Engineered heterodimeric protein domains

ES 2 395 969 T3

DESCRIPTION

Genetically modified heterodimeric protein domains

Invention area.

The present invention relates to heterodimeric immunoglobulin domains and methods of producing the same.

Background of the invention

Nature provides a large number of heterodimeric proteins and protein domains that fall into related protein families. Such proteins and domains often form homodimers with each other, but do not form heterodimers with other members of the family. On the other hand, heterodimeric or heteromultimeric proteins are often useful. They provide novel research and therapeutic tools. For example, bispecific antibodies (BsAbs) capable of binding to at least two different antigens present significant potential in a wide range of clinical applications as targeting agents, for immunodiagnosis and in vitro therapy. and in vivo, and for diagnostic immunoassays. In the diagnostic area, BsABs have been very useful in hybridizing the functional properties of cell surface molecules, and in defining the ability of the different Fc receptors to mediate cytotoxicity (Fanger et al. (1992) Crit. Rev. Immunol, 12: 101-124, the contents of which are incorporated herein by reference). However, when BsAbs are generated simply by the co-expression of multiple components that can interact without specificity, a large number of species are generated, and it is often difficult to separate the desired species from the undesired species. Therefore, it is desirable to have techniques to produce heteromultimers efficiently. It is particularly desirable to generate subunits of antibodies that form heterodimers in a manner preferential to the formation of homodimers, so that BsAbs can be recovered directly from recombinant cell cultures.

Methods of making heterodimeric proteins have been reported. For example, Stahl and Yancopoulos have described the use of fusion proteins that include two different receptor subunits to form soluble heterodimeric receptors that could bind to a given cytokine in circulation, and thus block the activity of that cytokine (see US Pat. No. 6,472,179). Carter et al. have described a "cavity bulge" approach to generate a heterodimeric Fc fraction (see US Patent No. 5,807,706).

These existing methods allow the constructions of individual heterodimers, but do not provide general techniques for the construction of multimeric proteins that involve multi-domain interactions. Therefore, there is a need for a general system for the design of heterodimeric pairs that can be specifically assembled in an environment containing multiple different potential assembly partners.

Summary of the invention

The present invention provides a novel approach for designing immunoglobulin domains that preferentially heterodimerize. In particular, the present invention uses a "Strand Exchange Engineered Domain" (SEED) strategy to engineer the protein-protein interaction interface within said heterodimerization. of immunoglobulin domains. The invention also provides immunoglobulins containing domains engineered using the method of the present invention.

In one aspect, the present invention features a multidomain heterodimeric immunoglobulin, including at least one non-identical modified first and second domain, each containing a protein-protein interaction interface containing amino acid sequence segments derived from two or more natural homologous parental domains, thereby granting, to the modified first and second domains assembly specificities other than the assembly specificities of the parent domains, where the modified first and second domains form heterodimers for each other, preferentially to the formation of homodimers (for example, heterodimers constitute more 55%, 65%, 75%, 80%, 85%, 90%, or 95% of the total amount of dimers). The modified first and second domains are not antibody variable domains. In some embodiments, the multidomain immunoglobulin of the invention includes a first subunit that contains the first modified domain, and a second subunit that contains the second modified domain. As used in the present patent, an "amino acid sequence segment" includes any sequence segment that contains two or more amino acids (eg, three or more, four or more, five or more, six or more, seven or more. more, eight or more, nine or more, or ten or more).

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In preferred embodiments, the multidomain immunoglobulin includes non-identical domains engineered from native homologous parent domains, eg, CH3 domains of antibodies. In particular, the modified domains are derived from the CH3 domains of IgG and IgA.

In some embodiments, the multidomain immunoglobulin of the invention includes modified domains that are part of polypeptide chains that are connected by a disulfide bridge.

In one embodiment, one of the modified domains contained in the multi-domain immunoglobulin of the invention includes at least two non-adjacent sequence segments derived from the same parental domain. In another embodiment, each of the first and second domains includes at least two, three, or four or more non-adjacent sequence segments, derived from the same parent domain. In another embodiment, at least one of the modified domains includes sequence segments from each parent domain that are at least two amino acids in length. In another embodiment, at least one of the modified domains includes sequence segments from each parent domain that are at least three, four, five, or six amino acids in length.

In some embodiments, the multidomain immunoglobulin of the invention includes a first bioactive domain. The first bio-active domain can occupy an N-terminal or C-terminal position with respect to the first modified domain.

In additional embodiments, the multidomain immunoglobulin may additionally include a second bio-active domain in addition to the first bio-active domain. In one embodiment, the second bioactive domain is associated with the second modified domain, and can occupy an N-terminal or C-terminal position with respect to the second modified domain. In an alternative embodiment, the second bio-active domain is also associated with the first modified domain, and may occupy a position opposite the first bio-active domain. For example, the first and second bioactive domains can occupy N-terminal and C-terminal positions, respectively, relative to the first modified domain.

The multidomain immunoglobulin of the present invention can be used to generate bispecific antibodies. For example, the multidomain protein can include a first bio-active domain that contains an antibody variable domain, and a second bio-active domain that contains a second antibody variable domain with different specificity.

In another aspect, the invention provides a multidomain immunoglobulin, wherein the first bioactive region contains two or more variable domains of antibodies of a first specificity, or of a first combination of specificities. The multidomain protein can also contain a second bio-active region that includes two or more antibody variable domains of a second specificity or a second combination of specificities. For example, the multidomain protein may include one or more single chain Fv moieties, a single chain diabody [a VH (1) - VL (2) ----- VH (2) - VL (1)], or other repeats fused to single chain Fv (of the same or different specificities).

In another aspect, the invention provides a multidomain immunoglobulin wherein the first bioactive region comprises two or more variable domains of antibodies of a first specificity or of a first combination of specificities. The multidomain immunoglobulin further comprises a second bioactive region comprising two or more antibody variable domains of a second specificity or second combination of specificities, which are substantially different from the first combination of specificities.

The present invention further describes a method of colocalization of bioactive domains when administered to a biological system. The method includes the step of administering to the biological system the heterodimeric immunoglobulin inculcating the first and second bioactive domains, as described above in various embodiments. In one embodiment, the biological system is a mammal. In a more preferred embodiment, the biological system is a human.

In another aspect, the present invention provides a multidomain heterodimeric immunoglobulin that includes at least a non-identical first and second domain that converge at an interface. The interface of the first modified domain contains at least two segments of amino acid sequences, where each segment is derived from a different natural homologous parental domain, thereby conferring an assembly specificity distinct from the assembly specificity of the parent domains, wherein the first and second engineered domains form heterodimers. In a preferred embodiment, the modified second domain also contains at least two amino acid sequence segments, where each segment is derived from a different natural homologous parental domain, thereby conferring an assembly specificity different from that of assembly specificity of parental domains, wherein the second engineered domains form heterodimers.

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In yet another aspect, the present invention provides a multidomain immunoglobulin that includes at least non-identical engineered first and second domains that converge at an interface, wherein (1) the engineered first and second domains are derived of two or more natural homologous parental domains, (2) the interface of the first modified domain comprises at least one amino acid sequence segment that interacts with an amino acid sequence segment, over the interface of the second engineered domain derived from the same parent domain, and (3) the domains first and second engineered to form heterodimers.

In a further aspect, the present invention features an engineered immunoglobulin domain containing a protein-protein interaction interface that includes amino acids from two or more parent immunoglobulin domains, in such a way that the protein-protein interaction interface gives the genetically modified immunoglobulin domain assembly specificities that are different from the assembly specificities of the parental immunoglobulin domains, where the genetically modified immunoglobulin domain is not a variable domain of antibody. In preferred embodiments, the genetically modified immunoglobulin domain of the invention is assembled with a parental domain with increased specificity, compared to the parental domains. In some embodiments, the parent domain is an engineered immunoglobulin domain of the invention.

In yet another aspect, the present invention provides a genetically modified immunoglobulin superfamily domain containing a protein-protein interaction interface, including amino acids from two or more domains of a parent immunoglobulin superfamily, in such a way that the protein-protein interaction interface imparts to the domain of the genetically modified immunoglobulin superfamily interaction properties that are different from the interaction properties of the domains of a parent immunoglobulin superfamily.

The invention further provides a multidomain immunoglobulin comprising a genetically modified domain with the following properties. First, the genetically modified domain comprises a protein-protein interaction interface. Second, the genetically modified domain is homologous to a family of natural domains, preferably in such a way that the amino acid sequence of the genetically modified domain can be aligned with the amino acid sequences of the natural domains, which can be further aligned between Yes. Preferably, the alignment of the amino acid sequences of the natural domains corresponds to an alignment of the three-dimensional structures of the natural domains. Third, the engineered domain interaction interface comprises amino acids from corresponding sequence positions of two or more natural parent domains. Fourth, not all amino acids at the interface of the genetically modified domain, considered as a group, are found at the corresponding interface of any individual member of the homologous natural domains. Fifth, the interaction interface of the genetically modified domain confers assembly properties distinct from any of the parent domains. Preferably, the assembly properties of the genetically modified domain are distinctive in that the interaction interface has amino acids from two or more parents that make specific contacts with assembly partners, thereby acquiring an assembly specificity that is a hybrid between the specificities. assembly of parental domains.

Furthermore, the present invention provides a nucleic acid encoding a multidomain immunoglobulin, as described in various embodiments above. In particular, the present invention provides a nucleic acid encoding a multidomain protein that includes at least one bio-active domain. The present invention further provides cells containing the nucleic acid of the invention.

In another aspect, the present invention provides a method of designing a multidomain immunoglobulin with heterodimerizing domains. The method includes the following steps: (a) selecting a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first and third polypeptides dimerize with each other but not with the second or fourth polypeptides, and wherein said second and fourth polypeptides dimerize with each other , (b) constitute an amino acid sequence with a first domain of the first and second polypeptides comprising at least one assembly element of the first polypeptide, and (c) constituting an amino acid sequence of a second domain of the third and fourth polypeptides that comprise at least one assembly element of the third polypeptide, such that the assembly elements of the first and third polypeptides assemble together. , promoting the heterodimerization of the first and second domains.

Other features, objects, and advantages of the present invention are apparent from the detailed description that follows. It should be understood, however, that the detailed description, while indicating the preferred embodiments of the present invention, is provided by way of illustration only, not by way of limitation. Various changes and modifications within the scope of the invention will be apparent to those skilled in the art from the detailed description.

To summarize, the invention refers to:

ES 2 395 969 T3 • A multidomain heterodimeric immunoglobulin comprising at least a first and a second non-identical genetically modified domain, where each of the first and second modified domains contains a protein-protein interaction interface comprising amino acid sequence segments derived from two or more natural homologous parental domains, thereby granting, to the genetically modified first and second domains assembly specificities other than the assembly specificities of the parental domains, wherein the genetically modified first and second domains form heterodimers.

• A corresponding multidomain immunoglobulin, wherein the multidomain immunoglobulin comprises a first subunit comprising the first modified domain, and a second subunit comprising the second genetically modified domain;

• A corresponding multidomain immunoglobulin, wherein the immunoglobulin domains are antibody CH3 domains;

• A corresponding multidomain immunoglobulin, wherein the CH3 domains comprise CH3 domains of IgG and IgA;

• A corresponding multidomain immunoglobulin, where the first and second genetically modified domains are part of polypeptide chains that are associated by a disulfide bridge;

• A corresponding multidomain immunoglobulin, wherein one of the first and second modified domains comprises at least two non-adjacent sequence segments derived from the same parent domain;

• A corresponding multidomain immunoglobulin, wherein each of the first and second genetically modified domains comprises at least two non-adjacent sequence segments derived from the same parental domain;

• A corresponding multidomain immunoglobulin, wherein each of the amino acid sequence segments comprises two or more amino acids;

• A corresponding multidomain immunoglobulin, wherein the protein-protein interaction interface of the first genetically modified domain comprises at least two amino acids from each parent domain.

• A corresponding multidomain immunoglobulin, wherein the multidomain immunoglobulin comprises a first bio-active domain;

• A corresponding multidomain immunoglobulin, wherein the first bio-active domain occupies an N-terminal position of the first genetically modified domain;

• A corresponding multidomain immunoglobulin, wherein the multidomain immunoglobulin further comprises a second bio-active domain;

• A corresponding multidomain immunoglobulin, wherein the second bio-active domain occupies a C-terminal position of the first genetically modified domain;

• A corresponding multidomain immunoglobulin, wherein the first bioactive domain comprises an antibody variable domain;

• A corresponding multidomain immunoglobulin, wherein the multidomain immunoglobulin further comprises a second bioactive domain comprising a second antibody variable domain with different specificity.

• A multidomain immunoglobulin comprising, at least, non-identical genetically modified first and second domains that come together in an interface, where said interface of each of the genetically modified first and second domains comprises at least two amino acid sequence segments, each derived from a different natural homologous parental domain, thereby conferring an assembly specificity distinct from the assembly specificity of the parental domains, where the first and second genetically modified domains form heterodimers.

• A corresponding multidomain immunoglobulin comprising at least non-identical first and second genetically modified domains that converge at an interface, wherein (1) the modified first and second domains are derived from two or more natural homologous parental domains, (2) the interface of the first genetically modified domain comprises at least one amino acid sequence segment that interacts with an amino acid sequence segment on the interface of the second genetically modified domain

ES 2 395 969 T3 derived from the same parental domain, and (3) the first and second genetically modified domains form heterodimers.

• A genetically modified immunoglobulin domain that contains a protein-protein interaction interface that comprises amino acids from two or more parent immunoglobulin domains, such that the protein-protein interaction interface gives the modified immunoglobulin domain assembly specificities that are distinct of the assembly specificities of the parental immunoglobulin domains, wherein the genetically modified immunoglobulin domain is not an antibody variable domain;

• A corresponding genetically modified immunoglobulin domain, wherein the genetically modified immunoglobulin domain is assembled with a companion domain with increased specificity, compared to said parent domains.

• A constant domain of the genetically modified immunoglobulin superfamily that contains a protein-protein interaction interface that comprises amino acids from two or more parental immunoglobulin domains, such that the protein-protein interaction interface gives the genetically modified immunoglobulin domain properties of interaction that are distinct from the interaction properties of the parental immunoglobulin domains.

• A multidomain immunoglobulin comprising a genetically modified domain that comprises a protein-protein interaction interface, where said domain is homologous to a family of natural domains, where said interface comprises amino acids found in corresponding sequence positions in two or more of said natural domains, wherein not all of said amino acids are found at corresponding sequence positions in any individual member of said family of natural domains;

• A corresponding multidomain immunoglobulin, where the interaction interface of the genetically modified domain confers assembly properties distinct from any of the parental domains;

• A heterodimeric genetically modified immunoglobulin molecule, comprising a first natural immunoglobulin chain derived from a first member of the immunoglobulin superfamily, and a second natural immunoglobulin chain derived from a second different element of the immunoglobulin family, where each of immunoglobulin chains comprise a bio-active domain, which is not an antibody variable region, and comprises a protein-protein interface domain comprising complementary amino acid segments of the other chain of the immunoglobulin superfamily; Said protein-protein interface of the first chain is found by interacting with the domain of the protein-protein interface of the second chain by dimerization, preferably homodimerization of the corresponding amino acid segments derived from the same immunoglobulin superfamily within said interaction domains. ;

• A heterodimeric genetically modified immunoglobulin molecule, wherein the bio-active domain is an antibody CH3 domain, a CH2-CH3 domain, or a CH1-CH2-CH3 domain;

• A nucleic acid encoding a multidomain immunoglobulin comprising at least a non-identical first and second genetically modified domain, where each of the first and second genetically modified domains contain a protein-protein interaction interface comprising segments of amino acid sequence derived from two or more natural homologous parental domains, thereby granting assembly specificities of said modified first and second domains, different from assembly specificities of parental domains, wherein (1) the genetically modified first and second domains form heterodimers for each other preferentially to the formation of homodimers, and (2) the first and second genetically modified domains are not antibody variable domains;

• A cell comprising the nucleic acid as described;

• A nucleic acid encoding multimeric immunoglobulin as described;

Brief description of the drawings

Drawings are provided by way of illustration, not by way of limitation.

Figure 1A schematically depicts an exemplary method for designing SEED constructs. Two related parental domains X and Y are aligned. The sequences of the two SEED subunits (XY and YX) are then generated by choosing for each SEED subunit alternate sequence segments from the two parental sequences, and choosing the complementary sequence segments to generate the other SEED subunit sequence. SEEDs genetically modified by this method are called "Complete" SEEDS.

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Figure 1B depicts, schematically, a second exemplary method for designing SEED constructs, which is similar to Figure 1A, except that only amino acids are chosen that form the dimerization interface of one of the parent sequences. SEEDs genetically modified by this method are also called "Surface" SEEDS.

Figure 1C represents, in diagram form, exemplary configurations of a SEED heterodimer, comprised of a first child SEED (blank oval) and a second child SEED (black oval), and a fusion partner, such as a bio-active domain (white diamonds connected). The SEED moiety and the fusion partner can be coupled via a connecting segment (not shown). In configurations with more than one fusion partner, the fusion partners can be identical to each other or different from each other, although in the diagrams they are shown generically as connected white diamonds. The fusion partner can be N-terminal (A) or C-terminal (B) with respect to the SEED moiety. There may be multiple fusion partners concatenated at one end of a SEED, as in (C), or the fusion partners can be located at opposite ends of a SEED (D). A fusion partner can be located N-terminal to a first child SEED and a second fusion partner can be located N-terminal (F) or C-terminal (G) to a second child SEED. The SEED heterodimer can contain three (H) or four (I) fusion partners.

Figure 2 represents the structural alignment of the domains CH3 (SEQ ID NO: 51) of human IgG1 and CH3 (SEQ ID NO: 52) of human IgA. Residue numbers are shown at the top and bottom of the sequences. IgG1 is numbered according to Kabat's EU numbering, while IgA is numbered sequentially, as in the structure of the Protein Data Bank (PDB) with the reference PDB 1OW0. The bold letters designate the backbone positions that were included in the alignment described in Table 2, in Example 1. The diamonds designate the residues that make contact or approach the dimerization interface in the IgG1 homodimers and of IgA.

Figure 3A depicts the sequence alignments and secondary structure of human IgA (SEQ ID NO: 52), IgG1 (SEQ ID NO: 51), and AG SURF daughter "Surface" SEED sequences (SEQ ID NO: 10 ) and GA SURF (SEQ ID NO: 11), while Figure 3B represents the alignments and secondary structure of the human IgA, IgG1 and sequences of "Complete" sEeD son AG SEED (SEQ ID NO: 3) and GA SEED ( SEQ ID NO: 6). IgG1 is numbered according to the Kabat EU numbering, while IgA is numbered sequentially as in the structure with the reference PDB 1OW0 (native numbers in the center of the alignment). For the purposes of this figure, the sequential numbering of the SEED is interrupted at the residues of the extra loop, which are designated by the letters "A", "B", and the like. (eg, 18A), to illustrate the structural alignment of the molecules. The exchange points are designated by the bold letters of the sequence. The two interchange points that do not contain common residues are in italics. The modeled secondary structures (arrows at the top and bottom of the sequences) of the two SEEDs, illustrate the chain changes, and are colored to indicate the way in which the domain was divided, as shown in the Figures. 6B and 6C. The blank segments □ are from IgA; the gray segments are from IgG, and the black segments are common residues at the exchange sites. Twelve (12) residues in the IgA segments are underlined. These are residues that remained IgG due to their proximity to the CH2 / CH3 interface region. These residues are not involved in CH3 dimerization, but are potentially important for interaction with CH2, and / or with the FcRn complex. Because CH2 is from human IgG for both SEEDs, these residues were allowed to maintain both the native CH2 / CH3 interaction, as well as the different and well-known advantages conferred by binding to FcRn.

Figure 4 is a representation of an IgG antibody molecule illustrating the symmetry of the CH3 homodimer. The vertical bar designates the axis of double rotational symmetry.

Figure 5 is a representation of a bispecific molecule, similar to an antibody having two different Fab domains, paired by the heterodimeric SEED analog of the CH3 domain. The gray part with padding represents the portion derived from IgG, while the part in white □ represents the portion derived from IgA. The symmetry of the CH3 complex is broken at the AG / AG heterodimer, as represented by the "X" in the vertical bar designating the axis of double rotational symmetry.

Figures 6A-C are schematic representations of the CH3 secondary structure of IgG and the two CH3-based SEEDs. Figure 6A depicts the wild-type CH3 secondary structure.

Figure 6B represents the secondary structure of "GA SEED", and shows the chain exchange pattern. Gray represents the sequence of IgG; the □ represents the sequence of IgA; and the shows the exchange points, with a broader black band indicating residues that are conserved in both IgA and IgG.

Figure 6C represents the secondary structure of "AG SEED", which contains a pattern opposite to the pattern of "GA SEED".

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Figures 7A-C are tape diagram representations of the three-dimensional structure of the CH3 domains of "GA SEED" and "AG SEED" and their putative heterodimeric structure depicting interactions of the CH3 domain and the interchange crossover point. In all diagrams, the light gray or white ribbons represent the sequence and structure of IgA, the dark gray color corresponds to the sequence and structure of IgG, and the black sections denote where the sequence from G to A or vice versa. Apart from the two exchange points referenced at 55-56 and 101-102 (numbered according to Figure 3B), all residues in black are shared by IgA and IgC, in sequence and in basic structure.

Figure 7A represents the "GA SEED", where the N-terminus begins as an IgG sequence and ends as IgA after being exchanged seven times. In this structure, the upper layer of the β chains is found in the outer lamina, while the layer behind it forms the interface with the other CH3 domain.

Figure 7B represents the "AG SEED", starting with the sequence of IgA. Here, the front β chains form the interface, while the rear β chains lie on the outside of the dimer.

Figure 7C depicts the putative heterodimeric structure of "GA SEED" and "AG SEED". The translation of the structure shown in Figure 7A onto the structure shown in Figure 7B groups the interface surfaces together. The black residues form an approximate plane that is oriented vertically and perpendicular to the page. All residues on the left are dark gray (IgG), while all residues on the right are white (IgA). Thus, with white versus white and gray versus gray, the entire interface is well formed, as a fusion of the IgA and IgG interfaces. The alternative homodimers, (AG / AG and GA / GA) would each have their IgA side juxtaposed to their IgG side (on both sides of the cleavage plane), and are therefore not favored.

Figures 8-10 diagrammatically show a series of protein molecules that can be produced using the SEED fractions described in the present patent. For all these figures, the different fractions are indicated as follows. In Figure 8 and Figure 9, the polypeptide chains that include GA SEED are colored black, while the polypeptide chains that include AG SEED are colored white. Within these polypeptide chains, the antibody V regions that are part of the polypeptide chain that contains GA SEED are black with white stripes, while the antibody V regions that are part of the polypeptide chain that contains AG SEED They are white with fine black stripes. Light chain constant regions are shown in a checkerboard pattern. The antibody hinge regions are shown as thin ovals connected by an "SS" and a thick line to represent the disulfide bridges between the hinge regions. Polypeptide linkers are represented by dashed lines.

The different parts of Figure 8, Figure 9 and Figure 10 are labeled numerically as follows. In some cases, to simplify the figures, the numerical labels are not shown, but the identity of the various domains and regions can be inferred from the figures with the corresponding domains and regions.

"1" indicates a group of GA-associated heavy and light chain V regions.

"2" indicates a group of AG-associated heavy and light chain V regions.

"3" indicates a GA-associated light chain V region.

"4" indicates a Fab region.

"5" indicates a GA-associated heavy chain V region.

"6" indicates an AG-associated heavy chain V region.

"7" indicates a light chain V region associated with AG.

"8" indicates a light chain constant region.

"9" indicates an Fc region comprising a SEED pair.

"10" indicates a SEED pair.

"11" indicates an artificial connector.

ES 2 395 969 T3 "12" indicates a camelid V region or GA-associated single domain.

"13" indicates a camelid V region or AG-associated single domain.

"14" indicates a single chain fused diabody or diabody that is incorporated into the polypeptide chain comprising GA SEED.

"15" indicates a single chain fused diabody or diabody that is incorporated into the polypeptide chain comprising AG SEED.

"16", "17", "18", or "19" refers to any protein or peptide, such as a non-Ig domain. Such domains can include, for example, cytokines, hormones, toxins, enzymes, antigens, and extracellular domains of cell surface receptors.

"20" indicates a canonical homodimeric Fc region.

"21" indicates a canonical homodimeric pair of CH3 domains.

Figure 8 illustrates types of antibody-like SEED configurations comprising fractions with essentially natural V regions, such as the Fab regions (Figure 8A and Figure 8F), single chain Fab (Figure 8C and Figure 8D), and V regions of camelid single domain or single domain (Figure 8E and Figure 8F). Figure 8A, Figure 8C, and Figure 8E show molecules that comprise an essentially intact Fc region, including cH2 domains, in addition to a hinge region. Figure 8B, Figure 8D, and Figure 8F show molecules lacking a CH2 domain, in which the hinge region is optionally replaced by a linker that optionally possesses or lacks cysteine ​​residues capable of disulfide bonding. .

Figure 9 illustrates types of antibody-like SEED configurations comprising fractions with artificially configured V regions, such as single chain Fvs (Figure 9A and Figure 9B), diabodies (Figure 9C and Figure 9D), and chain Fvs. unique with additional fractions coupled to the N- and / or C-terminals of the two polypeptide chains (Figure 9E and Figure 9F). Figure 9A, Figure 9C, and Figure 9E show molecules that comprise an essentially intact Fc region, including cH2 domains, in addition to a hinge region. Figure 9B, Figure 9D, and Figure 9F show molecules lacking a CH2 domain, in which the hinge region is optionally replaced by a linker optionally possessing or lacking cysteine ​​residues capable of binding via disulfide bridge.

Figure 10 diagrammatically illustrates a molecule in which the GA / AG SEED pair essentially replaces the CH1-CL pairing in an antibody. Additional fractions, indicated by X and Y, can be located at the N terminals of the SEED GA and AG. Fraction X and fraction Y can be, for example, a Fab region, a single chain Fab, a camelid single domain V region, a single chain Fv, a single chain diabody such as illustrated referenced "14" and "15" in Figure 9C and Figure 9D. Additional fractions can be fused to the C-terminals of the CH3 domains indicated with the reference "21".

Figure 11: Figure 11A shows a heterodimer produced as described in Example 5, in which a fraction of AG SEED has a fraction of IL-2 fused to its C-terminus. The CH2 and hinge fractions are identical in this case. Figure 11B shows an antibody produced as described in Example 7, in which the AG SEED fraction has an IL-2 fraction fused to its C-terminus. Each antibody domain is represented by an oval, and the IL-2 fraction is represented by a blank square. The CH2, CH1, hinge, VH, VL, and CL fractions are identical in this case. The hinge regions are coupled by disulfide bridges represented by "SS" in the figure. The light chain constant region is represented by a checkerboard pattern. The heavy chain V region is represented by a vertical line pattern. The vH, CH1, and CH2 regions are black.

Figure 12: Figures 12A-C depict the preferred assembly of AG / GA SEEDs in heterodimers, as represented by the results of the expression of Fc and Fc-IL2 in the same cell. Figure 12A represents the possible configurations of the molecules that are the result of the co-expression of Fc-IL2, in such a way that each dimeric species has a different molecular weight. Figure 12B depicts a non-reducing SDS gel run into which the following samples were loaded: lane 1 - molecular weight standards; lane 2-4 about 1, 2, and 4 micrograms of "Complete" Fc (GA SEED) / Fc (AG SEED) -IL2 total protein expressed from NS / 0 cells; lanes 5-7 - approximately 1, 2, and 4 micrograms of total Fc (GA SEED) / Fc (AG SEED) -IL2 "Surface" protein expressed from NS / 0 cells; Lanes 8-10-1, 2, and 4 micrograms of total IgG Fc / Fc-IL2 protein expressed from NS / 0 cells. Figure 12C is a reducing gel analysis showing the expression ratio of Fc and Fc-IL2 derived from IgG.

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Figures 12D-E represent a Western blot analysis of non-reduced (panel D) and reduced (panel E) samples of the Fc / Fc-IL2 proteins of Figures 12B-C. Duplicate samples of Fc (GA SEED) / Fc (AG sEeD) -Il2 "complete" (lanes 1 and 4), Fc (GA SEED) / Fc (AG SEED) -IL2 "surface" (lanes 2 and 5), and parental Fc / Fc-IL2 (lanes 3 and 6) were loaded, and the blot was hybridized using anti-human IgG Fc (lanes 1-3) and anti-human IL-2 (lanes 4-6) antibodies.

Detailed description of the invention

The present invention provides methods for designing heterodimeric immunoglobulin domains that preferably heterodimerize or heteromultidimerize. In particular, the invention utilizes a "Chain Exchange Modified Domain" (SEED) strategy to engineer a protein-protein interaction interface that promotes heterodimerization or heteromultimerization. The invention further provides multidomain proteins containing domains engineered using this approach. Thus, the present invention represents a significant advance in protein engineering.

Various aspects of the invention are described in greater detail in the following subsections. The use of subsections is not intended to limit the invention. Each subsection can be applied to any aspect of the invention.

As used in the present patent, a "multidomain immunoglobulin" includes any immunoglobulin that contains two or more domains. The domains can be found in a single polypeptide; they can also be found in different polypeptides. "Heteromultidimerization" refers to non-identical domains that form a multimeric complex mediated by domain interactions. A "heteromultidimeric protein" is a protein molecule that comprises at least a first subunit and a second subunit, each subunit containing a non-identical domain. The heteromultimer may include a "heterodimer" made up of the first and second subunits or may form higher order structures (eg, ternary), where polypeptides of the subunits are present in addition to the first and second subunits. Typically, each subunit contains a domain. Exemplary structures for the heteromultimer include heterodimers, heterotrimers, heterotetramers (eg, a bispecific antibody), and additional oligomeric structures.

As used in the present patent, a "domain" includes any region of a polypeptide that is responsible for selective assembly with an assembly partner of interest (eg, another domain, ligand, receptor, substrate, or inhibitor). Exemplary domains include a constant domain from an immunoglobulin superfamily, such as a CH2 or CH3 domain, a receptor-binding domain, a ligand-binding domain, an enzymatic domain, or any polypeptide that has been genetically modified and / or selected. to join a target. When two domains assemble together, they come together at a protein-protein interaction interface. As used in the present patent, a "protein-protein interaction interface", an "interaction interface", or an "interface" includes those "contact" residues (amino acid residues or other non-amino acids such as carbohydrate groups , NADH, biotin, FAD or heme group) in the first domain that interacts with one or more "contact" residues (amino acid or other non-amino acid groups) at the interface of the second domain. As used in the present patent, a "contact" residue refers to any amino acid or non-amino acid residue in one domain that interacts with another amino acid or non-amino acid residue in a different domain, through van der Waals forces, linkages of hydrogen, water-mediated hydrogen bonds, salt bridges or other electrostatic forces, attractive interactions between aromatic side chains, the formation of disulfide bridges, or other forces known to a person skilled in the art. Typically, the distance between the alpha carbons of the two interacting contact amino acid residues at the interaction interface is not greater than 12 A. More commonly, the distance between the alpha carbons of the two contact amino acid residues that interact in the interaction interface is not greater than 11 A.

As used in the present patent, a "parental domain" refers to any existing assembly domain, as described above, that can be used as a parental sequence for the design of a genetically modified domain using the strategy chain exchange. Suitable parental domains are usually related or homologous, and have a particular assembly specificity. "Homologous" means two domains that share at least 35%, 40%, 45%, 50%, 55%, 60%, 62%, 65%, 68%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. If the parent domains are present in a common solution, they may tend to homodimerize rather than heterodimerize with each other. As used in the present patent, "existing assembly domains" include natural or wild-type sequences from organisms such as a human, mouse, yeast, bacteria, to name a few, as well as derived sequences that have been modified from wild-type sequences, such as, for example, sequences that have been stabilized; they have become less immunogenic; have been endowed with modified, increased or decreased assembly specificity, with modified enzymatic properties, with modified solubility, or increased expression; they have been truncated; or have been fused to another polypeptide. The "existing assembly domains" can further be fully or partially synthetic sequences that are synthesized based on molecular design, in vitro or in vivo selection methods (eg, yeast two-hybrid system, phage display (or phage display )), or combinations thereof.

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A "genetically modified domain" refers to a domain modified from at least two non-identical parental domains. A genetically modified domain is also called a child domain. Typically, a genetically modified domain of the present invention contains amino acid sequence segments derived from two or more existing homologous parent domains. Preferably, the interface of a genetically modified domain includes amino acids derived from more than one parent domain. The presence of amino acids from different parental domains confers an assembly specificity different from the assembly specificities of the parental domains. For example, the presence of amino acids from different parental domains promotes or increases heterodimerization or heteromultimerization.

A strand swap modified domain (SEED) is a genetically modified domain that has been modified from at least two non-identical parental domains, by the strand swap genetic engineering method described in detail below.

As used in the present patent, a "polypeptide" refers generally to any polypeptide or protein that has more than about ten amino acids. Preferably, mammalian polypeptides (polypeptides that were originally derived from a mammalian organism) are used for the genetic engineering of SEED, more preferably those that are directly secreted into the medium. Examples of bacterial polypeptides include, for example, alkaline phosphatase and β-lactamase. Examples of mammalian polypeptides include molecules such as renin, a growth hormone, including human growth hormone; bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; α-1-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VIIIC, factor IX, thrombokinase, and von-Willebrand factor; anticoagulation factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor -α and -β; enkephalinase; RANTES chemokine (Regulated on Activation Normal T-cell Expressed and Secreted, expressed and secreted by normal T lymphocytes and regulated according to their degree of activation); human macrophage inflammatory protein (MIP-Ι-α); a serum albumin, such as human serum albumin; Müllerian inhibitory substance; relaxin A chain; relaxin B chain; prorelaxin; Mouse Gonadotropin Associated Peptide; DNase; inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; protein A or D; rheumatoid factors; a neurotrophic factor, such as bone derived neurotrophic factor (BDNF); neurotrophin-3, -4, -5, or -6 (NT-3, NT4, NT-5, or NT-6), or a nerve growth factor such as NGF-beta; platelet derived growth factor (PDGF); fibroblast growth factor, such as AFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF), such as TGF-α and TGF-β, including TGF-βΙ, TGF-e2, TGF-e3, TGF-e4, or TGF-e5; Insulin-like growth factors I and II (IGF-I and IGF-II); des (1-3) -IGF-I (brain IGF-I), insulin-like growth factor binding proteins; CD proteins, such as CD-3, CD-4, CD-8, and CD-19; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon, such as interferon -alpha, beta, and -gamma; colony stimulating factors (CSFs), eg, M-CSF, GM-CSF, and G-CSF; interleukins (ILs), eg IL-1 to IL-10; superoxide dismutase; T lymphocyte receptors; surface membrane proteins; accelerating factor of degradation; transport proteins; homing receivers; adresins; regulatory proteins; immunoglobulins (antibodies); and fragments of any of the polypeptides listed above.

As used in the present patent, the "first polypeptide" or "first subunit" is a polypeptide that is found associated with a second polypeptide through the interaction between genetically modified domains. The "second polypeptide" or "second subunit" is any polypeptide that is found associated with the first polypeptide through the interaction between the genetically modified domains. In addition to the genetically modified domains, the first and / or second polypeptide can include one or more additional bioactive domains, such as, for example, an antibody variable domain, receptor-binding domain, ligand-binding domain, or enzymatic domain or other "binding domains", such as antibody constant domains (or parts thereof), including CH3 and CH2 domains. As an example, the first polypeptide can include at least one genetically modified domain of the invention, such as a genetically modified cH3 domain of an immunoglobulin, and can interface with the first polypeptide. The first polypeptide may further include other antibody heavy chain binding domains (eg, CH1, CH2, or CH4), and additional bioactive domains, such as receptor polypeptides (especially those that form dimers with another polypeptide receptor, eg, integrin heterodimers and interleukin-8 receptor, eg, LFA1 or GBIIIb / IIIa), ligand polypeptides (eg, cytokines, nerve growth factor, neurotrophin-3, and brain derived neurotrophic factor - see Arakawa et al. (1994) J. Biol. Chem. 269 (45): 27833-27839 and Radziejewski et al (1993) Biochem. 32 (48): 1350), and antibody variable domain polypeptides (eg, diabodies and BsAbs).

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As used in the present patent, "assembly" refers to a protein-protein interaction that occurs during the production of a multi-subunit protein. For example, during antibody production, light and heavy chains are synthesized from ribosomes associated with the endoplasmic reticulum. The individual chains are then folded, and then assembled into mature antibodies through the appropriate association of light and heavy chains. For example, in the case of IgG antibodies, the assembly of the Fab portion is initially driven primarily by the interactions between the CH1 and CL domains, and also by the interactions between the VH and VL regions. In the case of two heavy chains, the initial assembly reaction is the association of two CH3 domains. These initial assembly reactions are usually, but not always, followed by the formation of a disulfide bridge between the assembled subunit polypeptides. As used in the present patent, "assembly" is different from "joining"; assembly refers to the protein interaction events that take place during the production of a mature protein, such as an antibody before it is secreted from a cell, while binding refers to the protein interaction events that take place after secretion, such as the interaction of an antibody with an antigen or with an Fc receptor. In an operational sense, the assembly of a diagnostic or therapeutic protein takes place during the preparation of the therapeutic protein up to and including the placement of a product in a vial, and the binding of a diagnostic or therapeutic protein refers to the events that take place after a therapeutic protein is administered to a patient or when a diagnostic protein is used in a diagnostic test.

By "binding" is meant the interaction of a protein with a target protein subsequent to protein synthesis and assembly.

Chain exchange genetic engineering

The present invention utilizes the fact that the natural protein domains that mediate protein-protein interactions are often homologous or, in the case of homodimers, identical, and that such proteins and domains often only homodimerize each other. , but they do not usually heterodimerize with members of other families, or do not heterodimerize with members of other families with an affinity equal to or greater than their affinities for homodimerization. According to the invention, such proteins can be used to design heterodimeric or heteromultimeric proteins using strand exchange genetic engineering methods, described in detail below. Such genetically modified domains are also known as "Chain Exchange Genetically Modified Domains" ("SEEDs"). Multidomain proteins containing such genetically modified domains are also known as chain exchange genetically modified proteins.

Strand exchange genetic engineering usually begins with a structural model of a dimeric parent protein domain. Two parental domains that can homodimerize or dimerize each with its assembly partner, but do not heterodimerize with each other, are structurally aligned. Parental domains can dimerize so that they face each other in a face-to-face position, that is, the dimer's partners can be related by 180 degree rotational symmetry. Parental domains can also dimerize so that they are in a face-to-back position.

Due to the rotational symmetry geometry of homodimeric proteins, there is usually an amino acid line on the interacting surface that interacts in a homotypic manner. In other words, there are amino acids that interact with their equivalents in the other subunit. For example, in the CH3 domain of IgG1, these amino acids include L351, P352, T366, T394, P395, and Y407. This amino acid line is generally parallel to the axis of rotational symmetry of the dimer. When choosing parental domains, it is often useful to choose proteins that homodimerize in such a way that the long axis of the dimerization interface is not clearly parallel to the axis of rotational symmetry. For example, SEEDs based on members of the leucine zipper family are difficult to construct, since the dimerization interface is parallel to the axis of symmetry, and many of the amino acid interactions are homotypic. Accordingly, in some preferred embodiments, the genetically modified domains of the invention are not leucine zipper domains. In contrast, the CH3 family domains are particularly useful, since a significant portion of the interaction surface lies outside the line of symmetry. However, it will be recognized by those skilled in the art that the line of symmetry (ie, a line of homotypically interacting amino acids), may be an oversimplification. For example, amino acid side chains on the line of symmetry can point toward the hydrophobic core of the domain.

A new dimerization interface is conceptually designed and divided into at least two regions that typically lie on each side of the homotypic interaction line (ie, the line of symmetry). New domains are then designed by chain exchange where two daughter domain linear amino acid sequences are constructed from two aligned parental domain amino acid sequences, taking complementary segments from each parental sequence. As a result, in the dimerization interface regions, the two daughter domains (ie, two SEEDs) have complementary amino acid segments of parent domains. This concept is illustrated in Figures 1A and 1B. As shown in Figure

ES 2 395 969 T3

1A, two daughter SEED sequences, 1 and 2, are genetically modified from two parental sequences, A and B, in an entirely complementary manner. If Daughter 1 has an amino acid segment from Parental A in a given region of the interaction interface, Daughter 2 will have the corresponding amino acid segment from Parental B. The interaction interface is designed such that at least one segment of an amino acid sequence in Daughter 1 interacts with a segment of amino acid sequence in Daughter 2 that was derived from the same parent domain. In Figure 1B, the child SEED domains are derived primarily from a parent domain. However, the amino acids at the dimerization interface are derived from either one parent or the other in a complementary manner.

It should be noted that Figure 1A and Figure 1B represent two extreme examples of the invention, and that SEEDs can be genetically modified by methods of the invention that have intermediate designs between Figure 1A and Figure 1B. For example, as described in the Examples in more detail, it is possible to construct a SEED based on parental domains from the immunoglobulin CH3 domain family. The daughter SEEDs can be derived primarily in a complementary manner from IgG and IgA, but amino acids that interact with FcRn are derived from IgG to preserve interaction with FcRn.

Therefore, SEEDs are genetically modified by combining two or more homologous parental domains. Parental domains are polypeptides that differ from each other by at least four amino acids. When producing a SEED, the sequences of the original polypeptides are aligned based on their homologies, theoretical structural models, crystal structures or structures from modeling solutions, or a combination thereof. There is at least one different amino acid in one or more aligned sequence positions, or a different number of amino acids in at least one original aligned pair of sequences. The parental sequences are then divided into at least two segments that include at least one amino acid each. A SEED sequence can be composed by choosing, from among the original sequences, the desired one for each divided segment. A SEED often differs from each individual parental sequence by at least two consecutive amino acids, and sometimes by three, four, or more consecutive amino acids. In addition to selecting sequences from the parent parent polypeptides, a SEED can contain any desired amino acid at any position, such as positions outside of the designed interface, to meet other design needs.

There are positions in the SEED sequence where the parental sequence changes from one parent to a second parent. These positions are called trading points or trading positions. The exchange points or exchange positions can include one or more amino acids whose identity can be shared by both parents. Typically, the exchange points are chosen from amino acids on or near the line of symmetry, although the points can also be chosen. The exchange points can include amino acids not shared by the parents. In this case, the sequence changes abruptly from one parent to another. Furthermore, the exchange sites can include one or more amino acids that do not belong to any of the parents. In this case, different parental sequences usually appear on each side of the new amino acids. If there are multiple exchange points in the sequence of a SEED, the total number of parental segments can be greater than two, up to a number greater than the number of exchange points. These parental segments can be selected from different parental domains. Therefore, the present invention contemplates SEEDs that are genetically modified from more than two parental domains.

For convenience, each SEED is identified according to the order of its parental sequences, beginning with the N-terminus of the SEED. In the examples below, a SEED AG has an IgA1 sequence segment at the N-terminus, which then changes to an IgG1 sequence segment at the first exchange point. A GA SEED has an IgG1 sequence segment at the N-terminus, which then changes to an IgA1 sequence segment at the first exchange point.

Thus, the SEED interaction interface of the invention includes segments of amino acid sequences derived from two or more parent domains. As a result, the SEED interface has different interaction properties from the interaction properties of the parental domains. In particular, the presence of amino acids from different parental domains confers an assembly specificity different from the assembly specificity of any of the parental domains. For example, the specificity of heterodimerization or heteromultimerization is increased by the presence of amino acids from different parent domains in the interface of a SEED. As a result, a pair of SEEDs form heterodimers for each other preferentially over homodimer formation. Therefore, when a pair of SEEDs are expressed in an expression system, heterodimers of the SEEDs can assemble in a specific way, in such a way that the heterodimeric SEEDs can be directly recovered from the cell culture system without the need to elaborate separation steps. to extract the homodimers.

CH3-based SEEDs

Homology and backbone differences between the dimerization interfaces of the parental domains are important for the creation of SEEDs. Therefore, according to an embodiment of the invention, the

ES 2 395 969 T3 classes of immunoglobulin proteins are a useful source for parental domains. SEEDs can be created using parental sequences from two different immunoglobulin classes. For example, SEEDs can be genetically modified from domains of the CH3 family by the method of the invention. CH3 family domains suitable for the design of SEEDs include, but are not limited to, the CH3 domains of IgG1, IgG2, IgG3, IgG4, IgA, and IgD, and the CH4 domains of IgE and IgM.

The CH3 domains of human IgG1 and IgA form homodimers but do not form heterodimers with each other. Therefore, SEED pairs (for example, an AG SEED and a GA SEED), can be genetically modified from the CH3 domains of IgG1 and IgA, in such a way that they can heterodimerize with each other but their ability to homodimerize is minimal. According to one embodiment, the assembly interface in the CH3 domain is divided into two regions, which lie on each side of the line of homotypic interactions. Homotypic interactions for the CH3 domains of IgA and IgG1 can be determined by observing and hybridizing the crystal structure with a 1.4A sphere, to determine whether or not the two side chains are close enough to avoid water. If the surfaces are joined across the interface, this implies that the side chains are interacting closely. For example, in the wild-type CH3 domain of IgG1, homotypically interacting amino acids include, but are not limited to, L351, P352, T366, T394, P395, and Y407. For the wild-type CH3 domain of IgA1, homotypically interacting amino acids include, but are not limited to, L352, P353, T368, W398, A399, and T414. In an exemplary SEED subunit, those amino acids with outward pointing side chains that lie to the left of the homotypic interaction line are taken from the CH3 of IgA, and those with outward pointing side chains outer to the right of the homotypic ineraction line to the right of the homotypic interaction line, are taken from the CH3 of IgG1. The choice of amino acids along the homotypic interaction line is based on structural considerations and is made on a case-by-case basis, although it is likely that amino acids from either of the two parent domains can be selected for a region of a SEED. in particular.

For example, a CH3-based AG SEED may have a polypeptide sequence as shown in SEQ ID NO: 1, where X₁, X₂, or X₃ they can be any amino acid. In some embodiments, X₁ is K or S, X₂ is V or T, and X₃ is T or S. Preferably, X₁ is S, X₂ is V or T, and X₃ is S. A CH3-based GA SEED may have a polypeptide sequence as shown in SEQ ID NO: 2, where X₁, X₂, X₃, X4, X₅, or X₆ they can be amino acids. In some embodiments, X₁ is L or Q, X₂ is A or T, X₃ is L, V, D, or T, X₄ is F, A, D, E, G, H, K, N, P, Q, R, S ', or T, X₅ is A or T, and X₆ is E or D. Preferably, X₁ is Q, X₂ is A or T, X₃ is L, V, D, or T, X₄is F, A, D, E, G, H, K, N, P, Q, R, S, or T, X₅ is T, and Xg is D. Exemplary SEED heterodimers may include a SEED subunit selected from AG (F0) SEED (SEQ ID NO: 3), AG (f) SEED (SEQ ID NO: 4), or AG (f2) SEED (SEQ ID NO: 5), and the other SEED subunit selected from GA (F0) SEED (SEQ ID NO: 6), GA (f1) SEED (SEQ ID NO: 7), GA (f2) SEED (SEQ ID NO: 8), or GA (f3) SEED (SEQ ID NO: 9). For example, a SEED heterodimer can include AG (F0) SEED (SeQ ID NO: 3) and GA (f0) SeEd (SEQ ID NO: 6) subunits. In another example, a SEED heterodimer can include AG (f2) SEED (SEQ ID NO: 5) and GA (f2) SEED (SEQ ID NO: 8) subunits. In yet another embodiment, a SEED heterodimer may include AG (s0) SEED (SEQ ID NO: 10) and GA (s0) SEED (SEQ ID NO: 11) subunits.

Bio-active domains

SEEDs according to the present invention are particularly useful when coupled with a fusion partner. A fusion partner (X) can fuse to the N-terminal of the SEED (X-SEED), it can also fuse to the C-terminal of the SEED (SEED-X). Furthermore, a fusion partner can fuse to the N-terminal and the C-terminal of the SEED at the same time (X-SEED-X). Two different merge partners can be merged into one SEED (XSEED-Y).

Since two SEED sequences usually form heterodimers, it is possible that at least one, two, three, or four fusion partners may be contemplated in the SEED heterodimer. For example, according to one embodiment, the first child SEED has a fusion partner, and the second child SeEd has no fusion partner, resulting in the following exemplary configurations: SEED-X heterodimerized to SEED; or X-SEED heterodimerized to SEED. In an additional example, the first child SEED has two different fusion partners (X, Y), and the second child SEED has two different fusion partners (W, Z) that differ from the first child SEED's fusion partners. Possible exemplary configurations include, but are not limited to: X-SEED-Y heterodimerized to W-SEED-Z; X-SEED-Y heterodimerized to Z-SEED-W; Y-SEEDX heterodimerized to W-SEED-Z; or Y-SEED-X heterodimerized to Z-SEED-W. According to the invention, a SEED can also have two or more fusion partners (X) sequentially fused to, for example, the N-terminus (XX-SEED). Alternatively, in another embodiment of the invention, the first child SEED has a fusion partner (X), and the second child SEED has a fusion partner (Y), resulting in the following exemplary configurations : X-SEED heterodimerized to Y-SEED; X-SEED heterodimerized to SEED-Y; or SEED-X heterodimerized to SEED-Y. In yet another embodiment of the present invention, the first child SEED has one fusion partner (X), and the second child SEED has two fusion partners (Z, Y).

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Possible exemplary configurations include, but are not limited to: X-SEED heterodimerized to Y-SEED-Z; X-SEED heterodimerized to Z-SEED-Y; SeED-X heterodimerized to Z-SEED-Y; or SEED-X heterodimerized to YSEED-Z. Exemplary configurations are illustrated in Figure 1C.

In particular, a fusion partner can be one or more bioactive domains, including any biologically active protein or a biologically active portion thereof. For example, a bioactive domain can include an antibody constant or variable region, including, but not limited to, a VL domain, a VH domain, an Fv, a single chain Fv, a diabody, a Fab fragment, a Single chain Fab, or an F (ab ')₂.

According to the invention, the fusion partners can be coupled to the SEED fractions directly or indirectly. For example, a fusion partner can be linked to a SEED moiety via a linker peptide, such as described in US Patent Nos. 5,258,498 and US 5,482,858 to Huston et al., Or US Patent Nos. 5,856,456 and US 5,990,275 to Whitlow et al., The contents of which are incorporated herein by reference. Typically, a suitable linker peptide may contain glycine and serine residues. Typically, a suitable linker peptide may also have different properties. For example, in some embodiments, a linker may further include a cleavage site for the protease, such as a matrix metalloproteinase recognition site.

Thus, the present invention provides a novel method for producing multispecific antibodies based on SEED technology. A multispecific antibody is a molecule that has binding specificities for at least two different antigens. While such molecules usually bind only two antigens (ie BsAbs), antibodies with additional specificities, such as trispecific or tetra-specific antibodies are encompassed by this term when used in the present patent. Examples of BsAbs include those that bind to different antigens on the same cell surface, or those that bind to a cell surface antigen and a non-cell surface antigen. A non-cell surface antigen includes, but is not limited to, an intracellular or extracellular antigen, a soluble or insoluble antigen. Multispecific antibodies can bind to different antigens simultaneously, although simultaneous binding is not required for the function of multispecific antibodies. In some applications, antigens are preferably functionally related, such as EGFR and HER2. In particular, useful types of multispecific antibodies include, but are not limited to, anti-EGFR / anti-HER2; antiEGFR / anti-HER2 / anti-HER3; anti-EGFR / anti-HER3; anti-EGFR / anti-HER2 / anti-IGF1 R; anti-EGFR / anti-HER2 / antiHER3 / anti-IGF1R; anti-EGFR / anti-HER3 / anti-IGF1R; anti-EGFR / anti-IGF1R; and anti-HER2 / anti-IGF1R. Other combinations of specificities involving the EGFR, HER, and IGF1R family are within the scope of the present invention.

Additional examples of BsAbs include those with one arm directed against a tumor cell antigen and the other arm directed against a cytotoxic trigger molecule, such as anti-FcyRI / anti-CD 15, anti-p185HER2 / FcyRIII (CD 16), anti- CD3 / anti-malignant B cell (1D10), anti-CD3 / anti-p 185^HER2, anti-CD3 / anti-p97, anti-CD3 / anti-renal cell carcinoma, anti-CD3 / anti-OVCAR-3, anti-CD3 / L-D1 (anti-colon carcinoma), anti-CD3 / anti -melanocyte stimulating hormone analog, anti-EGF / anti-CD3 receptor, anti-CD3 / anti-CAMA1, anti-CD3 / anti-CD19, antiCD3 / MoV18, anti-neuronal cell adhesion molecule (NCAM, for its acronym in English) / anti-CD3, anti-folate binding protein (FBP) / anti-CD3, anti-pan carcinoma associated antigen (AMOC-31) / anti-CD3; BsAbs with one arm that specifically binds to a tumor antigen and one arm that binds to a toxin such as antisaporin / anti-id-1, anti-CD22 / anti-saporin, anti-CD7 / anti-saporin, anti- CD38 / anti-saporin, anti-CEA / anti-ricin A chain, anti-interferon-α (IFN-a) / anti-hybridoma idiotype, anti-CEA / anti-vinca alkaloid; BsAbs to convert enzyme-activated drugs, such as anti-CD30 / anti-alkaline phosphatase (which catalyzes the conversion of the prodrug mitomycin phosphate to mitomycin alcohol); BsAbs that can be used as fibrinolytic agents, such as anti-fibrin / anti-tissue plasminogen activator (tPA), anti-fibrin / anti-urokinase-type plasminogen activator (uPA); BsAbs to target immune complexes to cell surface receptors, such as anti-low-density lipoprotein (LDL) / anti-Fc receptor (eg, FcyFI, FcyRII, or FcyRIII); BsAbs for use in infectious disease therapy, such as an anti-CD3 / anti-herpes simplex virus (HSV), anti-T-cell receptor: CD3 / anti-influenza complex, anti-FcyR / anti-HIV, BsAbs for in vitro or in vivo tumor detection, such as anti-CEA / anti-EOTUBE, anti-CEA / anti-DPTA, anti-p185^HER2/ anti-hapten; BsAbs as vaccine adjuvants; and BsAbs as diagnostic tools, such as anti-rabbit IgG / anti-ferritin, anti-horseradish peroxidase (HRP) / anti-hormone, anti-somatostatin / anti-substance P, anti-HRP / anti-FITC, anti-CEA / anti-p-galactosidase. Examples of trie-specific antibodies include anti-CD3 / anti-CD4 / anti-CD37, anti-CD3 / anti-CD5 / anti-CD37, and anti-CD3 / anti-CD8 / anti-CD37.

According to the invention, other bioactive domains include hormones, cytokines, chemokine, secreted enzymes, ligands, extracellular portions of transmembrane receptors, or receptors. Hormones include, but are not limited to, growth hormones, or glucagon-like peptide (GLP-1). Cytokines include, but are not limited to, interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-14 , IL-15, IL-16, IL-18, IL-21, IL-23, IL-31; hematopoietic factors such as granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor or G-CSF and erythropoietin; tumor necrosis factors

ES 2 395 969 T3 (TNF), such as TNFa; lymphokines such as lymphotoxin; regulators of the metabolic process such as leptin; and interferons (IFN) such as INF-α, INF-β, and IFN-γ.

Thus, the genetically modified heterodimeric immunoglobulins of the present invention allow the colocalization of different bioactive domains in a biological system. This can be achieved, for example, in the context of a multimeric protein incorporating two different antibody variable domains, where an antibody variable domain is fused to a genetically modified domain, and a second antibody variable domain is fused to a second domain. genetically modified that preferentially joins with the first genetically modified domain. Administration of such a genetically modified protein causes two distinct activities - in this case, binding activities - to be present on the same molecule in the biological system, co-localizing the activities within the biological system. If the activities involve binding to other molecules (such as an antibody / antigen variable domain interaction, a ligand / receptor interaction, etc.), enzymatic activities, or a combination thereof, the present invention provides a system that requires the activities are present in the same place allowing, for example, the targeting of a therapeutic activity to a particular cell or location; the crossover of different receptors or cells; the co-localization of an antigen and adjuvant; etc. This can be achieved by direct administration of a genetically modified heteromeric protein to a biological system or by expression of nucleic acid encoding the subunits within the biological system. Nucleic acid expression allows for the engineering of additional levels of control in the system. For example, the expression of each subunit can be regulated differentially, in such a way that the complete heteromeric protein and the resulting co-localization of the activities take place only in the presence of all the conditions that are required for expression. of each subunit.

Genetically modified domains with reduced immunogenicity

In another embodiment of the invention, the SEED sequences can be modified to reduce their potential immunogenicity. Because SEED polypeptides are hybrids between two different natural human sequences, they include sequence segments at their junctions that are not found in natural human proteins. In an organism, these sequence segments can be processed into non-self T lymphocyte epitopes.

Methods for analyzing peptide sequences for their potential to create T lymphocyte epitopes are well known in the art. For example, ProPred (http://www.imtech.res.in/raghava/propred; Singh and Raghava (2001) Bioinformatics 17: 1236 - 1237), is a publicly available web-based tool that can be used for prediction of peptides that bind HLA-DR alleles. ProPred is based on a matrix prediction algorithm described by Sturniolo for a set of 50 HLA-DR alleles (Sturniolo et al., (1999) Nature Biotechnol. 17: 555-561). Using such an algorithm, several peptide sequences were discovered within the AG SEED and GA SEED polypeptide sequences, which are predicted to bind to multiple MHC class II alleles with significant binding strength and thus therefore, potentially immunogenic.

For example, in one embodiment, the AG SEED and GA SEED sequences are modified to remove one or more T cell epitopes present in the SEED sequence. This modification can include the substitution, deletion, or modification of one or more amino acid residues in order to remove the T-lymphocyte epitope. Table 1 presents a list of peptide sequences that are potential T cell epitopes in AG SEED and GA SEED, and possible amino acid substitutions predicted to reduce or eliminate the T cell epitope.

<td colspan="3">AG (f0) SEED</td>

<td>Pos</td><td>Peptide</td><td>Amino Acid Substitution</td>

<td> 32</td><td>FYPKDIAVE (SEQ ID NO: 12)</td><td>K35S</td>

<td> 67 69</td><td>FAVTSKLTV (SEQ ID NO: 13) VTSKLTVDK (SEQ ID NO: 14)</td><td>V75T</td>

<td> 99</td><td>YTQKTISLS (SEQ ID NO: 15)</td><td>T103S</td>

ES 2 395 969 T3

Table 1 (continued)

<td colspan="3">GA (f0) SEED</td>

<td>Pos</td><td>Peptide</td><td>Amino Acid Substitution</td>

<td> 18</td><td>LALNELVTL (SEQ ID NO: 16)</td><td></td>

<td> 20</td><td>LNELVTLTC (SEQ ID NO: 17)</td><td>L23Q</td>

<td> 23</td><td>LVTLTCLVK (SEQ ID NO: 18)</td><td></td>

<td> 54</td><td>YLTWAPVLD (SEQ ID NO: 19)</td><td>A58T</td>

<td> 55</td><td>LTW APVLDS (SEQ ID NO: 20)</td><td>L61V, D, T</td>

<td> 61</td><td>LDSDGSFFL (SEQ ID NO: 21)</td><td>L61V, D, T</td>

<td> 67</td><td>FFLYSILRV (SEQ ID NO: 22)</td><td>F67A, D, E, G, H, K, N, P, Q, R, S, T</td>

<td> 68</td><td>FLYSILRVA (SEQ ID NO: 23)</td><td></td>

<td> 69</td><td>LYSILRVAA (SEQ ID NO: 24)</td><td>A76T</td>

<td> 70</td><td>YSILRVAAE (SEQ ID NO: 25)</td><td>E78D</td>

<td> 72</td><td>ILRVAAEDW (SEQ ID NO: 26)</td><td></td>

Table 1 shows the peptides in AG (f0) SEED or GA (f0) SEED that are predicted to bind to HLA5 DR alleles and are potential T cell epitopes, and amino acid substitutions at specific residues (indicated in bold. ) within peptides predicted to reduce binding to HLA-DR alleles. "Pos" indicates the position of the peptide within the sequence. Amino acid numbering is sequential and relative to the first amino acid of the SEED molecule.

The parent polypeptides of AG SEED (AG (f0) SEED (SEQ ID NO: 3)) and GA SEED (GA (f0) SEED (SEQ ID

NO: 6)) "whole", and some exemplary polypeptide variants, including AG (f1) SEED (SEQ ID NO: 4),

AG (f2) SEED (SEQ ID NO: 5), GA (f1) SEED (SEQ ID NO: 7), GA (f2) SEED (SEQ ID NO: 8), and GA (f3) SEED (SEQ ID NO: 9), are shown in the following alignments.

Alignment of AG SEEDs (the dot indicates the identity of the residue)

GQPFRPEVHLLPPSREEMTKNQVSLTCLARGFYPKDIAVEWESNGQPENNYKTTPSRQEP AG (fO) SEED

.................................. S ............... .......... AG (fl) SEED

................................ .S. ........................ AG (f2) SEED

SQGTTTFÁVTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKTISL

......................... S ...

...... T ........................... S ...

AGÍfO) SEED AG (fl) SEED

AG (f2) SEED

Alignment of GA SEEDs (the dot indicates the identity of the residue)

GQPREPQVYTLPPPSEELALNELVTLTCLVKGFYPSDIAVEWLQGSQELPREKYLTWAPV GAffO) SEED

...................... Q ........................... ....... T-. GAÍfl) SEED

..................... .Q ............................... GAÍf2} SEED

....... OR .................................. T. . GA (f3) SEED

LDSDGSFFLYSILRVAAEDWKKGDTFSCSVMHEALHNHYTQKSLDR

V

D

T

T. D

<td>..... H. . . .</td><td>. . . .T.</td><td>, D ............................</td>

<td>..... D. . . .</td><td>. . . -T.</td><td>D ............................</td>

GAffO) SEED GA (fl) SEED

GA (f2) SEED GA &lt;f3) SEED

Additional exemplary embodiments according to the invention are detailed in the examples below.

Examples

EXAMPLE 1: Identification of homologous structures to become parents of a SEED.

ES 2 395 969 T3

In this group of examples, the aim is to produce two different CH3 homologous SEEDs that will form dimers that favor the formation of one heterodimer over the formation of two possible homodimers, thereby resulting in a predominance of homologous CH3 heterodimers. . The first task is to identify two or more CH3 domains that can produce this result when used as parents of a pair of SEEDs. The CH3 homodimer forms a dimerization interface between the sheets. It is important to find two CH3 domains that present significant differences at this interface, in order to produce an effective pair of SEEDs that preferentially heterodimerize.

The CH3 domains of IgG are structurally highly conserved throughout the animal kingdom, containing a classic Π-sandwich fold of the immunoglobulin domain. While there are significant interspecies differences in the identities of amino acids seen on the outer surface, the interface of the dimerization surface that is buried by dimerization is mostly conserved.

Each different class of immunoglobulin has its own Fc, and in particular has its own equivalent of the CH3 sequence and structure of IgG. Examination of the CH3 domain in the crystal structure of the Fc portion of a human IgA1 (PDB number 1OW0, resolution 3.1A), revealed that the global folding was homologous to the CH3 of human IgG. The principal RMSD (mean squared deviation) of the single CH3 domain alignment of IgA Fc 1OW0 and IgG Fc 1L6X, excluding turns where the alignment had different lengths, was approximately 0.99 A. (See Table 2). However, the interface of the IgA CH3 homodimer is significantly different from that of IgG1. Therefore, two SEEDs produced from the CH3 of human IgA1 and the CH3 of human IgG1 each contain some portion of the IgA1 interface, and some of the IgG1, and are designed not to dimerize between them, nor with any other parental CH3, but to dimerize, preferentially, with the other complementary SEED.

Table 2. Structural alignment of the CH3 domains of IgG and IgA *

<td>Human IgG</td><td>Human IgA</td>

<td>Q342 - M358</td><td>N343 - L359</td>

<td>N361 - P387</td><td>E363 - E389</td>

<td>N390 - S400</td><td>K394 - P404</td>

<td>G402 - L443</td><td>T409 - R450</td>

<td colspan="2">* Portions of IgG and IgA sequences were used to overlap structures and determine the RMSD of the main chain. The Insightll program (Accelrys, San Diego, CA) superimposed the backbone atoms of the Included residues into the structurally homologous sequences, numbered above. The RMSD between the two main chains (within the ranges in the table) was 0.99A.</td>

For example, the CH3 domain of human IgA1 and the CH3 domain of human IgG1 were used as parental polypeptides. For alignment and structural modeling, the IgG1 PDB entries 1DN2 (resolution 2.7A) and 1L6X (CH3 sequence highly homologous to 1DN2, with two minor differences, resolution 1.65 A), and the entry of the IgA1 1OW0 PDB (Resolution 3.1A). Figure 2 shows the structural alignment of the two sequences. The CH3 domain of IgG1 is numbered according to the numbering of the Kabat EU index (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242), while the CH3 domain IgA is sequentially numbered as in the PDB 1OW0 structure. The bold letters designate the main chain positions that were included in the alignment described in Table 2, which were additionally used to design junction crossing points in the SEED design.

EXAMPLE 2: Choice of exchange points

Once the structural alignment is determined and the interface residues are identified, the exchange points are ready to be chosen to create the SEEDs. The CH3 homodimer has a 180 ° rotational symmetry around an axis that runs between the domains approximately perpendicular to the beta chains (Figure 4). Each domain has the N-terminal and the C-terminal on opposite sides of the axis of simteria. Therefore, CH3 domains dimerize in a handshake-like manner, where, only in a line through the center of the interface along the axis of symmetry, residues on one side come into contact with the same residue. in the other partner. Residues on either side of that line come into contact with the partner's domain in an opposing manner: for example, residues on the Nterminal side of the first domain make contact with residues in the second domain that are on the Cterminal, and vice versa.

ES 2 395 969 T3

In one embodiment, a CH3-based SEED is designed to break symmetry, making the two sides different. For example, chain swapping will make one side of the dimer more similar to IgA1, and the other side more similar to IgG. This approach creates two different CH3-based SEEDs that are approximately complementary in their use of amino acids derived from IgG and IgA. As shown in Figures 3A and 3B, the linear polypeptide sequence runs back and forth between the IgG and IgA sequences in order to produce one physical side of the dimer structure similar to IgA and the other side similar to IgG. Thus, each sequence of a final SEED contains multiple exchange points, at each of which the linear sequence changes from IgA to IgG or from IgG to IgA (Figures 3A and 3B).

In general, there are several potential multiple exchange points in the polypeptide sequence that can be chosen to alternate between IgA and IgG1 sequences. An important consideration is that the final structure should have good structural characteristics (eg, stability, folding, expression, homology to the original). This can be accomplished by inspection, simple modeling, extensive calculation, trial and error, selection, or by other means. In the specific embodiment described herein, the sequence homology between the CH3 domains of IgA and IgG1 was used to decide the exchange points. Alignment of the CH3 crystal structures of IgG1 and IgA revealed roughly parallel lines of amino acids along an approximate angled plane through the mid-domain area. In-plane residues were identical in both CH3 classes in all but two strands in the IgG1 / IgA structural alignment. Furthermore, the alignment of structures generally showed the side chains of those amino acids in the same orientations of the rotamers, in particular in the hydrophobic nucleus. The hypothesis was then made that these residues could be used as trade-off points, and the residues on one side or the other could be modified without altering the overall structure. Figures 3A and 3B show sequence alignment with swapping points highlighted in bold type. Figures 5 and 6A-C show the molecular structure illustrating the three-dimensional locations of the exchange points.

In the two cases where the residues are not the same in a junction region, the exchange point options were based on structural considerations. In one case, Pro395 and Pro396 in IgG1 structurally correspond to Ala399 and Ser400 in IgA1. The division was made between these two residues. The other location is near the C-terminus, Leu441 and Ser442 in IgG1 structurally correspond to IIe448 and Asp449 in IgA1. Again, the division was made between these two residues.

Protein-protein interactions are mediated by the complementarity of the two interacting surfaces. The dominant factor for interaction is the composition and shape of those surfaces. Since the underlying backbone structures and the hydrophobic interiors of the CH3 domains of IgA and IgG1 are similar, it was contemplated according to the principles of the invention that only the surface would have to be modified, while the rest of the domain could contain IgG sequences. In this case, the exchange points were designed in the chains that form the interface and were very close to each other, allowing only the residues crucial for dimerization to be exchanged. Therefore, as an alternative, it is possible that the rest of the structure could stabilize the assembly domain, and thus CH3 SEEDs with a single exchange point in each of the seven chains could have advantages.

Therefore, two types of SEED can be designed and designated as "Complete" for SEEDs in which most or all residues in the domain were involved in chain exchange (corresponding to Figure 1A), or "Surface" for SEEDs in which the only modified residues are at the CH3 dimerization interface (corresponding to Figure 1B).

Based on this Example, it will be appreciated by those skilled in the art that a variety of strategies can be used to generate SEEDs based on the constant domains of the immunoglobulin superfamily.

EXAMPLE 3: Design of the “Complete” AG and GA SEED sequences

As an example, the simplest way to produce a "Complete" SEED would be to use a pure IgA sequence on the first side of the exchange site, and a pure IgG1 sequence on the second side of the exchange site. If the exchange point is chosen appropriately, this would result in a SEED that should fold appropriately and have a dimerization surface similar to IgGA1 on one side (eg, about half) of the domain, and a surface dimerization similar to IgG on the other side. A 'spectral image' SEED can be produced in a similar way, in which the first side is made up of IgG1 sequence and the second side is made up of IgA sequence. When these two SEEDs are expressed together, they will preferentially form heterodimers, since only in the heterodimer will each surface be making contact with a surface in the other domain that adapts to its class: that is, the first half of the first SEED, which is similar to IgA, will make contact with the second half of the second SEED, which is also similar to IgA, while the second half of the first SEED, which is similar to IgG1 , will make contact with the first half of the second SEED, which is also similar to IgG1. Since both sides of the interface are highly complementary, the association should be strong. On the other hand, when

ES 2 395 969 T3 any of the SEED tries to form a homodimer, each half of the dimerization surface will make contact with a surface on the companion SEED that comes from a different class: that is, the first half of a SEED, which is similar IgA will make contact with the second half of the partner domain, which is similar to IgG; and the second half of the first SEED, which is similar to IgG1, will make contact with the first half of the partner domain, which is similar to IgA. Because these surfaces are not very complementary, their affinity will be diminished, resulting in a thermodynamics that favors the formation of fewer homodimers and more heterodimers.

In this example, CH3 is the only part of the Fc or antibody that was modified. The rest of the Fc or immunoglobulin comes from human IgG1. Modifying the amino acid sequence where CH3 contacts or interacts with CH2 could potentially create problems with the interface between CH3 SEEDs and the CH2 domains of IgG1. Furthermore, this interface contains the binding site for FcRn, which confers important properties to Fc that it is desirable to maintain. Therefore, structural information (Martin et al. (2001) Molec. Cell 7: 867) was used to identify the CH3 residues involved in the interactions between CH3 and CH2, and between Fc and FcRn. Human IgG1 sequences were used for those residues in all SEEDs. Molecular modeling was also used to help choose adjacent residues to avoid modifying the structure of the FcRn interaction surface. The portion of CH3 that interacts with CH2 and FcRn is not part of the dimerization interface, therefore it was unlikely that these modifications would hinder the formation of heterodimers.

Figure 3B has the "Complete" SEED sequences aligned with the IgG1 and IgA sequences in structural alignment. The residues that reside at the exchange points are highlighted in bold. The residues that were not modified due to their importance in maintaining the interaction with CH2 and / or with FcRn are underlined.

EXAMPLE 4: Construction of Heterodimeric Fc and Antibody Molecules Containing CH3-Based SEEDs

The following general approach was used to produce HuFc and HuFc-IL2 constructs, in addition to antibody and antibody-IL2 constructs, which contain CH3 domains from SEED instead of CH3 domains from IgG1. The CH3 domain of IgG1 is contained almost entirely in a fragment of genomic DNA Ngo MIV / Sma I of approximately 0.4 kb, which is present in the expression plasmids pdCs or pdHL that express the constant region of a heavy chain of the IgG1. Exemplary expression plasmids are, for example, pdCs-huFc-IL2 (see, for example, Lo et al., Protein Engineering [1998] 11: 495), or pdHL7-KS-IL2 (see, for example, US Patent US Patent 6,696,517). The Ngo MIV site is found within the intron sequence immediately 5 'of the exon encoding the CH3 of IgG1, and the Sma I site is found in a sequence encoding Ser₄₄₄Pro₄₄₅Gly₄₄₆ near the C-terminus of IgG1 (EU Kabat index). An exemplary DNA sequence of mature human IgG1 Fc, expressed from a pdCs vector, is shown in SEQ ID NO: 27. Replacement of the parental Ngo MIV / Sma I fragment with a Ngo MIV / Sma I fragment encoding a CH3 SEED of the invention, generates upon expression a polypeptide containing a constant region with a CH3 SEED.

SEQ ID NO: 27

DNA sequence in pdCs encoding mature human IgG1 Fc including a terminal lysine residue

ES 2 395 969 T3

GAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGGTAAGCCAGCCCAGGCCTC

GCCCTCCAGCTCAAGGCGGGACAGGTGCCCTAGAGTAGCCTGCATCCAGGGACAGGCCCCAGCCG

GGTGCTGACACGTCCACCTCCATCTCTTCCTCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTT

CCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTGACATGCGTGG

TGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTG

CATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCT

CACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAAGCCC

TCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGTGGGACCCGTGGGGTGCGAGGGCCA

CATGGACAGAGGCCGGCTCGGCCCACCCTCTGCCCTGAGAGTGACCGCTGTACCAACCTCTGTGC ctacagggcagccccgagaaccacaggtgtacaccctgcccccatcacgggaggagatgaccaag

AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGA gagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctcct

TCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGC

TCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAA ATGA

Standard techniques were used to obtain DNA sequences encoding the CH3 SEEDs of the invention.

For example, DNA molecules with the following sequences as shown in SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID No: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 53 , were synthesized de novo and propagated on a pUC-derived carrier plasmid (Blue Heron Biotechnology, Bothell, WA).

SEQ ID NO: 28

Ngo MIV / Sma I DNA fragment, containing sequence encoding AG (f0) SEED (underlined corresponding to Figure 3B, "Complete AG SEED"):

gccggctcggcccaccctctgccctgagagtgac-cgctgtaccaacctctgtccctacaGGGCAG

CCCTTCCGGCCAGAGGTCCACCTGCTGCCCCCATCACGGGAGGAGATGACCAAGAACCAGGTCAG

CCTGACCTGCCTGGCACGCGGCTTCTATCCCAAGGACATCGCCGTGGAGTGGGAGAGCAATGGGC

AGCCGGAGAACAACTACAAGACCACGCCTTCCCGGCAGGAGCCCAGCCAGGGCACCACCACCTTC gctgtgacctcgaagctcaccgtggacaagagcagatggcagcaggggaacgtcttctcatgctc

CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGACCATCTCCCTGtc c ccg gg

SEQ ID NO: 29

Ngo MIV / Sma I DNA fragment, containing sequence encoding AG (s0) SEED (underlined corresponding to Figure 3A, "Surface AG SEED"):

qccqqctcqgcccaccctctqccctqaqaqtqaccqctqtaccaacctctqt-ccctacaGGGCAG

CCCTTCGAACCAGAGGTCCACACCCTGCCCCCATCACGGGAGGAGATGACCAAGAACCAGGTCAG cctgacctgcctggtccgcggcttctatcccagcgacatc-gccgtggagtgggagagcaatgggc

AGCCGGAGAACAACTACAAGACCACGCCTTCCCGGCTGGAGCCCAGCCAGGGCACCACCACCTTC

GCTGTGACCTCGAAGCTCACCGTGGACAAGAGCAGATGGCAGCAGGGGAACGTCTTCTCATGCTC

CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGtCCCCqqq

ES 2 395 969 T3

SEQ ID NO: 30

Ngo MIV / Sma I DNA fragment, containing sequence encoding GA (f0) SEED (underlined corresponding to Figure 3B, "Complete GA SEED"):

gccggctcggcccaccctctgccctgagagtgaccgctgtaccaacctctgtccctacaGGGCAG

CCCCGAGAACCACAGGTGTACACCCTGCCCCCACCGTCGGAGGAGCTGGCCCTGAACGAGCTGGT

GACGCTGACCTGCCTGGTCAAAGGGTTCTATCCCAGCGACATCGCCGTGGAGTGGCTGCAGGGGT

CCCAGGAGCTGCCCCGCGAGAAGTAGCTGACTTGGGCACCCGTGCTGGACTCCGACGGCTCCTTC ttcctctatagtatactgcgcgtggcagccgaggactggaagaagggggacaccttctcatgctc CGTGATGCATGACCAGGCTCGTCGTCGACGACCAGGcc

SEQ ID NO: 31

Ngo MIV / Sma I DNA fragment, containing sequence encoding GA (s0) SEED (underlined corresponding to Figure 3A, "Surface GA SEED"):

gccggctcggcccaccctctgccctgagagtgaccgctgta-ccaacctctgtccctacaGGGCAG

CCCCGAGAACCACAGGTGTACACCCTGCCCCCACCGTCGGAGGAGCTGGCCCTGAACAACCAGGT

GACGCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATG

GGCAGCCGGAGCCCCGCGAGAAGTACCTGACTTGGGCACCCGTGCTGGACTCCGACGGCTCCTTC

TTCCTCTATTCGATACTGCGCGTGGACGCAAGCflGGTGGCAGCAGGGGAAGGTCTTCTCATGCTC CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTGTCCCTG t ccccggg

SEQ ID NO: 32

Ngo MIV / Sma I DNA fragment, containing sequence encoding GA (f1) SEED (underlined):

gccggctcggcccaccctctgccctgagagtgaccgctgtaccaacctctgtccctacaGGGCAG

CCCCGAGAACCACAGGTGTACACCCTGCCCCCACCGTCGGAGGAGCTGGCCCTGAAGGAGCaGGT

GACGCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGCTGCAGGGGT

CCCAGGAGCTGCCCCGCGAGAAGTACCTGACTTGGaCcCCCGTGgTGGACTCCGACGGCTCCTTC

TTCCTCTATAGTATACTGCGCGTGaCAGCCGAtGACTGGAAGAAGGGGGACACCTTCTCATGCTC

CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCGACCGCtccccggg

SEQ ID NO: 53

Ngo MIV / Sma I DNA fragment, containing sequence encoding GA (f2) SEED (underlined):

ES 2 395 969 T3 qccggctcggcccaccctctgccctgagagtgaccgctgtaccaacctctgtccctacaGGGCAG

CCCCGAGAACCACAGGTGTACACCCTGCCCCCACCGTCGGAGGAGCTGGCCCTGAACGAGCaGGT

GACGCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGCTGCAGGGGT

CCCAGGAGCTGCCCCGCGAGAAGTACCTGACTTGGgCaCCCGTGgacGACTCCGACGGCTCCcaC TTCCTCTATAGTATACTGCGCGTGaCAGCCGAtGACTGGAAGAAGGGGGACACCTTCTCATGCTC CGTGATGCATGACGACc

In addition, a polypeptide containing GA (f3) SEED can be encoded by the following DNA sequence:

SEQ ID NO: 54

Ngo MIV / Sma I DNA fragment, containing sequence encoding GA (f3) SEED (underlined):

gccggctcggcccaccctctgccctgagagtgaccgctgtaccaacctctgtccctacaGGGCAG

CCCCGAGAACCACAGGTGTACACCCTGCCGCCACCGTCGGAGGAGCTGGCCCTGAACGAGCaGGT

GACGCTGACCTGCCTGGTCAAAGGCTTGTATCCCAGCGACATCGCCGTGGAGTGGCTGCAGGGGT CCCAGGAGCTGCCCCGCGAGAAGTACCTGACTTGGaCcGCCGTGaCCGACTGCGACGGCTCCgae TTCCTCTATAGTATACTGCGCGTGaCAGCCGAtGACTGGAAGAAGGGGGACACCTTCTCATGCTC CGTGATGCATGAGGCTGTGCACAACCACTACACGCAGAAGAGCCTCGACCGCtccccggq

These synthetic sequences were further extended at their 3 'end with a random DNA stretch of approximately 50 bp, in order to allow easy separation of the desired Ngo MIV / Sma I insert fragment taken by excision and a fragment of a plasmid vector of similar size during fragment purification. The purified Ngo MIV / Sma I fragments were then ligated to a similarly treated pdCs vector, which could contain either an Fc fraction or a Fc-IL2 fraction, or alternatively, to a similarly treated pdHL vector that could contain either a DI-KS moiety or a DI-KS-IL2 moiety. Thus, for example, pdCs-HuFc (AG (f0)) -IL2 containing the Ngo MIV / Sma I fragment for AG (f0) SEED (SEQ ID NO: 28), and pdCs-HuFc (GA (f0 )), containing the Ngo MIV / Sma I fragment for GA (f0) SEED (SEQ ID NO: 30). pdCs-HuFc (AG (f0)) - IL2 and pdcs-HuFc (GA (f0)) encode a polypeptide chain Fc (AG (f0) sEeD) -IL-2, and a polypeptide chain Fc (GA (f0) SEED) , respectively. Exemplary sequences of Fc (AG (f0) SEED) -IL-2 and of Fc (GA (f0) SEED) are shown as SEQ ID NO: 33 and SEQ ID NO: 34, respectively, below. A diagram of the resulting heterodimeric protein is shown in Figure 11A. To obtain the simultaneous expression of both polypeptide chains from a host cell, the transcription units for these Fc polypeptides were combined into a single expression vector, as described below in Example 5.

SEQ ID NO: 33

Polypeptide sequence of an Fc (AG (f0) SEED) -IL2:

EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVD

GVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPFR

PEVHLLPPSREEMTKNQVSLTCLARGFYPKDIAVEWESNGQPENNYKTTPSRQEPSQGTTTFAVT

SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKTISLSPGKAPTSSSTKKTQLQLEHLLLDLQMIL NGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNELFHLRPRDMFCNCENCELISTKEFSIGRPRDMFCNCENCELISTKEFSIGRPRDMFCNCENCELISTKEFSIGRPRDMFCNCENCELISTFHLRPRDMFCNCE

SEQ ID NO: 34

ES 2 395 969 T3

Polypeptide sequence of an Fc (GA (f0) SEED):

EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVD

GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK-GQPRE PQVYTLPPPSEELALNELVTLTCLVKGFYPSDIAVEWLQGSQELPREKYLTWAPVLDSDELPREKYLTWAPVLDSD

SILRVAAEDWKKGDTFSCSVMHEALHNHYTQKSLDRSPGK.

Similarly, pdHL-DI-KS (AG (f0)) - IL2, containing the Ngo MIV / Sma I fragment for AG (f0) SEED (SEQ ID NO: 28), and pdHL-DI-KS ( GA (f0)), containing the Ngo MIV / Sma I fragment for GA (f0) SEED 5 (SEQ ID NO: 30). pdHL-DI-KS (AG (fD)) - IL2 and pdHL-DI-KS (GA (f0)) encode the heavy chain of DI-KS (AG (f0)

SEED) -IL-2 (SEQ ID NO: 35), the heavy chain of DI-KS (GA (f0) SEED) (SEQ ID NO: 36), respectively. Both expression vectors also encode the DI-KS light chain (SEQ ID NO: 37).

SEQ ID NO: 35

DI-KS (AG (f0) SEED) -IL2 heavy chain polypeptide sequence:

qiqlvqsgpelkkpgssvkisckasgytftnygmnwvrqapgkglkwmgwintytgeptyaddfk grftitaetststlylqlnnlrsedtatyfcvrfiskgdywgqgttvtvssastkgpsvfplaps

SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSÜGTQ

TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC

VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK

ALPAPIEKTISKÁKGQPFRPEVHLLPPSREEMTKNQVSLTCLARGFYPKDIAVEWESNGQPENNY

KTTPSRQEPSQGTTTFAVTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKTISLSPGAAPTSSS

TKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

SEQ ID NO: 36

DI-KS heavy chain polypeptide sequence (GA (f0) SEED):

QIQLVQSGPELKKPGSSVKISCKASGYTFTNYGMNWVRQAPGKGLKWMGWINTYTGEPTYADDFK

GRFTITAETSTSTLYLQLNNLRSEDTATYFCVRFISKGDYWGQGTTVTVSSASTKGPSVFPLAPS

SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQ

TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMTSRTPEVTC

VWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK

ALPAPIEKTISKAKGQPREPQVYTLPPPSEELALNELVTLTCLVKGFYPSDIAVEWiX2GSQELPR

EKYLTWAPVLDSDGSFFLYSILRVAAEDWKKGDTFSCSVMHEALHNHYTQKSI.DRSPGK

SEQ ID NO: 37

DI-KS light chain polypeptide sequence:

ES 2 395 969 T3

QIVLTQSPASLAVSPGQRATITCSASSSVSYILWYQQKPGQPPKPWIFDTSNLASGFPSRFSGSG

SGTSYTLTINSLEAEDAATYYCHQRSGYPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS vvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacev

THQGLSSPVTKSFNRGEC

To obtain a single expression vector that expresses both the DI-KS (AG (f0) SEED) -IL-2 and DI-KS (GA (f0) SEED) heavy chain transcription units, in addition to the unit For common light chain transcription, a construct was prepared essentially as follows: a Sal I / Mfe I fragment of approximately 3.9 kb containing the sequence encoding KS (AG (f0) SEED) -IL-2 was taken by excision of the expression construct pdHL-10 (pdHL-10 is an expression vector of a subsequent generation of pdHL containing a single Sal I site outside the transcription unit) and ligated to a Sal I / Bam HI digested pBS plasmid, together with a Bam HI / Mfe I double linker fragment. This double linker fragment is composed of Oligo 11 (SEQ ID NO: 38) and Oligo12 (SEQ ID NO: 39) and contains an internal Sal I site.

Oligo11 (SEQ ID NO: 38)

AATTGCCGGGTCGACATACG

Oligo12 (SEQ ID NO: 39)

GATCCGTATGTCGACCCGGC

The 3.9 kb fragment was then excised from pBS as a Sal I fragment and inserted into the unique Sal I site of a pdHL-10 expression construct that already contains the transcription units encoding the DI-KS heavy chain. (GA (f0) SeEd) and the DI-KS light chain.

EXAMPLE 5: Assay to Determine Heterodimeric Fc Molecules Containing CH3-Based SEEDs

The examples described in the present patent involve the dimerization of CH3, which is an important step in the nucleation of the formation of the Fc heavy chain dimers and of the immunoglobulin. In theory, if two distinct Fc moieties (for example, named A and B) containing CH3 domains are expressed simultaneously in a cell, they could pair up and form dimeric Fc molecules in the following configurations: A: A, A: B , and B: B. If the CH3 domains and the hinge domains are identical, the A: A, A: B, and B: B configurations are expected to occur in a 1: 2: 1 ratio if A and B are expressed in equal amounts. The relative quantities, kinetics, and thermodynamics of AA, AB, and BB interactions are important regulating factors for the observed ratio of these final three species, as are the expression levels. In general, when protein A and protein B are expressed in relative amounts [A] and [B], where [A] + [B] = 1, and homodimers and heterodimers are produced in relative concentrations [AA], [AB ], and [BB], if there is an unbiased association, these dimeric species will be respectively present in a ratio of [A]²: 2 * [A] * [B]: [B]². If the relative concentration is [AB]&gt; 2 * [A] * [B], then heterodimerization is facilitated, while if the relative concentration is [AB] &lt;2 * [A] * [B], then the homodimerization. For a preferred SEED pair, the ratio [AB] / 2 * [A] * [B] is greater than 2, and preferably greater than 3, and more preferably greater than 5.

To determine the ratios of different species, you need a way to distinguish them by testing. An easy way to do this is to attach a fusion partner to one of the Fc subunits (eg, "A"), which would result in each of the final three species having a significantly different molecular weight. Accordingly, constructs were prepared to express both human Fc (HuFc) and human Fc fused to human IL-2 (HuFc-IL-2) in a cell. The constructs were prepared as follows: The gene for HuFc was taken by excision of a vector containing an Fc fraction (see, for example, Lo et al., Protein Engineering [1998] 11: 495) by enzymatic restriction in a 5 'Xbal site and a 3' Xhol site. The 1.4 kb fragment containing the HuFc gene was gel pufirified and subcloned into a second vector, pdCS-MuFc-KS-kappa, replacing its muFc with HuFc. The HuFc gene was flanked by two Sall sites on the outside of the promoter region.

A third vector containing a gene encoding HuFc-IL-2 and a unique Sall site was chosen to receive the HuFc gene. The vector was cut with Sall, treated with Calf Intestine Phosphatase (CIP) and gel purified. The second vector was digested with Sall and a 2.5 kb fragment was gel purified. This fragment contained the HuFc gene and a promoter, and was inserted into the third gel purified vector. The final resulting vector contained two different transcription units with duplicate versions of the same regulatory elements, where one

ES 2 395 969 T3 transcription unit controlled expression of wild-type HuFc and the other controlled expression of wild-type HuFcIL-2. Expression constructs containing SEED-based HuFc and SEED-based HuFc-IL-2 were produced in a similar manner.

This final vector was expanded using Qiagen maxi-prep. 10 mg of DNA was used to transiently transfect baby hamster kidney (BHK) cells, using a Lipofectamine TM2000 kit (Invitrogen). The cells were divided, one half was cultured in usual medium, the other half in serum-free medium, for two days. Supernatants (eg, 100 µl) were harvested. 10 microliters of protein A granules were added and mixed overnight at 4 ° C to bind the protein. After washing 3 times with PBS containing 1% Triton-X100, the samples were loaded on Bi- and Tri- gels with 4-12% gradient electrophoresis on Un-Page (Invitrogen), both under reducing conditions and not. reducers. The gels were stained with colloidal blue (Invitrogen) for direct visualization of proteins.

Typical control results are shown in lanes 8-10 on the gels shown in Figure 12. Reducing gel analysis in Figure 12C shows the ratio of the HuFc and HuFc-IL-2 subunits. Non-reducing gel analysis in Figure 12B shows that HuFc and HuFc-IL-2 molecules dimerize randomly, with no preference for heterodimerization compared to homodimerization.

The gels were transferred to nitrocellulose membranes for analysis by Western blot. In Western blots, the protein was detected in two ways in order to measure both Fc and IL-2. Antibodies against human IgG Fc (Jackson Immunolabs) conjugated to horseradish peroxidase (HRP) were used to detect Fc. The blots were detected with Enzyme Chemiluminescence Substrate or ECL (for its acronym in English) and by film exposure. A biotinylated antibody against human IL-2 (R&amp;D systems) was used to detect IL-2, and the signal was developed by the addition of HRP-conjugated avidin, and detection with ECL substrate and film exposure. These experiments confirmed the identity of the bands shown in Figure 12.

To measure the levels of heterodimers and homodimers formed during protein expression of GA SEED / AG SEED "Full" and GA SEED / AG SEED "Surface", similar experiments were carried out. Single expression expression vector constructs expressing an AG SEED-IL2 fusion protein and a GA SEED protein were constructed as described above for Fc / Fc-IL2 expression. As shown in lanes 2-4 in Figures 12B and 12C, when the proteins GA SEED (Fc (GA (f1)) SEED) "Complete" and AG SEED-IL-2 (Fc (AG (f0)) SEED) -IL2) "Complete" were co-expressed in Ns / 0 cells, heterodimerization was clearly preferred, with non-detectable Fc (AG SEED) -IL2 homodimers, and only small amounts of Fc homodimers (GA SEED ) were detected. Similarly, as shown in lanes 5-7 in Figures 12B and 12C, when GA SEED (Fc (GA (s0) SEED) "Surface" and AG SEED-IL-2 (Fc (AG (s0) SEED) -IL2) "Surface" were co-expressed in NS / 0 cells, heterodimerization was clearly favored, with non-detectable Fc (AG SEED) -IL-2 homodimers, and only small amounts of homodimer of Fc (GA SEED) detected. Heterodimers were estimated to constitute approximately&gt; 90% of the total amount of assembled proteins in the cell.

EXAMPLE 6. Construction, expression, and heterodimerization properties of SEED molecules with reduced immunogenicity.

Because the AG and GA SEED protein sequences are hybrids between two natural human sequences, these sequences include peptide segments that are not seen in normal human proteins and that can be processed on non-MHC Class II T-cell epitopes. own. Therefore, the following sequences were designed to reduce the number of potential non-self T lymphocyte epitopes in the AG SEED and GA SEED sequences, represented by the polypetide sequence shown in SEQ ID NO: 1 and SEQ ID NO : 2, respectively, where X₁, X₂, X₃, X4, X₅, or X₆ they can be any amino acid. In some embodiments, in SEQ ID NO: 1, X₁ is S, X₂ is V or T, and X₃ is S. In some embodiments, in SEQ ID NO: 2, X₁ is Q, X₂ is A or T, X₃ is L, V, D, or T, X₄ is F, A, D, E, G, H, K, N, P, Q, R, S, or T, X₅ is T, and X₆is D.

SEQ ID NO: 1

AG SEED polypetide sequence, with amino acid variants X₁ - X₃:

GQPFRPEVHLLPPSREEMTKNQVSLTCLARGFYPX₁DIAVEWESNGQPENNYKTTPSRQEPSQGTT

TFAVTSKLTX₂DKSRWQQGNVFSCSVMHEALHNHYTQKX₃ISL

SEQ ID NO: 2

ES 2 395 969 T3

GA SEED polypetide sequence, with amino acid variants X₁ - X₆:

gqprepqvytlpppseelalnex₁vtltclvkgfypsdiavewlqgsqelprekyltwx₂pvx₃ds dgsx₄flysilrvx₅ax₆dwkkgdtfscsvmhealhnhytqksldR.

The DNA molecule (SEQ ID NO: 32) encoding the exemplary SEED variant GA (f1) SEED (SEQ ID NO: 7) was created by de novo synthesis and introduced into the expression vector pdCs, as described in Example 4, producing the Fc polypeptide (GA (f1) SEED).

SEQ ID NO: 7

GA (f1) SEED polypeptide sequence:

GQPREPQVYTLPPPSEELALNEQVTLTCLVKGFYPSDIAVEWLQGSQELPREKYLTWTPWDSDG

SFFLYSILRVTADDWKKGDTFSCSVMHEALHNHYTQKSLDR

Mutations were introduced in the variants of the SEED fractions by way of example, AG (f1) SEED (SEQ ID NO: 4), AG (f2) SEED (SEQ ID NO: 5), and GA (f2) SEED (SEQ ID NO: 8), by a two-step PCR (polymerase chain reaction) approach, in which two partially overlapping mutagenized PCR fragments from a first round of PCR amplification are combined in a second round PCR amplification to generate the final full-size fragment, using standard methods familiar to those skilled in the art. Essentially, two PCR reactions were carried out in the first round, each with a PCR primer incorporating the mutant sequence paired with a suitable flanking primer containing appropriate restriction sites, Ngo MIV for the upstream primer, and Sma I for the downstream primer, and a template DNA encoding the appropriate parental SEED moiety. The same flanking PCR primers were used in the second PCR amplification reaction, using the products of the first PCR amplification as templates. The resulting fragment was cloned into a vector pCR2.1 (Invitrogen) and its sequence was verified. Finally, the 0.4 kb Ngo MIV / Sma I DNA fragment was excised from the vector, gel purified, and ligated to a similarly treated recipient expression plasmid, as described in Example 4 .

Specifically, for AG (f1) SEED, the primer pairs Oligo 1 (SEQ ID NO: 40) / Oligo2 (SEQ ID NO: 41) and Oligo3 (SEQ ID NO: 42) / Oligo4 (SEQ ID NO: 43 ), with the model pdCs-Fc (AG (f0) SEED) -IL2, were used in the first round of PCR reactions. Oligo1 (SEQ ID NO: 40) / Oligo4 (SEQ ID No: 43) were used in the second round of PCR reactions, generating the DNA fragment shown in SEQ ID NO: 44, which was introduced into pdCs- Fc (AG (f0) SEED) -IL2. For AG (f1) SEED, the Oligo1 (SEQ ID NO: 40) / Oligo5 (SEQ ID NO: 45) and Oligo6 (SEQ ID NO: 46) / Oligo4 (SEQ ID NO: 43) primer pairs were used, with model pCR2.1 containing the sequence shown in SEQ ID NO: 44 in the first round of PCR reactions. Oligo1 (SEQ ID NO: 40) / Oligo4 (SEQ ID NO: 43) were used in the second round of PCR reactions, generating the DNA fragment shown in SEQ ID NO: 47, which was introduced into pdCs- Fc (AG (f0) SEED) -IL2. For For GA (f2) SEED, the Oligo1 (SEQ ID NO: 40) / Oligo10 (SEQ iD NO: 48) and Oligo7 (SEQ ID NO: 49) / Oligo9 (SEQ ID NO: 50) primer pairs were used with the pUC model carrier plasmid containing the sequence shown in SeQ ID NO: 32, in the first round of PCR reactions. Oligo1 (SeQ ID NO: 40) / Oligo9 (SEQ ID NO: 50) were used in the second round of PCR reactions, generating the DNA fragment shown in SEQ ID NO: 47, which was introduced into pdCs- Fc (GA (f2) SEED). All the sequences referenced above are shown below.

Oligo1 (SEQ ID NO: 40)

GCCGGCTCGGCCCACCCTCT

Oligo2 (SEQ ID NO: 41)

CGGCGATGTCGCTGGGATAGAA

Oligo3 (SEQ ID NO: 42)

TTCTATCCCAGCGACATCGCCG

Oligo4 (SEQ ID NO: 43)

ES 2 395 969 T3

CCCGGGGACAGGGAGATGGACTTCTGCGTGT

Oligo5 (SEQ ID NO: 45)

GCTCTTGTCTGTGGTGAGCTT

Oligo6 (SEQ ID NO: 46)

AAGCTCACCACAGACAAGAGC

Oligo7 (SEQ ID NO: 49)

CCTGACTTGGGCACCCGTGGACGACTCCGACGGCTCCCACTTCCTCTATA

Oligo9 (SEQ ID NO: 50)

CCCGGGGAGCGGTCGAGGCTC

Oligo10 (SEQ ID NO: 48)

TATAGAGGAAGTGGGAGCCGTCGGAGTCGTCCAGGGGTGCCCAAGTCAGG

SEQ ID NO: 44

Ngo MIV / Sma I DNA fragment, containing a sequence encoding AG (f) SEED (underlined):

gccggctc gcccaccctct £ £ £ tccotacaGrGGCAGCCCTTCCGGC cagaggtccacctgctgcccccatcacgggaggagatgaccaagaaccaggtcagcgtgacctgc ccctgagagtgaccECtetaccaacctct CTGGCACGCGGCTTCTATCCCAgcGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAA CAACTACAAGACCACGCCTTCCCGGCAGGAGCCCAGCCAGGGCACCACCACCTTGGCTGTGACCT

CGAAGC T CAC CGT GGACAAGAGCAGATGGCA G CAGGGGAACGTCTTCTCATGCTCCGTGATGCAT

GAGGCTCTGCACAACCACTACACGCAGAAGtCCATCTCCCTGtccccggg

SEQ ID NO: 47

Ngo MIV / Sma I DNA fragment, containing a sequence encoding AG (f2) SEED (underlined):

qccqqctcqqcccaccctctqccctqaqaqtqaccqctgtaccaacctctqtccctacaGGGCAGCCCTTCC ggccagaggtccacctgctgcccccatcacgggaggagatgaccaagaaccaggtcagcctgacctgcctgg

CACGCGGCTTCTATCCCAgcGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCGGGAGAACAACTACAAGA

CCACGCCTTCCCGGCAGGAGCCCAGCCAGGGCACCACCACCTTCGCTGTGACCTCGAAGCTCACCacaGACA AGAGCAGATGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGC

AGAAGtCCATCTCCCTGtccc cg gg

The sequences Fc (AG (f1) SEED), Fc (AG (f2) SEED), Fc (GA (f1) SEED) -IL2 and Fc (GA (f2) SEED) -IL2 were expressed individually and in combinations in HEK 293T cells, and the resulting secreted proteins were partially purified based on the binding of Fc to Staphylococcal protein A and characterized by SDS-PAGE gel. When the samples were subjected to a reducing SDS gel, it was apparent that the Fc (AG (f1) SEED) and Fc (AG (f2) SEED) proteins were very poorly expressed by them, which is similar to the case of the parental Fc (AG (f0) SEED) protein. Without wishing to be bound by theory, the deficient expression is most likely the result of proteolysis of the monomeric protein that has no dimerization partner. The Fc protein (GA (f1) SEED) -IL2 was expressed at a high level, while the Fc protein (GA (f2) SEED) -IL2, which differs in additional amino acid substitution Va175Thr, was expressed at a very level

ES 2 395 969 T3 low. Again, without wishing to be bound by theory, the deficient expression may be the result of proteolysis of the monomeric protein that has no dimerization partner. The combinations Fc (AG (f1) SEED) plus Fc (GA (f1) SEED) -IL2, Fc (AG (f2) SEED) plus Fc (GA (f1) SEED) -IL2, Fc (AG (f1) SEED) plus Fc (GA (f2) SEED) -IL2, and Fc (AG (f2) SEED) plus Fc (GA (f2) SEED) -IL2, were tested and all expressed at high levels. The same samples were run on non-reducing gel analysis and these results were confirmed. This analysis indicated that, for the pools, basically all of the expressed protein was heterodimeric. These results indicate that certain GA and AG SEED protein variants with reduced immunogenicity retain their preference for heterodimerization.

EXAMPLE 7. Expression of an antibody-cytokine fusion protein using SEED Fc regions.

To further demonstrate the versatility of SEED-based Fc regions, an intact antibody with a single IL-2 moiety was constructed as described in Example 4. A diagram of this protein is shown in Figure 11B. Specifically, the protein contains antibody V regions that bind EpCAM, and that have the sequences as described in US Patent 6,696,517, CH2 and cH1 domains of human IgG1, human Ckappa, GA and AG SEED domains, and human IL-2 fused to the C-terminus of the heavy chain containing AG SEED.

The protein was expressed in mammalian cells according to standard techniques, producing a protein with the polypeptide chains shown in SEQ ID NO: 37, SEQ ID NO: 36, and SEQ ID NO: 35.

The resulting protein was characterized to determine the degree to which heterodimeric forms were secreted from mammalian cells. For example, the secreted protein was characterized by non-reducing SDS polyacrylamide gel electrophoresis. In principle, three bands could be identified, corresponding to antibodies with none, one or two IL-2 fractions. Actual non-reducing gel analysis predominantly showed a single band with a molecular weight corresponding to an antibody with a single IL-2 fraction. A much less intense band with a molecular weight corresponding to non-IL-2 fractions was observed, and a band with a molecular weight corresponding to two IL-2 fractions was not detectable. When samples were reduced prior to gel analysis, approximately equal amounts of protein corresponding to an antibody heavy chain and a heavy chain IL-2 were detected.

The foregoing description of the present invention provides illustration and description, but is intended to be exhaustive or to limit the invention to that precisely disclosed. Modifications and variations consistent with the contents expressed above can be acquired from the practice of the invention. Therefore, it is seen that the scope of the invention is defined by the claims and their equivalents.