with the heavy chain constant region of a different class or subclass of antibody. Preferably, the heavy chain constant region of the antibody produced is from an IgG antibody and the hinge region is substituted with one or more amino acids of the IgA, IgD, IgM or IgE antibody hinge region. In another embodiment, the heavy chain constant region of the antibody produced is from an IgG antibody, e.g., an IgG4 antibody, and the hinge region is substituted with one or more amino acids of the hinge region of a different antibody species (e.g., IgG1, IgG2, and IgG3 antibodies).

In another embodiment, the heavy chain constant region of the antibody prepared is from an IgG4 antibody, and the serine residues of the hinge region may be replaced with proline residues. For example, the serine residue at amino acid number 241 of the hinge region of an IgG4 antibody can be replaced with a proline residue.

In another embodiment, the milk of the transgenic mammal is substantially free of a half-molecule form of the exogenous antibody. Preferably, the ratio of assembled exogenous antibody to half-molecular form antibody present in the milk of the transgenic mammal is at least 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1 or higher (e.g., 20: 1). In a preferred embodiment, the hinge region has been altered such that at least 70%, 75%, 80%, 85%, 90%, 95% of the exogenous antibodies present in the milk of the transgenic mammal are in assembled form.

The present invention encompasses all methods known to those skilled in the art for introducing antibody coding sequences into transgenic animals. For example, coding sequences encoding portions of the antibody (e.g., heavy chain variable region, light chain variable region, heavy chain constant region, light chain constant region, etc.) can be introduced as separate constructs under the control of separate promoters, e.g., separate promoters that control expression in mammary epithelial cells. The individual promoters may be the same type of mammary epithelial promoter (e.g., both constructs include a casein promoter) or different types of mammary epithelial promoters (e.g., one construct includes a casein promoter and the other includes a beta-lactoglobulin promoter). Thus, in a related embodiment, the invention provides a method of making a transgenic mammal capable of expressing an assembled exogenous antibody or portion thereof in its milk, comprising the steps of: introducing into a mammal a construct comprising a sequence encoding a light chain of an exogenous antibody linked to a promoter that directs expression in mammary epithelial cells, and introducing into a mammal a construct comprising a sequence encoding a heavy chain of a mutated exogenous antibody or a portion thereof linked to a promoter that directs expression in mammary epithelial cells. In another embodiment, the construct comprises a sequence encoding a mutated heavy chain and sequences encoding a light chain variable region or antigen-binding fragment thereof and a light chain constant region or functional fragment thereof. The sequence encoding the mutated heavy chain and the sequence encoding the light chain or a portion thereof are operably linked to, or placed under the control of, different promoters that direct expression in mammary epithelial cells. For example, the modified antibody coding sequence may be polycistronic, e.g., the heavy chain coding sequence and the light chain coding sequence may have an Internal Ribosome Entry Site (IRES) between them. When under the control of a separate promoter, the promoters may be under the control of the same type of mammary epithelial cell promoter (e.g., the sequences are all under the control of a beta-casein promoter) or each under the control of a different type of mammary epithelial cell promoter (e.g., one sequence is under the control of a beta-casein promoter and another sequence is under the control of a beta-lactoglobulin promoter).

In another embodiment, the invention provides a method of making a transgenic mammal capable of expressing an assembled exogenous antibody in its milk, the method comprising the steps of: providing cells from a transgenic mammal whose somatic and germ cells comprise a coding sequence for a light chain of an exogenous antibody operably linked to a promoter that directs expression in mammalian epithelial cells and introducing into the cells a construct comprising a coding sequence for a mutated heavy chain of the exogenous antibody or a portion thereof operably linked to the promoter that directs expression in mammalian epithelial cells, wherein the heavy chain or portion thereof comprises a hinge region that is different from the hinge region normally associated with the constant region of the heavy chain. In another embodiment, the invention provides a method of making a transgenic mammal capable of expressing an assembled exogenous antibody in its milk, comprising the steps of: providing cells from a transgenic mammal whose somatic and germ cells comprise a coding sequence for a mutated heavy chain of an exogenous antibody or a portion thereof operably linked to a promoter that directs expression in mammalian epithelial cells, and introducing into the cells a construct comprising a coding sequence for a light chain of an exogenous antibody operably linked to a promoter that directs expression in mammalian epithelial cells.

In another aspect, the invention features a transgenic mammal capable of expressing an exogenous antibody in milk, wherein somatic and germ cells of the transgenic mammal include a modified antibody coding sequence encoding an exogenous heavy chain variable region or antigen-binding fragment thereof, at least one heavy chain constant region or fragment thereof, and a hinge region, operably linked to a promoter that directs expression in mammary epithelial cells, wherein the hinge region has been altered from the hinge region normally associated with the heavy chain constant region of the antibody produced.

The promoter used may be any promoter known in the art to direct expression in mammary epithelial cells, such as a casein promoter, a whey protein promoter, a beta lactoglobulin promoter, or a whey acidic protein promoter. In a preferred embodiment, the transgenic animal may be, for example, cattle, goats, mice, rats, sheep, pigs and rabbits.

In a preferred embodiment, the hinge region has been modified such that one or more amino acids of the hinge region naturally associated with the heavy chain constant region are substituted with amino acids at positions corresponding to the hinge region associated with the heavy chain constant region of a different class or subclass of antibody. Preferably, the heavy chain constant region of the antibody produced is from an IgG antibody and the hinge region is substituted with one or more amino acids of the IgA, IgD, IgM or IgE antibody hinge region. More preferably, the heavy chain constant region of the antibody produced is from an IgG antibody, such as an IgG4 antibody, and the hinge region is substituted with one or more amino acids of the hinge region of a different antibody species (e.g., IgG1, IgG2, and IgG3 antibodies).

In a preferred embodiment, the hinge region has been altered such that at least 70%, 75%, 80%, 85%, 90%, 95% of the exogenous antibody present in the milk of the transgenic mammal is in assembled form. In another embodiment, the modified antibody coding sequence further comprises sequences encoding a light chain variable region or antigen binding fragment thereof and a light chain constant region or functional fragment thereof. The light chain variable region or antigen-binding fragment thereof and the light chain constant region or functional fragment thereof may be operably linked to a promoter that directs expression in mammary epithelial cells or under the control of the same promoter as the exogenous heavy chain variable region, heavy chain constant region (or portion thereof) and hinge region coding sequences. For example, the modified antibody coding sequence may be polycistronic, e.g., having an Internal Ribosome Entry Site (IRES) between both the heavy chain coding sequence and the light chain coding sequence.

In another aspect, the invention provides a composition comprising a milk component and an antibody component as described herein. Preferably, at least 70%, 75%, 80%, 85%, 90%, 95% of the exogenous antibody is in assembled form. In another embodiment, the hinge region has been altered such that at least 70%, 75%, 80%, 85%, 90%, 95% of the exogenous antibody present in the composition is in assembled form.

In another preferred embodiment, the composition is substantially free of milk components, e.g., less than 10%, 5%, 3%, 2%, 1%, 0.5%, 0.2% by weight of one or more milk components by volume. Examples of milk components include casein, lipids (e.g., soluble lipids and phospholipids), lactose and other small molecules (e.g., galactose, glucose), small peptides (e.g., microbial peptides, anti-biological peptides), and other milk proteins (e.g., whey proteins such as beta-lactoglobulin and alpha-lactalbumin, lactoferrin (lactoferrin), and serum albumin).

In another aspect, the invention provides a nucleic acid comprising a coding sequence encoding a heavy chain variable region or antigen-binding portion thereof, a heavy chain constant region or fragment thereof, and a hinge region, operably linked to a promoter that directs expression in mammary epithelial cells, wherein the hinge region has been altered from the hinge region normally associated with the heavy chain constant region.

The promoter used may be any promoter known in the art to direct expression of mammary epithelial cells, such as a casein promoter, a whey protein promoter, a beta lactoglobulin promoter, or a whey acidic protein promoter. The heavy chain variable region or antigen-binding portion thereof, the heavy chain constant region or fragment thereof, and the hinge region may be from any antibody of any antibody class, for example IgA, IgD, IgM, IgE, or IgG or fragments thereof. In a preferred embodiment, the antibody is an IgG antibody, such as an IgG1, IgG2, IgG3, or IgG4 antibody. In another preferred embodiment, the antibody is an IgG4 antibody.

The present invention encompasses various alterations of the hinge region. For example, in one embodiment, all or part of the hinge region is modified. In another embodiment, all or part of the hinge region is replaced, e.g., replaced with a hinge region or part thereof different from the hinge region normally associated with the heavy chain constant and/or variable region. In a preferred embodiment, the hinge region of an antibody having an IgG antibody heavy chain constant region or portion thereof may be replaced with a hinge region or portion thereof of an antibody other than an IgG antibody. For example, the hinge region or portion thereof of an IgG antibody (e.g., an IgG1, IgG2, IgG3, or IgG4 antibody) can be replaced with a hinge region or portion thereof derived from an IgA, IgD, IgM, IgE antibody. In another embodiment, the hinge region or portion thereof of an antibody having an IgG antibody (e.g., an IgG1, IgG2, IgG3, or IgG4 antibody) heavy chain constant region or portion thereof can be replaced with a hinge region or portion thereof derived from another IgG antibody, e.g., the hinge region of an IgG1, IgG2, IgG3, or IgG4 antibody can be replaced with a hinge region derived from another IgG subclass. In yet another preferred embodiment, the hinge region of an antibody having the constant region of the heavy chain of the IgG4 antibody may be replaced with a hinge region derived from IgG1, IgG2, or IgG 3.

In yet another embodiment, the hinge region has been modified such that at least one nucleic acid residue of the nucleic acid sequence encoding the hinge region of the antibody differs from the naturally occurring nucleic acid sequence of the hinge region normally associated with the heavy chain constant region. In another embodiment, at least one amino acid residue of the amino acid sequence of the hinge region differs from the amino acid sequence of the hinge region naturally found in the constant region of a heavy chain of the antibody.

In a preferred embodiment, the hinge region has been modified such that one or more amino acids of the hinge region naturally associated with the heavy chain constant region are substituted with amino acids at positions corresponding to the hinge region associated with the heavy chain constant region of a different class or subclass of antibody. Preferably, the heavy chain constant region of the antibody produced is from an IgG antibody and the hinge region is substituted with one or more amino acids of the IgA, IgD, IgM or IgE antibody hinge region. In another preferred embodiment, the heavy chain constant region of the antibody produced is from an IgG antibody, such as an IgG4 antibody, and the hinge region is substituted with one or more amino acids of the hinge region of a different antibody species (e.g., IgG1, IgG2, and IgG3 antibodies).

In certain embodiments, the nucleic acid may be polycistronic, e.g., the heavy chain coding sequence and the light chain coding sequence are under the control of the same promoter, e.g., by having an Internal Ribosome Entry Site (IRES) between the two.

Brief Description of Drawings

FIG. 1 shows a schematic representation of a method for producing cloned animals by nuclear transfer.

FIG. 2 shows an overview of the analysis of the hinge region modification using KMK 917.

FIG. 3A shows a CEx-HPLC plot of an isolated KMK antibody sample.

Fig. 3B shows a CEx-HPLC plot of the isolated KMK antibody sample.

Fig. 3C shows a CEx-HPLC plot of the isolated KMK antibody sample.

Fig. 3D shows a CEx-HPLC plot of the isolated KMK antibody sample.

Fig. 3E shows a CEx-HPLC plot of the isolated KMK antibody sample.

Fig. 3F shows a CEx-HPLC plot of the isolated KMK antibody sample.

Fig. 3G shows a CEx-HPLC plot of the isolated KMK antibody sample.

FIG. 4A shows a CEx-HPLC plot of KMK wild-type samples + -endoglycosidase F treatment, wild-type.

FIG. 4B shows a CEx-HPLC plot of KMK wild-type samples + -endoglycosidase F treatment, wild-type.

FIG. 4Cc shows CEx-HPLC profiles, hinges and CH2 mutants of KMK wild-type samples + -endoglycosidase F treatment.

FIG. 4D KMK wild type sample + -CEx-HPLC profile of endoglycosidase F treatment, hinge and CH2 mutant.

FIG. 5A shows a CEx-HPLC plot of the characteristics of KMK 9171099/2010 sugar, wild type.

FIG. 5B shows a CEx-HPLC plot of the characteristics of KMK 9172012/2014 sugar, hinge + Ch2 mutant.

FIG. 5C shows a CEx-HPLC plot, full range, of KMK917 sugar properties.

Detailed Description

The following abbreviations have the indicated meanings in the specification:

abbreviations:

somatic Cell Nuclear Transfer (SCNT)

Cultured Inner Cell Mass Cells (CICM)

Nuclear Transfer (NT)

Synthetic Ovoid Fluid (SOF)

Fetal Bovine Serum (Fetal bone Serum, FBS)

Polymerase Chain Reaction (PCR)

Bovine Serum Albumin (Bovine Serum Albumin, BSA)

High Pressure Liquid Chromatography (HPLC)

Interpretation of terms:

cattle-different species that belong to or are related to cattle.

Goat-different species that belong to or are related to goats.

Cell couplet (couplet) -enucleated oocytes and somatic or embryonic cell nucleosomes prior to fusion and/or activation.

Cytochalasin B, a metabolite of some fungi, selectively and reversibly blocks cytokinesis without affecting nuclear division.

Cytoplast-the cytoplasmic material of eukaryotic cells.

Fusion sheet-glass sheets of parallel electrodes placed at a fixed distance apart. The cell couplet is placed between electrodes to obtain an electrical current for fusion and activation.

Nucleome-the nucleus of a cell obtained from the cell by enucleation, surrounded by a narrow ring of cytoplasm and plasma membrane.

Nuclear transfer-or "nuclear transfer" refers to a cloning procedure in which nuclei are transferred from a donor cell to an enucleated oocyte.

Sheep-belongs to or pertains to sheep.

Parthenogenesis-the development of an embryo from an oocyte without the penetration of sperm.

Pig-belongs to, pertains to, or is similar to a pig.

Reconstituted embryo-a reconstituted embryo is an oocyte that has had its genetic material removed by an enucleation procedure. Adult or embryonic somatic genetic material is "reconstituted" by the incorporation of the genetic material into the oocyte by a fusion event. Selection agent-a compound, composition or molecule that can act as a cell selection marker, capable of killing and/or arresting the growth of a living organism or cell that does not contain an appropriate resistance gene. According to the present invention, such agents include, but are not limited to, neomycin, puromycin, zeocin, hygromycin, G418, 9- [1, 3-dihydroxy-2-propoxymethyl ] guanine (gancyovir) and FIAU. Preferably, the present invention increases the dose of the selection agent to kill all cell lines (e.g., heterozygote animals and/or cells) that contain only one integration site.

Somatic cell-any cell of an organism other than a germ cell.

Somatic cell nuclear transfer-also known as therapeutic cloning, by which a somatic cell is fused with an enucleated oocyte. Somatic cell nuclei provide genetic information, while oocytes provide nutrients and other energy-producing substances required for embryonic development. Once fusion has occurred, the cells are totipotent and eventually develop into blastocysts, at which time the inner cell mass is isolated.

Transgenic organisms-organisms that have been experimentally transformed with the genetic material of another organism such that the host chromosome acquires the genetic information of the transgene in addition to the genetic information already present in its genetic complement. Ungulates-mammals belonging to or related to typical ungulate quadrate, including but not limited to sheep, pigs, goats, cattle and horses.

Xenotransplantation-refers to any procedure using living cells, tissues and organs of one animal origin for transplantation or implantation into another animal species (typically a human) or for clinical ex vivo perfusion.

Detailed Description

The present invention relates to the production of antibodies in the milk of transgenic mammals. Various aspects of the invention relate to antibodies and antibody fragments, methods of making antibodies or fragments thereof in the milk of transgenic mammals, and methods of making transgenic mammals whose somatic and germ cells include modified antibody coding sequences. Also provided are nucleic acid sequences for expressing the modified antibody coding sequences in mammary epithelial cells.

To make the invention easier to understand, certain terms are defined. Definitions are set forth throughout the detailed description.

Antibodies and fragments thereof

As used herein, an antibody "class" refers to the five major antibody isotypes, including IgA, IgD, IgE, IgG, and IgM. Antibody "subclass" refers to the subclass of a particular class of antibodies based on amino acid differences between members of that class, e.g., classes identified as IgG can be classified, e.g., as subclasses IgG1, IgG2, IgG3, and IgG4, while classes identified as IgA can be classified, e.g., as subclasses IgA1 and IgA 2.

The term "antibody" refers to a protein comprising at least one and preferably two heavy chain (H) variable regions (abbreviated herein as VH), at least one and preferably two light chain (L) variable regions (abbreviated herein as VL), and at least one and preferably two heavy chain constant regions. The VH and VL regions can be further subdivided into hypervariable regions, termed "complementary determining regions" (CDRs), interspersed with more conserved regions, termed "framework regions" (FRs). The extent of the framework regions and CDRs has been precisely defined (see Kabat, E.A. et al (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. department of Health and Human Services, NIH publication No.91-3242 and Chothia, C.et al (1987) J.mol.biol.196: 901-917, incorporated herein by reference). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4.

The antibody may additionally comprise a light chain constant region, thereby forming an immunoglobulin heavy chain and light chain. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are interconnected by, for example, disulfide bonds. The heavy chain constant region consists of three domains, CH1, CH2, and CH 3. The light chain constant region consists of one domain CL. The heavy and light chain variable regions comprise a binding domain that interacts with an antigen. Antibody constant regions generally mediate the binding of antibodies to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

The antibody may additionally include a hinge region, as described in detail below. As used herein, an "assembled" antibody is one in which the heavy chains of the antibody are joined to each other, for example, by disulfide bonds. Each heavy chain hinge region includes at least one, and typically several cysteine residues. In assembled antibodies, the cysteine residues of the heavy chain are adjusted so as to form a disulfide bond between cysteine residues in the hinge region, covalently binding the heterodimers of the two heavy-light chains together. Thus, a fully assembled antibody is bivalent because it has two antigen binding sites. The term "antibody" (or "immunoglobulin") as used herein also refers to fragments of full length antibodies, such as F (ab') 2 fragments, which are bivalent fragments comprising two Fab fragments linked by a disulfide bridge in the hinge region. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and useful fragments are screened in the same manner as intact antibodies.

An "antigen-binding fragment" (or "functional fragment") of an antibody refers to one or more portions of an antibody that retain the ability to specifically bind to an antigen. The term "antigen-binding fragment" of an antibody encompasses examples of binding fragments that include one or more Complementarity Determining Regions (CDRs).

As used herein, a "chimeric antibody heavy chain" means an antibody heavy chain having a portion (e.g., the variable region) of the antibody heavy chain that is at least 85%, preferably 90%, 95%, 99% or more identical to the corresponding amino acid sequence of an antibody heavy chain of a particular species, or is part of a particular antibody species or type, while the remaining portion (e.g., the constant region) of the antibody heavy chain is substantially identical to the corresponding amino acid sequence of another antibody molecule. For example, the heavy chain variable region has substantially the same sequence as the heavy chain variable region of an antibody of one species (e.g., a "donor" antibody, such as a rodent antibody), while the constant region is substantially the same as the constant region of an antibody of another species (e.g., an "acceptor" antibody, such as a human antibody). The donor antibody may be an antibody produced in vitro, for example by phage display.

The term "humanized" or "CDR-grafted" light chain variable region refers to an antibody light chain comprising one or more CDRs, or having no more than one or two amino acid residues different from the corresponding one or more CDRs of a species, or class or type of antibody, e.g., a "donor" antibody (e.g., a non-human (usually mouse or rat) immunoglobulin, or an in vitro generated immunoglobulin); and amino acid sequences in which the framework regions have about 85% or greater, preferably 90%, 95%, 99% or greater identity to the corresponding portion of an acceptor antibody framework (e.g., a naturally occurring immunoglobulin framework (e.g., a human framework) or consensus framework) of a different species, or class or type of antibody. In certain embodiments, the framework region comprises at least about 60, more preferably about 70 amino acid residues identical to a recipient antibody light chain variable region framework (e.g., a naturally occurring antibody framework (e.g., a human framework) or consensus framework).

A "heterologous antibody" or "foreign antibody" is an antibody that is not normally produced by a mammal, or is not normally produced in the mammary gland (e.g., an antibody present only in serum), or is produced in the mammary gland but has an increased or enhanced level of expression in its production.

Any antibody described herein, e.g., a chimeric, humanized or human antibody, may include further modifications to its sequence, e.g., by addition, deletion or substitution (e.g., conservative substitution) to modify the sequence.

Antibody hinge region

The methods of the invention involve, for example, the production of an antibody in the milk of a transgenic animal in which the hinge region has been altered from that normally associated with the constant region of the heavy chain of the antibody. Such constant regions are also referred to herein as "mutated heavy chain constant regions". The term "generally related" refers to the association of the hinge region and the heavy chain constant region in a naturally occurring antibody. The term "naturally occurring" as used herein refers to an antibody that can be found in nature, e.g., in a natural organism. For example, an antibody or fragment thereof that is present in a natural organism and that has not been intentionally modified by man is naturally-occurring. The term also refers to the association between the hinge region of an antibody and at least a portion of the heavy chain constant region (e.g., the CH1 region), if this heavy chain constant region portion is found to be "naturally occurring" with the hinge region in an antibody. The term is not limited to only the heavy chain constant regions found in nature. The chain constant region may include modifications such as substitutions, insertions or deletions of one or more amino acids. Examples of IgG hinge and heavy chain constant regions (or portions thereof) that are typically associated with each other include: the hinge region of an IgG1 antibody and the heavy chain constant region (or portion thereof) of the same IgG1 antibody; the hinge region of an IgG2 antibody and the heavy chain constant region (or portion thereof) of the same IgG2 antibody; the hinge region of an IgG3 antibody and the heavy chain constant region (or portion thereof) of the same IgG3 antibody; the hinge region of an IgG4 antibody and the heavy chain constant region (or portion thereof) of the same IgG4 antibody. These examples are non-limiting and the term also applies to other types of antibodies.

As used herein, the "hinge region" of an antibody refers to a peptide sequence between the CH1 and CH2 domains of the antibody. The hinge region is present between the Fab and Fc portions of the antibody. The hinge region is typically encoded by a unique exon and contains a disulfide bond that links two heavy chain fragments of an antibody. See Paul et al Fundamental Immunology, 3^rdEd. (1993). The amino acid sequence of the hinge region is generally rich in proline, serine and threonine residues.For example, the extended peptide sequence between the CH1 and CH2 domains of IgG, IgD and IgA is proline-rich. IgM and IgE antibodies include a domain of about 110 amino acids with hinge-like characteristics (Ruby, j., Immunology (1992)), which is included in the term "hinge region" as used herein.

The amino acid sequence of the hinge region may include cysteine residues. Cysteine residues play a role in interchain disulfide bond formation. Depending on the antibody species, there may be 2-11 inter-heavy chain disulfide bonds in the antibody hinge region. These disulfide bonds are responsible for holding the two parts of the intact antibody molecule together. The hinge region of each class and subclass of antibodies is known in the art.

Change of

Standard molecular biology techniques can be used to provide antibodies with altered hinge regions. These techniques can be used to create changes, such as deletions, insertions, or substitutions, in the known amino acid sequence of the hinge region of an antibody (or other portion of the antibody sequence). The term "altered" refers to any change made in the hinge region of an antibody or portion thereof. Such changes include, but are not limited to, deletions, insertions, and substitutions/replacements of one or more or all amino acids of the hinge region. It will be appreciated by the skilled practitioner that any suitable technique, such as directed or random mutagenesis techniques, may be used to provide specific sequences or mutations in the hinge region. Such techniques may also be used to alter other regions of an antibody, such as the heavy and/or light chain constant and/or variable regions.

For example, oligonucleotide-mediated mutagenesis is a useful method for making DNA substitution, deletion and insertion variants, see, e.g., Adelman et al (DNA 2: 183, 1983). Briefly, a DNA of interest is altered by hybridizing an oligonucleotide encoding the mutation to a DNA template, wherein the template is a plasmid or phage comprising a single stranded form of the unaltered or native DNA sequence of the protein of interest. Following hybridization, the entire second complementary strand of the template is synthesized using a DNA polymerase, thereby incorporating the oligonucleotide primer and encoding the selected change in the protein DNA of interest. Generally, oligonucleotides of at least 25 nucleotides in length are used. The optimal oligonucleotide has 12-15 nucleotides on each side of the nucleotide encoding the mutation that are fully complementary to the template. This ensures that the oligonucleotide is strictly hybridized to the single-stranded DNA template molecule. Oligonucleotides are readily synthesized using techniques known in the art, e.g., as described by Crea et al (Proc. Natl. Acad. Sci. USA, 75: 5765[1978 ]).

For example, in one embodiment, the hinge region or hinge region fragment of an antibody is replaced with another hinge region or hinge region fragment from a different antibody (e.g., a different class or subclass of antibody). In a preferred embodiment, the IgG4 hinge region is replaced with a hinge region from a different subclass, such as the IgG2 hinge region. Such substitutions may be made, for example, using oligonucleotide-mediated mutagenesis using oligomers encoding exons containing the hinge region of IgG 2. In another embodiment, a single amino acid within the hinge region (e.g., the IgG4 hinge region) is replaced with a different amino acid, e.g., an amino acid found at a corresponding position in a hinge region of a different subclass (e.g., an amino acid in the IgG2 hinge region). For example, a serine found at amino acid position 241 may be replaced with a proline (the amino acid found at the corresponding position in the hinge region of IgG 2). Using oligos that cause amino acid changes (e.g., oligo S241P), substitutions can be made using oligonucleotide-mediated mutagenesis. In another embodiment, the glycosylation site of an antibody, such as an IgG4 antibody, is altered, e.g., changed, to no longer be a glycosylation site. For example, the N-linked glycosylation site can be altered to change asparagine to glutamine. Oligonucleotide-mediated mutagenesis can also be used to effect such changes, for example, by using oligomers that can be altered by amino acids.

Another example of a method for altering proteins, cassette mutagenesis, is provided based on the technique described by Wells et al (Gene, 34: 315[1985 ]). The starting material is a plasmid (or other vector) comprising the subunit DNA of the protein to be mutated. The codons in the subunit DNA of the protein to be mutated are identified. Unique restriction enzyme sites must be present on each side of the identified mutation site. If such restriction sites are not present, such sites must be formed by introducing appropriate positions of the subunit DNA of the protein of interest using the oligonucleotide-mediated mutagenesis method described above. After introduction of the restriction sites into the plasmid, the plasmid is linearized by digestion at these sites. Double-stranded oligonucleotides encoding DNA sequences between the restriction sites, but containing the desired mutation, are synthesized using standard methods. The duplexes are synthesized separately and then hybridized to each other using standard methods. This double-stranded oligonucleotide is called a cassette (cassette). The cassette is designed to have 3 'and 5' ends that are equivalent to the ends of the linearized plasmid and thus can be directly ligated to the plasmid. The plasmid thus contains the mutated subunit DNA sequence of the protein of interest.

The invention additionally encompasses that random mutagenesis of DNA encoding the antibody or fragment thereof can also be used to produce antibodies with altered hinge regions. Useful methods include, but are not limited to, PCR mutagenesis, saturation mutagenesis, and the preparation and use of a degenerate set of oligonucleotide sequences. These methods are known.

Transgenic mammals

As used herein, a "transgenic animal" is a non-human animal whose one or more, preferably substantially all, of the cells comprise a heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques known in the art. Transgenes may be introduced into the cell precursor, directly or indirectly, by elaborate genetic manipulation, such as microinjection or recombinant viral infection.

The term "transgene" refers to a nucleic acid sequence (encoding, for example, one or more antibody polypeptides or portions thereof) that is partially or completely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced; alternatively, it is homologous to a gene endogenous to the transgenic animal or cell into which it is introduced, but is designed to be inserted or inserted into the animal's genome such that the genome of the inserted cell is altered (e.g., inserted into a different location than the native gene). The transgene may include one or more transcription regulatory sequences and any other nucleic acids, such as introns, which may be necessary for optimal expression and secretion of the selected antibody-encoding nucleic acid, e.g., in the mammary gland, may be operably linked to the selected antibody nucleic acid, and may include enhancer sequences and/or insulator sequences. The antibody sequence may be operably linked to a tissue-specific promoter, for example a mammary gland-specific promoter sequence which causes secretion of a protein in the milk of a transgenic mammal.

The term "transgenic cell" as used herein refers to a cell comprising a transgene. Mammals are defined herein as all animals, except humans, that have mammary glands and produce milk. Any non-human mammal may be utilized with the present invention. Preferred non-human mammals are ruminants such as cattle, sheep, camels or goats. Other examples of preferred non-human animals include cattle, horses, camels and pigs. For example, methods of making transgenic goats are known in the art. Transgenes can be introduced into goat germ lines by microinjection, for example Ebert et al (1994) Bio/Technology 12: 699, which is hereby incorporated by reference. The particular strain of any animal used in the practice of the present invention is selected for good overall health, high embryo yield, good visibility of pronuclei in embryos, and reproductive health. In addition, haplotypes are an important factor.

Methods for producing non-human transgenic mammals are known in the art. The method can include introducing the DNA construct into a germline of a mammal to make a transgenic mammal. For example, it is possible to incorporate one or several copies of the construct into the genome of a mammalian embryo by standard transgenic techniques. In addition, a non-human transgenic mammal can be prepared using somatic cells as donor cells. Then the somatic cell genome is inserted into the oocyte, and the oocyte is fused and activated to form a reconstructed embryo. For example, methods for preparing transgenic mammals using somatic cells are described below, PCT publications WO 97/07669; baguisi et al NATURE BIOTECH, vol.17(1999), 456-461; campbell et al NATURE, vol.380(1996), 64-66; cibeli et al SCIENCE, vol.280 (1998); SCIENCE, Kato et al, vol.282(1998), 2095-; SCIENCE, Schnieke et al, vol.278 (1997), 2130-; wakayama et al NATURE, vol.394(1998), 369-374; well et al, BIOL.REPROD., vol.57 (1997): 385-393.

Transfected cell lines

Genetically engineered cell lines can be used to make transgenic animals. The genetically engineered constructs can be introduced into cells by conventional transformation or transfection techniques. The terms "transfection" and "transformation" as used herein include various techniques for introducing a transgene sequence into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE dextran-mediated transfection, lipofection, or electroporation. In addition, a biological vector, such as a viral vector, may be used as described below. In Sambrook et al molecular cloning: a Laboratory Manual, 2^nded., (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other suitable Laboratory manuals may find suitable methods for transforming or transfecting host cells.

Two useful methods are electroporation and lipofection. Respective brief examples are as follows.

The DNA construct can be stably introduced into the donor cell line by electroporation using the following procedure: somatic cells, e.g. fibroblasts, e.g. embryonic fibroblasts at about 4X 10⁶cells/mL were resuspended in PBS. 50 μ g of linearized DNA was added to 0.5mL of cell suspension and the suspension was placed in a small chamber with 0.4cm electrode gap (Biorad). Electroporation was performed using a Biorad GenePulser electroporator at 330V voltage, 25mA, 1000. mu.F and infinite resistance. If the DNA construct contains a neomycin resistance gene for selection, neomycin resistant clones are selected by incubation for 15 days with 350. mu.g/mL G418 (GibcoBRL).

The DNA construct may be introduced into the donor somatic cell line by lipid stabilization using, for example, the following procedures: will be about 2X 10⁵Cells were seeded in 3.5cm diameter microwells using "LipfectAMINE^TM"(GibcoBRL) transfects 2. mu.g of linearized DNA. 48 hours after transfection, cells were isolated at 1: 1000 and 1: 5000, and G418 was added to a final concentration of 0.35mg/mL if the DNA construct contained the neomycin resistance gene for selection. Neomycin resistant clones were isolated and expanded for cryopreservation and nuclear transfer.

DNAConstruct

Cassettes encoding the heterologous protein can be assembled into constructs that include a promoter for a particular tissue, e.g., mammary epithelial cells (e.g., a casein promoter, such as a goat beta casein promoter), a milk-specific signal sequence (e.g., a casein signal sequence, such as a beta-casein signal sequence), and DNA encoding the heterologous protein.

The construct also includes a 3' untranslated region downstream of the DNA sequence encoding the non-secreted protein. This region stabilizes the RNA transcript of the expression system and thus increases the yield of the protein of interest of the expression system. A3' untranslated region useful in a construct used in the present invention is a sequence that provides a polyadenylation signal. The sequence may be derived from, for example, SV40 small t antigen, casein 3 'untranslated region, or other 3' untranslated sequences known in the art. In one aspect, the 3' untranslated region is from a milk specific protein. The length of the 3' untranslated region is not critical, and the stabilizing effect of its polyadenylation transcript is important in stabilizing the RNA of the expressed sequence.

Alternatively, the construct may include a 5' untranslated region between the promoter and the DNA sequence encoding the signal sequence. The untranslated regions may be derived from the same control region from which the promoter is derived, or from different genes, for example, may be derived from other synthetic, semi-synthetic or natural sources. The specific length is not important yet, but seems to be beneficial for increasing expression levels.

The construct may also include an N-terminal coding region that expresses about 10%, 20%, 30% or more of the gene, preferably in mammary epithelial cells. For example, the N-terminal coding region may correspond to the promoter used, e.g., the goat beta casein N-terminal coding region.

Constructs may be made using methods known in the art. The construct may be prepared as part of a larger plasmid. Such preparation allows the correct construct to be cloned and selected in an efficient manner. The construct may be located between the conventional restriction sites of the plasmid so as to be readily separated from the remaining sequences of the plasmid for introduction into the mammal of interest.

Insulator (insulator) sequence

The DNA construct used to make the transgenic animal can include at least one insulator sequence. The terms "spacer", "spacer sequence" and "spacer element" are used interchangeably herein. An insulator element is a control element that isolates the transcription of a gene within its range of action without disrupting (either negatively or positively) the expression of the gene. Preferably, an insulator sequence is inserted on either side of the DNA sequence to be transcribed. For example, the insulator may be located 5 'to the promoter by about 200bp to 1kb and at least about 1kb to 5kb from the promoter at the 3' end of the gene of interest. One skilled in the art can determine the distance of the spacer sequence from the promoter and 3' end of the gene of interest, depending on the relative sizes of the gene of interest, promoter and enhancer used in the construct. Furthermore, more than one insulator sequence may be located 5 'to the promoter, or 3' to the transgene. For example, two or more insulator sequences may be located 5' to the promoter. One or more of the isolates at the 3 'end of the transgene may be located at the 3' end of the gene of interest, or at the 3 'end of a 3' regulatory sequence such as the 3 'untranslated region (UTR) or the 3' flanking sequence.

A preferred isolator is a DNA fragment comprising the 5 'end of the chicken beta-globin locus and corresponds to the chicken 5' constitutive hypersensitivity site described in PCT publication 94/23046, the contents of which are incorporated herein by reference.

Expression of proteins in mammary glands

It is desirable to express heterologous proteins, such as antibodies, in specific tissues or body fluids of the transgenic animal, such as milk. Recovering the heterologous protein from the tissue or body fluid in which it is expressed. For example, a heterologous protein (e.g., an antibody) of the invention can be expressed in the milk of a transgenic animal. Methods for producing heterologous proteins under the control of mammary gland-specific promoters are described below.

Mammary gland specific promoter and signal sequence

Useful transcription promoters are promoters which are preferentially activated in mammary epithelial cells, including promoters controlling genes encoding milk proteins, such as casein, beta lactoglobulin (Clark et al (1989) BIO/TECHNOLOGY 7: 487) 492), whey acidic protein (Gordon et al (1987) BIO/TECHNOLOGY 5: 1183) 1187), and whey protein (Soulier et al (1992) FEBS letters.297: 13). The casein promoter may be from the alpha, beta, gamma or kappa casein gene of any mammalian species; preferred promoters are derived from the goat beta casein gene (DiTullio, (1992) BIO/TECHNOLOGY 10: 74-77). The promoter may also be derived from lactoferrin or a lactophilin (butyrophilin). The mammary gland-specific protein promoter or promoters specifically activated in mammary tissue may be derived from cDNA or genomic sequences. Preferably of genomic origin.

The DNA sequence information of the above listed mammary gland-specific genes can be obtained in at least one, usually several organisms. See, e.g., Richards et al J.BIOL.CHEM.256, 526-Astro 532(1981) (rat α -lactalbumin); campbell et al NUCLEIC ACIDS RES.12, 8685-8697(1984) (rat WAP); jones et al J.BIOL.CHEM.260, 7042-7050(1985) (rat. beta. -casein); Yu-Lee & Rosen, J.BIOL.CHEM.258, 10794-10804(1983) (rat gamma-casein); hall, BIOCHEM.J.242, 735-742(1987) (human α -lactalbumin); stewart, NUCLEIC ACIDSRES.12, 389(1984) (bovine α s1 and kappa casein cDNA); gorodettsky et al GENE 66, 87-96(1988) (bovine beta-casein); alexander et al EUR.J.BIOCHEM.178, 395-401(1988) (bovine kappa-Casein); brignon et al FEBS LETT.188, 48-55(1977) (bovine α S2 casein); jamieson et al GENE 61, 85-90(1987), Ivanov et al BIOL.CHEM.Hoppe-Seyler 369, 425-429 (1988); alexander et al NUCLEIC ACIDS res.17, 6739(1989) (bovine beta-lactoglobulin); vilotte et al BIOCHIMIE 69, 609-620(1987) (bovine alpha-lactalbumin). Mercier & Vilotte, J.DAIRY Sci.76, 3079-3098(1993) reviewed the structure and function of various milk protein genes (incorporated herein by reference in their entirety). If other flanking sequences are available to optimize the expression of the heterologous protein, such sequences can be cloned using the existing sequences as probes. Mammary gland-specific regulatory sequences of different organisms can be obtained by screening a library of an organism using antibodies to known homologous nucleic acid sequences or homologous proteins as probes.

Useful signal sequences are milk-specific signal sequences or other signal sequences which cause the secretion of eukaryotic or prokaryotic proteins. Preferably the signal sequence is selected from a milk-specific signal sequence, i.e. a gene encoding a product from secretion into milk. Preferably, the milk-specific signal is associated with a mammary gland-specific promoter used in the construct, as described below. The size of the signal sequence is not critical. All that is required is a sequence of sufficient size to effectively secrete the recombinant protein of interest, for example, in breast tissue. For example, signal sequences of caseins such as α, β, γ or κ casein, β lactoglobulin, whey acidic protein and whey protein-encoding genes may be used.

Cassettes encoding heterologous antibodies, such as modified IgG4 antibodies, can be assembled into constructs. For example, the construct may include a promoter (e.g., a casein promoter) for a particular tissue (e.g., a mammary epithelial cell), a milk-specific signal sequence (e.g., a casein signal sequence), and DNA encoding a heterologous antibody (e.g., a modified IgG4 antibody). The constructs can be made using methods known in the art. This construct can be made part of a larger plasmid. Such preparation allows the correct construct to be cloned and selected in an efficient manner. The construct may be located between convenient restriction sites on the plasmid so as to be readily separated from the remaining sequences of the plasmid for introduction into the mammal of interest.

Oocyte

Oocytes may be obtained at various times during the reproductive cycle of the animal. Oocytes at various stages of the cell cycle may be obtained and then induced in vitro into a particular meiotic stage. For example, oocytes cultured in serum-deficient medium stagnate in metaphase. In addition, the arrested oocytes may be induced to enter the terminal phase by serum activation.

Oocytes may be matured in vitro prior to use to form reconstituted embryos. This process typically requires collecting immature oocytes from a mammalian ovary (e.g., goat ovary) and maturing the oocytes in culture medium prior to enucleation until the desired meiotic stage, e.g., metaphase or telase, is reached. In addition, oocytes matured in vivo may be used to form reconstituted embryos.

Oocytes may be collected during superovulation of a female mammal. Briefly, oocytes, such as goat oocytes, may be surgically recovered by flushing the oviduct of a female donor. Methods for inducing superovulation in goats and collecting goat oocytes are described herein.

Transfer of reconstituted embryos

The reconstituted embryo can be transferred to a recipient and allowed to develop into a cloned or transgenic mammal. For example, the reconstituted embryo is transferred into the tubal lumen of each recipient via the fimbria. In addition, methods for transferring embryos to recipient mammals are known in the art and are described, for example, in Ebert et al (1994) Bio/Technology 12: 699.

purification of proteins from milk

As used herein, a preparation refers to two or more antibody molecules. The preparation may be produced by one or more transgenic animals. Molecules of different glycosylation may be included or, for that matter, homogeneous.

As used herein, "purified preparation", "substantially pure antibody preparation" or "isolated antibody" refers to an antibody that is substantially free of material contained in the milk of a transgenic mammal. Preferably, the antibody is also separated from the material used for purification, for example a gel matrix such as polyacrylamide. In one embodiment, the term "substantially free" includes antibody preparations having less than about 30% (by dry weight) of non-antibody material (also referred to herein as "milk impurities" or "milk components"), more preferably less than about 20% of non-antibody material, more preferably less than about 10% of non-antibody material, and most preferably less than about 5% of non-antibody material. Non-antibody substances include casein, lipids (e.g., soluble lipids and phospholipids), lactose and other small molecules (e.g., galactose, glucose), small peptides (e.g., microbial peptides, anti-biological peptides), and other milk proteins (e.g., whey proteins such as lactoglobulin and beta-lactalbumin, lactoferrin, and serum albumin). The antibody preferably constitutes at least 10, 20, 50, 70, 80 or 95% of the dry weight of the purified preparation. Preferably the preparation comprises at least 1, 10 or 100 μ g of antibody; at least 1, 10 or 100mg of antibody. Furthermore, the purified preparation preferably comprises about 70%, 75%, 80%, 85%, 90%, 95%, 98% of the assembled antibody.

The antibody (or fragment thereof) can be isolated using standard protein purification methods known in the art. For example, the antibodies and/or fragments of the invention can be purified using the Kutzko et al method (U.S. Pat. No. 6,268,487).

Milk proteins are usually isolated using combinatorial methods. For example, raw milk is first fractionated to remove fat, for example by skimming, centrifugation, sedimentation (H.E. Swaisgood, Developments in Dairy Chemistry, in: CHEMISTRY OF MILK PROTEIN, Applied Science Publishers, NY, 1982), acid precipitation (U.S. Pat. No. 4,644,056) or enzymatic coagulation using chymosin or chymotrypsin (Swaisgood). The major milk proteins are then fractionated into clear solutions or loose precipitates, facilitating the purification of the specific protein of interest therefrom. As another example, french patent #2,487,642 describes the separation of milk proteins from skim milk or whey by membrane ultrafiltration combined with exclusion chromatography or ion exchange chromatography. Whey is first prepared by removing casein by rennet or lactic acid coagulation. Us patent #4,485,040 describes the separation of a product enriched in alpha-lactoglobulin from the retentate using two successive ultrafiltration steps. U.S. Pat. No. 4,644,056 provides a process for the purification of immunoglobulins from milk or colostrum by acidic precipitation at pH4.0-5.5 and continuous tangential flow filtration, first clarifying the product with a membrane with a pore size of 0.1-1.2 μm and then concentrating with a membrane with a separation limit of 5-80 kd. Us patent #4,897,465 teaches the use of continuous ultrafiltration using metal oxide membranes with pH change to concentrate and enrich proteins, such as immunoglobulins, from serum, egg yolk or whey. Filtration is first performed at a pH below the isoelectric point (pI) of the selected protein, removing most of the impurities from the protein retentate, and then at a pH above the pI of the selected protein, retaining the impurities, allowing the selected protein to pass through into the permeate. European patent EP 467482B 1 illustrates a different method of filtration concentration to lower skim milk to a pH of 3-4 below the pI of milk proteins to solubilize casein and whey proteins. The protein is then concentrated using three successive rounds of ultrafiltration or diafiltration to form a retentate containing 15-20% solids, 90% of which is protein.

Another example is to first clarify the milk. A typical clarification process comprises the following steps:

(a) diluting the milk with 2.0M arginine-HCl pH5.5 at a ratio of 2: 1;

(b) spinning the diluted sample in a centrifuge at 4-8 ℃ for about 20 minutes;

(c) the sample was cooled on ice for about 5 minutes to allow the fat to solidify on top;

(d) the upper layer was "aspirated" with the tip of a pipette to remove the fat pad; and

(e) the supernatant was poured into a clean tube.

Further purification of the protein is carried out using any protein purification method known in the art, such as the methods described above.

Examples

Example 1: antibody modification

Oligonucleotide mutagenesis can be used to modify the antibody heavy chain. Briefly, a DNA of interest is altered by hybridizing an oligonucleotide encoding the mutation to a DNA template, wherein the template is a plasmid or phage comprising a single stranded form of the unaltered or native DNA sequence of the protein of interest. Following hybridization, the entire second complementary strand of the template is synthesized using a DNA polymerase, thereby incorporating the oligonucleotide primer and encoding the selected change in the protein DNA of interest. Generally, oligonucleotides of at least 25 nucleotides in length are used. The optimal oligonucleotide has 12-15 nucleotides on each side of the nucleotide encoding the mutation that are fully complementary to the template. This ensures that the oligonucleotide is strictly hybridized to the single-stranded DNA template molecule. Oligonucleotides are readily synthesized using techniques known in the art, e.g., as described by Crea et al (Proc. Natl. Acad. Sci. USA, 75: 5765[1978 ]).

To achieve a serine to proline change at amino acid number 241 of the hinge region, oligonucleotide mutagenesis was performed using oligo S241P, which changed serine to proline. The resulting mutant forms can be used to generate transgenic mice. Transgenic mice can produce milk and be tested for the presence of antibodies and relative amounts of "half molecules" in the milk. The IgG4 antibody hinge region and oligonucleotide S241P sequences used for mutagenesis were as follows:

IGG4 hinge region

1668 TCTGCA GAG TCC AAA TAT GGT CCC CCA TGC CCA TCA TGC CCA

GGTAAGCCAACCCAGGCCT

1^R _S Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro

S241P OLIGO

GGT CCC CCA TGT CCT CCC TGC CCA GGT AAG CCA

^R _S Gly Pro Pro Cys Pro Pro Cys Pro Gly Lys Pro

Furthermore, the entire hinge region of an IgG antibody may be replaced with the hinge region of another antibody. To effect this change, oligonucleotides encoding exons containing alternative hinge regions may be used.

The sequences of the hinge region of the IgG4 antibody and of the oligonucleotide comprising the replacement hinge region of IgG2 are as follows:

IGG4 hinge region

1662 CTTCTCTCTGCA GAG TCC AAA TAT GGT CCC CCA TGC CCA TCA TGC CCA GGTCCGCCAACCCAGGC

1^R _S Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro

IGG2 hinge region

1729 CTTCTCTCTGCA GAG CGC AAA TGT TGT GTC GAG TGC CCA CCG TGC CCA GGTCCGCCAACCCAGGC

1^R _S Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro

The N-linked glycosylation site of IgG heavy chain CH2 can be removed by oligonucleotide mutagenesis using an oligo that causes asparagine to be changed to glutamine in the consensus site. The oligonucleotide sequences that achieve this change are as follows:

2014 GAG GAG CAG TTC CAG TCT ACT TAC CGA GTG GTC

1^R _S Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val

testing mutant antibodies

The light chain and mutant heavy chain were linked to a casein promoter for the generation of transgenic mice. The mice were then tested for expression of antibodies and half-antibodies.

Transgenic animals

The starting (fountain) transgenic goat can be made by transferring goat zygotes injected with the construct. The method followed in this section can be used to produce transgenic goats. The skilled practitioner will understand that these methods can be adapted for use with other animals.

Goat species and lines

Goats of swiss origin, such as Alpine, sannen and Toggenburg lines, can be used to produce transgenic goats.

The following section briefly describes the steps required to prepare the transgenic goat. These steps include superovulation of female goats, mating with prolific males, and collection of spermatozoa. The pronuclei of single-cell spermatozoa are microinjected with the DNA construct once collected. All embryos from one female donor are put together and transferred, if possible, to a single recipient female.

Superovulation of goat

Donor estrous cycle phases were synchronized on day 0 by subcutaneous 6mg norgestrel (norgestomet) ear implants (Syncromate-B, CEVA Laboratories, inc., Overland Park, KS). Prostaglandins were administered initially 7-9 days later, shutting off endogenous progesterone synthesis. A total of 18mg of follicle stimulating hormone (FSH-Schering corp., Kenilworth, NJ) was administered intramuscularly, twice daily, beginning on day 13 after insertion of the implant, for 3 days. The implant was removed on day 14. 24 hours after implant removal, donor animals were mated several times within two days with prolific males (Selgrath et al, Theriogenology, 1990. pp.1195-1205).

Embryo collection

On day 2 post-breeding (or 72 hours after implant removal), embryo collection surgery was performed. 36 hours before the operation, the superovulated female goat was deprived of food and water. The female goat was given 0.8mg/kg Diazepam (Diazepam, Valium)^*) IV, then immediately 5.0mg/kg Ketamine (Ketamine, Keteset), IV. Halothane (Halothane) was administered during surgery via endotracheal tube in 2L/min of oxygen (2.5%). The genital tract is exteriorized by a midline laparotomy incision. The corpus luteum, non-ruptured follicles greater than 6mm in diameter, and ovarian cysts were counted, the superovulation results were evaluated, and the number of embryos to be collected by tubal irrigation was predicted. The cannula was placed at the tubal ostium and secured with a single temporary 3.0Prolene ligature. A 20 gauge needle was placed in the uterus about 0.5cm from the uterotubal junction. Wash the cannula with 10-20mL sterile Phosphate Buffered Saline (PBS)And collected into a petri dish. The procedure is repeated contralaterally and the reproductive tract is then placed back into the abdomen. Before closure, 10-20mL of sterile glycerol salt solution is poured into the abdominal cavity to prevent adhesion. The leukorrhagia line was closed with a simple interrupted suture of 2.0Polydioxanone or Supramid and the skin closed with sterile suture clips.

Goat zygotes were collected from PBS oviduct washes under a stereomicroscope and washed with Ham's F12 medium purchased from Sigma containing 10% peptide bovine serum (FBS) (Sigma, st. If the pronuclei is visible, the embryo is immediately microinjected. If the pronuclei is not visible, the embryo is placed in Ham's F12 containing 10% FBS in 5% CO₂Short-term cultures at 37 ℃ in humidified air chambers with air until pronuclei is visible (Selgrath et al, Theriogenology, 1990. pp.1195-1205).

Microinjection procedure

Single cell goat embryos are placed in microdroplets of medium under oil on a glass coverslip. Fertilized eggs with two visible pronuclei were mounted on flame-polished, grasping micropipettes using a Normarski optical setup under a Zeiss upright microscope with mounting platform. Pre-nuclei, such as BC355 vector containing the coding sequence of interest operably linked to goat β -casein gene regulatory elements, were microinjected with a DNA construct of interest in injection buffer (Tris-EDTA) using fine glass microneedles (Selgrath et al, Theriogenology, 1990. pp.1195-1205).

Development of embryos

After microinjection, the surviving embryos were placed in Ham's F12 culture containing 10% FBS, then in 5% CO₂Incubation at 37 ℃ in a humidified air chamber with air until the recipient animal is ready for embryo transfer (Selgrath et al, THERIOGENOLOGY, 1990. p.1195-1205).

Receptor preparation

Synchronization of the estrous cycle of the recipient animal was induced using 6mg of norgestrel ear implant (Syncromate-B). On day 13 after implant insertion, animals were given a single non-superovulating injection (400i.u.) of Sigma pregnant women serum gonadotropin (PMSG). Female recipients are mated with vasectomized males to ensure estrus cycle synchronization (Selgrath et al, THERIOGENOLOGY, 1990. pp.1195-1205).

Embryo transfer

All embryos from the same female donor are put together and transferred, if possible, to a single recipient. The procedure was the same as described above for embryo collection, except that the oviduct was not cannulated and the embryo was in Ham's F12 containing 10% FBS in minimum volume, transferred to the oviduct lumen through the umbrella using a glass micropipette. Animals with ovarian ovulatory points greater than 6-8 are considered unsuitable as recipients. Incision closure and post-operative management are the same as for donor animals (see, e.g., Selgrath et al, Theriogenology, 1990. pp.1195-1205).

Monitoring pregnancy and delivery

Pregnancy was determined by ultrasound image examination 45 days after the first day of the resting estrus cycle. A second ultrasound examination was performed on day 110 to confirm pregnancy and to evaluate fetal sounds. Inoculating tetanus toxoid and Clostridium difficile C to pregnant recipient ewes on day 130&D. Selenium and vitamin E (Bo-Se) are administered IM and Ivermectin (Ivermectin) is administered SC. The ewes were moved to a clean barn on day 145 and allowed to acclimatize to the environment approximately before induction on day 147. 40mg of PGF2a (Lutalyse) on day 147^*Upjohn Company, Kalamazoo Michigan) induces labor. This injection was given as an IM in two doses, one 20mg dose, and then a 20mg dose 4 hours later. The first Lutalyse injection on the 147 th day^*The ewes were observed periodically throughout the day. The next morning, the observation was increased to 30 minute intervals. Parturition occurs 30-40 hours after the first injection. After delivery, the ewes milked to collect colostrum and confirm that the placenta delivered.

Confirmation F ₀ Transgenic Properties of animals

To screen transgenic F0 animals, genomic DNA was isolated from two different cell lines to avoid missing any chimeric (mosaic) transgenes. A chimeric animal is defined as any goat that does not have at least one copy of the transgene in every cell. Thus, take 2 days old F₀Animal ear tissue samples (mesoderm) and blood samples, genomic DNA was isolated (Lacy et al, ALABORATORY MANUAL, 1986, Cold Springs Harbor, NY; and Herrmann and Frischauf, METHODS ENZYMOLOGY, 1987.152: pp.180-183). DNA samples were analyzed BY polymerase chain reaction using primers SPECIFIC FOR HUMAN DECORIN GENE AND BY SOUTHERN BLOTONAALYSIS (THOMAS, PROC Natl.Acad.Sci., 1980.77: 5201-. Assay sensitivity was assessed by detecting one transgene copy in 10% of somatic cells.

Production herd creation and selection

The above method can be used to generate transgenic initiators (F)₀) Goats and other transgenic goats. Transgenic F₀Goat initiators, for example, are bred to produce milk if female, or to produce transgenic female offspring if male. The transgenic male initiator can be bred with a non-transgenic female to produce transgenic female offspring.

Delivery of transgenes and related characteristics

The transmission of the transgene of interest in the goat pedigree was analyzed by PCR and Southern blot analysis of ear tissues and blood. For example, Southern blot analysis of male initiators and 3 transgenic offspring showed no rearrangement and copy number variation between generations. Southern blots were probed with a human decorin cDNA probe. The blot was analyzed using Betasscope 603 and the copy number was determined by comparing the transgene to the endogenous gene for goat beta casein.

Evaluation of expression levels

The expression level of the transgenic protein in the milk of the transgenic animals was determined using enzymatic analysis or Western blotting.

Example 2: mouse model of antibody hinge region alteration

To check the feasibility of transgenic animals to produce recombinant therapeutic antibodies, the cDNA of antibody KMK917 was expressed in the mammary gland of transgenic mice. KMK917 was then purified from mouse milk and compared to KMK917 derived from fed-batch fermentation of Sp2/0 cells transfected with KMK 917. KMK917 transgenic mice were produced in GTC Biothereutics, Inc., Framingham, MA, USA. Subsequent purification and analytical identification were performed by sub-contractor.

Production of KMK917 transgenic mice

Generation of 3 KMK917 encoding constructs:

1.1099/2010 encoding KMK917 wild type

2.2012/2014 encoding a KMK917 hinge mutant (229 Ser → Pro)

3.2012/2017 encodes KMK917 hinge + Ch2 mutant (229 Ser → Pro, 236Leu → Glu)

The mutant construct was generated in order to reduce the half antibody fraction observed in substance KMK917 derived from the wild-type construct. A total of 15 transgenic mouse lines were obtained based on these constructs (see tables 1a-c for line profiles and markers). Table 1 contains the expression level of KMK917 in mouse lines evaluated by Western blot.

TABLE 1a transgenic mouse lines obtained using the wild type of construct 1099/2010

Mouse species (sex)	Milk-extracting substitute		Date of milk taking	Approximate volume (μ L)	mu.L of PBS	Experimental level of assay (mg/mL)
	Milk-extracting substitute						F0	F1	F2
	1-73F	1-73					F0	F1	F2		7913	175225100	700900400	＜1
		1-73			In all	500μl	2000μl				7913	175225100	700900400	＜1
		1-78M			In all	500μl	2000μl		2-119		10	150	600	10+
			3-150	81014	125250100	5001000400			2-119		10	150	600	10+
			3-150	81014	125250100	5001000400				In all	625μl	2500μl
1-46M			2-138		10	50ul	200ul			In all	625μl	2500μl		10+
			2-138		10	50ul	200ul	3-145	2/5/022/11/02	15050	600200			10+
					In all	250μl	1000μl	3-145	2/5/022/11/02	15050	600200

Table 1b Using construct 2012/2014 hinge mutant derived transgenic mouse line

Mouse species (sex)	Milk-extracting substitute			Date of milk taking	Approximate volume (μ L)	mu.L of PBS	Measured test level (mg/ml)
	Milk-extracting substitute							F0	F1	F2
	1-4F	1-4						F0	F1	F2		711	125125	500500	7-10
		1-4		2-120		71113	250150100	1000600400				711	125125	500500	7-10
				2-120		71113	250150100	1000600400			In all	750μl	3000μl
1-57F				1-57			101315	2002550	800100200		In all	750μl	3000μl		4-5
		2-141		1-57			101315	2002550	800100200	611	150100	600400			4-5
		2-141			2-143		99	200200	800800	611	150100	600400
		2-144			2-143		99	200200	800800	71012	150250250	60010001000
		2-144					In all	1575μl	6300μl	71012	150250250	60010001000
	1-62F		2-145				In all	1575μl	6300μl		61012	100125125	400500500		7-10(1-62F)
			2-145	2-147		6	75	300			61012	100125125	400500500		7-10(1-62F)
				2-147		6	75	300			In all	425μl	1700μl
1-65M					2-149		710	5050	200200		In all	425μl	1700μl
		2-150			2-149		710	5050	200200	71012	150100200	600400800
		2-150					In all	550μl	2200μl	71012	150100200	600400800
	1-76F	1-76					In all	550μl	2200μl		6991111	150250250200200	60010001000800800
		1-76				In all	1050μ1	4200μl			6991111	150250250200200	60010001000800800
		1-96F	1-96			In all	1050μ1	4200μl				6911	50250200	2001000800
			1-96		In all	500μl	2000μl					6911	50250200	2001000800

TABLE 1c transgenic mouse lines obtained with construct 2012/2017

Mouse species (sex)	Milk-extracting substitute			Date of milk taking	Approximate volume (μ L)	mu.L of PBS	Experimental level of assay (mg/mL)
	Milk-extracting substitute							F0	F1	F2
	1-7M		2-92					F0	F1	F2		911	200100	800400
			2-92	2-93		68	10075	400300				911	200100	800400
			2-94	2-93		68	10075	400300			579	12515075	500600300
			2-94			In all	825μl	3300μl			579	12515075	500600300
		1-13F				In all	825μl	3300μl		2-87		5711	175200125	700800500	～1(1-13F)
					In all	500μl	2000μl			2-87		5711	175200125	700800500	～1(1-13F)
			1-25F		In all	500μl	2000μl			2-108		6810	5010075	200400300	～1.5(1-25F)
	2-109			6812	15050125	600200500				2-108		6810	5010075	200400300	～1.5(1-25F)
	2-109			6812	15050125	600200500					In all	550μl	2200μl
1-30F		2-116			6812	250200125	1000800500				In all	550μl	2200μl		～1(1-30F)
		2-116			6812	250200125	1000800500	2-118		571112	200250150150	8001000600600			～1(1-30F)
					In all	1325μl	5300μl	2-118		571112	200250150150	8001000600600
			1-36F		In all	1325μl	5300μl	1-36			5911	125100125	500400500		10+
	2-126			5	50	200		1-36			5911	125100125	500400500		10+
	2-126			5	50	200			2-127		7	100	400
				In all	500μl	2000μl			2-127		7	100	400

TABLE 1c (next) transgenic mouse lines obtained with construct 2012/2017

Mouse germline (gender)	Milk-extracting substitute		Date of milk taking	About volume(μL)	mu.L of PBS	Measured test level (mg/ml)
	Milk-extracting substitute						F0	F1	F2
	1-61M	2-129					F0	F1	F2		881212	200200150150	800800600600
		2-129	2-131		661012	125125250200	5005001000800				881212	200200150150	800800600600
		2-133	2-131		661012	125125250200	5005001000800			66810	175175250150	7007001000600
		2-133			In all	2150ul	8600ul			66810	175175250150	7007001000600

Purification and characterization of KMK917 in transgenic mouse milk

Milk samples from a total of 15 transgenic mouse lines (F0, F1, and/or F2 passages) were collected and diluted with PBS (see table 1 for details). The sample was then purified and the KMK917 antibody was identified. See figure 2 for an overview of the analysis performed.

To remove the colloidal components of the milk, a pre-diluted sample of milk was centrifuged at high speed for 30 minutes (SS-34 rotor, 20,000 rpm) using a Sorval centrifuge, and the supernatant was aspirated from the pellet using a syringe and the upper fat layer was removed. The light milky-white supernatant was filtered through a 0.22 μm Millex-GV filter and loaded onto a 1mL protein A column (MabSelect, APB). Bound antibody was eluted using 20mM sodium citrate/citric acid pH 3.2.

The antibody fractions were adjusted to pH5.5, sterile filtered and stored at 4 ℃.

Determination of KMK917 content in transgenic mouse milk

The KMK917 concentration of a pre-diluted mouse milk sample was determined using a commercial ELISA kit that can detect human IgG 4. The KMK917 content values of the corresponding undiluted mouse milk are shown in table 2.

TABLE 2 milk KMK917 concentration of transgenic lines

Construct	Is a system	Content in milk (mg/mL)		Content in purified fraction (mg/mL)		KMK917 amount (mg)
		Content in milk (mg/mL)		Content in purified fraction (mg/mL)		KMK917 amount (mg)	IgG4 ELISA	Derived from SEC	SEC	IgG4ELISA	SEC
		1099/2010 wild type							SEC	IgG4ELISA	SEC
	1-73	1099/2010 wild type							3.2	-	-	-	-
	1-73		1-78	＞10	22.1	3.2	3.4	9.1	3.2	-	-	-	-
	1-46		1-78	＞10	22.1	3.2	3.4	9.1	8.5	7.7	0.8	1.0	1.7
	1-46	2012/2014 hinge mutation							8.5	7.7	0.8	1.0	1.7
	1-4	2012/2014 hinge mutation								4.5	0.9	1.1	1.8
	1-4		1-57	3.5	-	-	-	-		4.5	0.9	1.1	1.8
	1-62		1-57	3.5	-	-	-	-	16	-	-	-	-
	1-62		1-65	11	10.9	1.9	2.8	4.6	16	-	-	-	-
	1-76		1-65	11	10.9	1.9	2.8	4.6	0.8	-	-	-	-
	1-76		1-96	3.4	-	-	-	-	0.8	-	-	-	-
2012/2017 hinge and Ch2 mutation							-	-
2012/2017 hinge and Ch2 mutation								1-7	5.5	3.2	0.9	1.1	1.9
	1-13	2.3	-	-	-	-		1-7	5.5	3.2	0.9	1.1	1.9
	1-13	2.3	-	-	-	-		1-25	1.5	4.7	0.8	0.9	1.4
	1-30	4.5	-	-	-	-		1-25	1.5	4.7	0.8	0.9	1.4
	1-30	4.5	-	-	-	-		1-36	＞10	9.7	1.4	1.5	3.3
	1-61	1	-	-	-	-		1-36	＞10	9.7	1.4	1.5	3.3

KMK917 from selected mouse lines (2 or 3 per construct) was then purified using protein A chromatography as described in 3.2. KMK917 content was then determined in the antibody fraction by size exclusion HPLC (SEC) (Table 2). The total amount of KMK917 available for further analysis is shown in table 2.

SEC analysis showed that all antibody samples contained more than 95% monomeric antibody. KMK917 content of the mouse milk sample was calculated based on the KMK917 content of the antibody component measured and the volume used for protein a purification. The estimated mouse milk KMK917 concentration was found to be 3.2-22.1mg/mL, which correlates well with the value of the mouse milk directly measured by IgG4 ELISA (Table 2).

Presence of mouse antibody in purified KMK917 material

Protein a purification was used to enrich not only for human IgG isotypes but also for certain mouse antibody isoforms that may be present in milk, thus examining the presence of mouse immunoglobulin in purified KMK 917. Using the SPR technique (Biacore 3000) and immobilized anti-mouse IgG as a "capture molecule", no or only minimal mouse IgG subclass (< 0.1%) was detected in the purified KMK917 material. Concentration measurements of the purified material using SEC and human IgG4 ELISA both showed very similar results, also supporting the above findings (table 2). It is possible to measure a large number of mouse immunoglobulins at higher concentration levels as determined by SEC because this method measures not only KMK917 but also mouse antibodies. In contrast, ELISA was specific for human IgG4 and therefore only KMK917 was detected.

Presence and amount of "half antibody

The amount of half-antibody present in substance KMK917 from the transgenic mouse line was determined using SDS-PAGE and SDS-DSCE. SDS-PAGE revealed that samples from wild-type transfected mice had a higher half antibody fraction than samples from mutant construct transfected mice.

These results were confirmed by SDS-DSCE, showing 24 and 34% half antibodies in substance KMK917 derived from the transgenic line generated with the wild type construct. The half antibody fraction of KMK917 material from mutant constructs was found to be very low at 5%, especially material derived from a single mutant construct (see summary of table 4).

To evaluate the biological activity of KMK917 derived from different constructs, a fluorescence-based cellular assay was used in which KMK917 competes with one cellular receptor for binding to its receptor target molecule. KMK917 from both wild-type and mutant transfected mice was found to have intact biological activity compared to KMK917 from cell culture (Sp2/0) (see table 4).

For further characterization, the kinetic rate constants for binding and dissociation of KMK917 to its ligand target were determined using the SPR technique (Biacore 3000). The rate constant of transgenic mouse-derived material was found to be similar to Sp 2/0-derived KMK917 in all samples. This indicates that the binding affinity and biological activity of KMK917 was (1) similar whether expressed in transgenic mice or cell line Sp2/0 and (2) not affected by the introduction of cDNA mutations.

Table 4.

KMK917 analysis profile from transgenic mice

Analytical experiments		Wild type		Hinge snap		Hinge + Ch2 mutation
		Wild type		Hinge snap		Hinge + Ch2 mutation			Is 1-46HT560/1	Series 1-78HT557/4	Is 1-4HT557/2	Is 1-65HT560/2	Is 1-7HT560/3	Is 1-25HT557/1	Series 1-36HT557/3
		Measurement amount of mouse Ab (%)	Biacore	＜0.1	0	＜0.1	＜0.1	＜0.1	Is 1-46HT560/1	Series 1-78HT557/4	Is 1-4HT557/2	Is 1-65HT560/2	Is 1-7HT560/3	Is 1-25HT557/1	Series 1-36HT557/3	～0.1	＜0.1
Semi-molecular antibody (%)	SDS-DSCE	Measurement amount of mouse Ab (%)	Biacore	＜0.1	0	＜0.1	＜0.1	＜0.1	24.0	34.4	1.8	1.6	4.6	2.9	4.9	～0.1	＜0.1
	SDS-DSCE	SDS-PAGE	38.1	43.5	2.4	3.7	7.6	4.0	24.0	34.4	1.8	1.6	4.6	2.9	4.9	4.5
	Biological Activity (% relative efficacy)	SDS-PAGE	38.1	43.5	2.4	3.7	7.6	4.0	FACS	105	99	115	116	109	94/98	4.5	122
Biological affinities (binding and dissociation rate constants ka and kd; ka (KMK ref) ═ 4.1kd (KMK ref) ═ 4.7)	Biological Activity (% relative efficacy)	Biacoreka(10⁶(Ms)^-1)	5.3	4.2	4.3	4.8	4.9	4.7	FACS	105	99	115	116	109	94/98	4.4	122
	Biacorekd(10^-4s^-1)	Biacoreka(10⁶(Ms)^-1)	5.3	4.2	4.3	4.8	4.9	4.7	3.5	3.7	3.0	3.6	4.2	2.4	3.8	4.4
	Biacorekd(10^-4s^-1)	Heterogeneity of eluted forms (KMKref ═ +)	CEx-HPLC	++	++	+++	+++	+++	3.5	3.7	3.0	3.6	4.2	2.4	3.8	nd	+++

^*Western blot assay nd-unidentified

Glycosylation pattern

The purified KMK917 material was analyzed using cation exchange HPLC. The particular method used enables the isolation of the C-terminal des-Lys variant of the antibody (variant K0, variant K1 and variant K2) and also allows the discrimination of antibodies in different glycosylated forms, such as sialylated and non-sialylated glycoforms (glycoforms) and also mannoforms and complex glycoforms.

Figures 3a-3g show elution profiles of KMK reference samples from cell cultures and of antibodies from milk samples. The 3 main peaks of the reference correspond to the K0, K1 and K2 variants.

Samples from transgenic milk were more heterogeneous. The two wild-type samples showed additional peaks eluting earlier than the reference, probably due to sialylated glycoforms. The resulting antibody samples from the mutant lines showed very heterogeneous patterns, with the variants also eluting after the reference.

To illustrate how much of the observed heterogeneity was caused by different glycosylation patterns, wild type and mutant samples were deglycosylated by N-glycosidase treatment. FIGS. 4a-4d show CEx-HPLC profiles of wild-type samples before and after glycosidase treatment. Deglycosylated wild-type samples produced a more heterogeneous pattern. The two peaks obtained in the 4: 1 ratio most likely correspond to the K0 and K1 forms of the antibody. From this result, it can be concluded that the observed heterogeneity of wild-type antibodies is mainly due to glycoforms.

The mutant antibodies of lines 1-36 also produced approximately the same ratio of the two major peaks. However, these two peaks elute far apart with a set of secondary peaks (see fig. 3 b). This behavior may be explained by the presence of different antibody conformers in the mutant variants, possibly due to partial unfolding. Thus, the major heterogeneity observed in mutant antibody CEx-HPLC analysis appears to arise not only from different glycoforms, but also from other causes.

Further elucidation of the sugar side chain structure has been performed using purified KMK 917. After enzymatic cleavage using PNGase F, the sugar side chains were isolated and labeled with 2-aminobenzamide. Each sugar structure was separated using Glyco Sep N-column HPLC and quantified by fluorescence detection. FIGS. 5a-5c show the chromatograms analyzed for KMK917 from a) transgenic mice, wild type, b) transgenic mice, mutant, c) cell cultures.

The chromatogram showed that KMK917 from the transgenic mice had a significantly different sugar profile than the antibodies isolated from the cell culture. The pattern of the mutant was qualitatively the same as the wild type, showing only some quantitative differences. Given the comparison with other known sugar side chain structures, several peaks have been clearly assigned from the HPLC plot. The molecular structure is shown in table 3.

TABLE 3 molecular Structure of sugar side chains

Peak #	RT(min)	Candy structure
Peak #	RT(min)	Candy structure	1	31.4	？
2	34.3	G0	1	31.4	？
2	34.3	G0	3	37.1	Man 5
4+5	39.7+40.4	G1	3	37.1	Man 5
4+5	39.7+40.4	G1	6	43.1	Man 6
7	45.9	G2	6	43.1	Man 6
7	45.9	G2	8	47.5	？
9	50.2	？	8	47.5	？
9	50.2	？	10	52.9	？
11	Ca.56	？	10	52.9	？
11	Ca.56	？	12	59.2	？

To confirm the molecular structure obtained from HPLC and to obtain some additional information about delayed elution peaks, the sugar mixture was also analyzed by MALDI-MS. MALDI-MS in negative format suggested another structure, the sugar BiG2S1 containing sialic acid (sialinic acid).

Expression of KMK917 in the mammary gland of transgenic mice produced mouse milk KMK917 titers of 3.2-22.1 mg/mL. Further identification of KMK917 derived from 3 different KMK917 constructs revealed a higher amount of "half antibody" derived from wild type construct 1099/2010 material (24% and 34%, respectively). Introduction of the 229 Ser → Pro mutation (constructs 2012/2014 hinge and 2012/2017 hinge + Ch2) significantly reduced the "half antibody" number value, 2012/2014 was less than 2%, and 2012/2017 was less than 5%. The biological activity of the material obtained for all 3 constructs showed no difference compared to cell culture derived KMK 917.

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A method of producing antibodies in milk of a transgenic mammal comprising: transgenic mammals are provided whose somatic and germ cells comprise sequences encoding an exogenous heavy chain variable region or antigen-binding fragment thereof, at least one heavy chain constant region or fragment thereof, and a hinge region operably linked to a promoter that directs expression in mammary epithelial cells, wherein the hinge region has been altered from the hinge region normally associated with the heavy chain constant region.

2. The method of claim 1, wherein at least 70% of the antibodies present in the milk are in assembled form.

3. The method of claim 1, wherein the transgenic mammal further comprises sequences encoding a light chain variable region, or antigen-binding fragment thereof, and a light chain constant region, or functional fragment thereof, operably linked to a promoter that directs expression in mammary epithelial cells.

4. The method of claim 1, further comprising the step of obtaining milk from said transgenic mammal, thereby providing an antibody composition.

5. The method of claim 4, further comprising the step of purifying the exogenous antibody from milk produced by said transgenic mammal.

6. The method of claim 1, wherein the promoter is selected from the group consisting of: casein promoter, whey protein promoter, beta lactoglobulin promoter and whey acidic protein promoter.

7. The method of claim 1, wherein the transgenic mammal is selected from the group consisting of: cattle, goats, mice, rats, sheep, pigs and rabbits.

8. The method of claim 1, wherein the antibody is selected from the group consisting of: IgA, IgD, IgM, IgE or IgG.

9. The method of claim 1, wherein the antibody is an IgG antibody.

10. The method of claim 1, wherein the antibody is an IgG4 antibody.

11. The method of claim 10, wherein all or a portion of the hinge region of the antibody has been altered.

12. The method of claim 10, wherein all or part of the hinge region of the antibody has been replaced, for example by a hinge region or part thereof which is different from the hinge region normally associated with the heavy chain constant region.

13. The method of claim 10, wherein the amino acid sequence of the hinge region of the antibody differs from the amino acid sequence of the hinge region naturally associated with said heavy chain constant region by at least one amino acid residue.

14. The method of claim 1, wherein the nucleic acid sequence encoding the hinge region of the antibody differs from the naturally occurring nucleic acid sequence of the hinge region naturally associated with said heavy chain constant region by at least one nucleic acid residue.

15. The method of claim 12, wherein the hinge region of the antibody or portion thereof has been replaced with a hinge region or portion thereof of an antibody other than an IgG4 antibody.

16. The method of claim 12, wherein the hinge region of the antibody or portion thereof has been replaced with a hinge region or portion thereof derived from an antibody selected from the group consisting of IgG1, IgG2, and IgG 3.

17. The method of claim 12, wherein the hinge region of the antibody or portion thereof has been replaced with a hinge region or portion thereof derived from an antibody selected from the group consisting of IgA, IgD, IgM, and IgE.

18. The method of claim 12, wherein one or more amino acids of the hinge region have been replaced with an amino acid at a position corresponding to an antibody other than an IgG4 antibody.

19. The method of claim 15, wherein the antibody other than the IgG4 antibody is selected from the group consisting of: IgA, IgD, IgM and IgE.

20. The method of claim 15, wherein the antibody other than the IgG4 antibody is selected from the group consisting of IgG1, IgG2, and IgG 3.

21. The method of claim 10, wherein the serine residue of the hinge region has been replaced with a proline residue.

22. The method of claim 10, wherein the serine residue at amino acid number 241 of the hinge region has been replaced with a proline residue.

23. The method of claim 10, wherein at least one amino acid of the hinge region other than the cysteine residue is replaced with a cysteine.

24. The method of claim 10, wherein at least one glycosylation site of the antibody is altered.

25. The method of claim 24, wherein at least one glycosylation site of the heavy chain or light chain is altered.

26. The method of claim 24, wherein at least one glycosylation site of the hinge region of the heavy chain is modified.

27. The method of claim 1, wherein the antibody is a humanized antibody.

28. The method of claim 1, wherein the antibody is a chimeric antibody.

29. The method of claim 1, wherein the antibody is a human antibody.

30. The method of claim 1, wherein the milk of the transgenic mammal is substantially free of a half-molecule form of the exogenous antibody.

31. The method of claim 1, wherein the ratio of assembled exogenous antibody to half-molecular form antibody present in the milk of the transgenic mammal is at least 2: 1, 3: 1, 4: 1, or 5: 1.

32. A method of producing a transgenic mammal whose somatic and germ cells comprise a modified antibody coding sequence, wherein said modified antibody coding sequence encodes an antibody molecule or portion thereof expressible in milk comprising a modified hinge region, said method comprising the steps of: introducing into a mammal a construct comprising a sequence encoding a foreign heavy chain variable region or antigen-binding fragment thereof, at least one heavy chain constant region or fragment thereof, and a hinge region operably linked to a promoter that directs expression in mammary epithelial cells, wherein the hinge region has been altered from the hinge region normally associated with the heavy chain constant region.

33. The method of claim 33, wherein said hinge region has been altered such that at least 70% of the exogenous antibody present in the milk of the transgenic mammal is in assembled form.

34. The method of claim 33, wherein the modified antibody coding sequence further comprises a coding sequence for a light chain variable region or antigen binding fragment thereof and a light chain constant region or functional fragment thereof operably linked to a promoter that directs expression in mammary epithelial cells.

35. The method of claim 33, wherein the promoter is selected from the group consisting of a casein promoter, a whey protein promoter, a beta lactoglobulin promoter, and a whey acidic protein promoter.

36. The method of claim 33, wherein the transgenic mammal is selected from the group consisting of cattle, goats, mice, rats, sheep, pigs, and rabbits.

37. The method of claim 33, wherein the antibody is selected from the group consisting of: IgA, IgD, IgM, IgE or IgG.

38. The method of claim 33, wherein the antibody is an IgG antibody.

39. The method of claim 33, wherein the antibody is an IgG4 antibody.

40. The method of claim 40, wherein all or part of the hinge region of the antibody has been altered.

41. The method of claim 40, wherein all or part of the hinge region of the antibody has been replaced, for example with a hinge region or part thereof which is different from the hinge region normally associated with said heavy chain variable region or said constant region.

42. The method of claim 40, wherein the amino acid sequence of the hinge region of the antibody differs from the amino acid sequence of the hinge region naturally associated with said heavy chain constant region by at least one amino acid residue.

43. The method of claim 33, wherein at least one nucleic acid residue of the nucleic acid sequence encoding the hinge region of the antibody is different from the nucleic acid sequence of the hinge region naturally associated with said heavy chain constant region.

44. The method of claim 44, wherein the hinge region of the antibody or portion thereof has been replaced with a hinge region or portion thereof of an antibody other than an IgG4 antibody.

45. The method of claim 42, wherein the hinge region of the antibody or portion thereof has been replaced with a hinge region or portion thereof derived from an antibody selected from the group consisting of IgG1, IgG2, and IgG 3.

46. The method of claim 42, wherein the hinge region of the antibody or portion thereof has been replaced with a hinge region or portion thereof derived from an antibody selected from the group consisting of IgA, IgD, IgM and IgE.

47. The method of claim 42, wherein one or more amino acids of the hinge region have been replaced with an amino acid at a position corresponding to an antibody other than an IgG4 antibody.

48. The method of claim 48, wherein the antibody other than an IgG4 antibody is selected from the group consisting of: IgA, IgD, IgM and IgE.

49. The method of claim 48, wherein the antibody other than an IgG4 antibody is selected from the group consisting of IgG1, IgG2, and IgG 3.

50. The method of claim 40, wherein a serine residue of the hinge region has been replaced with a proline residue.

51. The method of claim 40, wherein the serine residue at amino acid number 241 of the hinge region has been replaced with a proline residue.

52. The method of claim 40, wherein at least one amino acid of the hinge region other than a cysteine residue is replaced with a cysteine.

53. The method of claim 40, wherein at least one glycosylation site of the antibody is altered.

54. The method of claim 54, wherein at least one glycosylation site of the heavy chain or light chain is altered.

55. The method of claim 40, wherein at least one glycosylation site of the hinge region of the heavy chain is modified.

56. The method of claim 33, wherein the antibody is a humanized antibody.

57. The method of claim 33, wherein the antibody is a human antibody.

58. The method of claim 33, wherein the antibody is a chimeric antibody.

59. The method of claim 33, wherein said hinge region has been altered such that the milk of the transgenic mammal is substantially free of a half-molecule form of the exogenous antibody.

60. The method of claim 33, wherein the ratio of assembled exogenous antibody to half-molecular form antibody present in the milk of the transgenic mammal is at least 2: 1, 3: 1, 4: 1, or 5: 1.

61. The method of claim 60, wherein the antibody is selected from the group consisting of: IgA, IgD, IgM, IgE or IgG.

62. A method of making a transgenic mammal capable of expressing an assembled exogenous antibody or portion thereof in its milk, the method comprising: introducing into a mammal a construct comprising an exogenous antibody light chain coding sequence operably linked to a promoter that directs expression in mammary epithelial cells; and introducing into the mammal a construct comprising a coding sequence for a mutated heavy chain of the exogenous antibody or a portion thereof operably linked to a promoter that directs expression in mammary epithelial cells, wherein the heavy chain or portion thereof comprises a hinge region that has been altered such that at least 70% of the exogenous antibody present in milk is in assembled form.

63. A method of making a transgenic mammal capable of expressing an assembled exogenous antibody in its milk, the method comprising: providing cells from a transgenic mammal whose somatic and germ cells comprise an exogenous antibody light chain coding sequence operably linked to a promoter that directs expression in mammary epithelial cells; and introducing into the cell a construct comprising a coding sequence for a mutated heavy chain of the exogenous antibody, or a portion thereof, operably linked to a promoter that directs expression in mammary epithelial cells, wherein the heavy chain or portion thereof comprises a hinge region that has been altered such that at least 70% of the exogenous antibody present in milk is in assembled form.

64. A composition comprising a milk component and an antibody component, wherein the antibody component comprises an exogenous antibody or portion thereof having a hinge region, wherein the hinge region has been altered from the hinge region normally associated with an antibody.

65. The composition of claim 63, wherein at least 70% of the exogenous antibodies present in the composition are in assembled form.

66. The composition of claim 63, wherein said hinge region has been altered such that at least 70% of the exogenous antibodies present in said composition are in assembled form.

67. The composition of claim 63, wherein the antibody is selected from the group consisting of: IgA, IgD, IgM, IgE or IgG.

68. The composition of claim 63, wherein the antibody is an IgG antibody.

69. The composition of claim 67, wherein the antibody is an IgG4 antibody.

70. The composition of claim 63, wherein all or part of the hinge region of the antibody has been altered.

71. The composition of claim 63, wherein all or part of the hinge region of the antibody has been replaced, for example with a hinge region or part thereof which is different from the naturally occurring hinge region normally associated with an antibody.

72. The composition of claim 63, wherein the amino acid sequence of the hinge region of the antibody differs from the amino acid sequence of the hinge region of a naturally occurring antibody by at least one amino acid residue.

73. The composition of claim 63, wherein the hinge region of the antibody or portion thereof has been replaced with a hinge region or portion thereof of an antibody other than an IgG4 antibody.

74. The composition of claim 72, wherein the hinge region of the antibody or portion thereof has been replaced with a hinge region or portion thereof derived from an antibody selected from the group consisting of IgG1, IgG2, and IgG 3.

75. The composition of claim 72, wherein the hinge region of the antibody or portion thereof has been replaced with a hinge region or portion thereof derived from an antibody selected from the group consisting of IgA, IgD, IgM and IgE.

76. The composition of claim 63, wherein one or more amino acids of the hinge region have been replaced with an amino acid at a position corresponding to an antibody other than an IgG4 antibody.

77. The composition of claim 75, wherein the antibody other than IgG4 is selected from the group consisting of: IgA, IgD, IgM and IgE.

78. The composition of claim 75, wherein the antibody other than an IgG4 antibody is selected from the group consisting of IgG1, IgG2, and IgG 3.

79. The composition of claim 63, wherein a serine residue of the hinge region has been replaced with a proline residue.

80. The composition of claim 63, wherein the serine residue at amino acid number 241 of the hinge region has been replaced with a proline residue.

81. The composition of claim 63, wherein at least one amino acid of the hinge region other than a cysteine residue is replaced with a cysteine.

82. The composition of claim 63, wherein at least one glycosylation site of the antibody has been altered.

83. The composition of claim 63, wherein at least one glycosylation site of the heavy chain or light chain of the antibody is altered.

84. The composition of claim 82, wherein at least one glycosylation site of the hinge region of the heavy chain of the antibody is modified.

85. The composition of claim 63, wherein the antibody is a humanized antibody.

86. The composition of claim 63, wherein the antibody is a human antibody.

87. The composition of claim 63, wherein said hinge region has been altered such that the composition is substantially free of a half-molecular form of exogenous antibodies.

88. The composition of claim 63, wherein the ratio of assembled foreign antibody to half-molecular form antibody present in the composition is at least 2: 1, 3: 1, 4: 1, or 5: 1.

89. A nucleic acid comprising a sequence encoding a heavy chain variable region and a heavy chain constant region operably linked to a promoter that directs expression in mammary epithelial cells, wherein the heavy chain or portion thereof comprises a hinge region that has been altered such that at least 70% of exogenous antibodies present in milk are in assembled form.