Heterodimeric multispecific antibody format

Abstract

The present invention relates to novel heterodimeric multispecific forms of multiple antibody variable domains, which comprise a core of two separate pairs of variable domains, wherein all two variable light domains and two homologous variable heavy domains are tandemly located on two separate protein chains, respectively.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201680048188.4, titled "Heterodimeric Multispecific Antibody Forms", with an application date of June 15, 2016. Technical Field

[0002] The present invention relates to novel heterodimeric multispecific forms of multiple antibody variable domains, which comprise a core of two separate pairs of variable domains, wherein all two variable light domains and two homologous variable heavy domains are tandemly located on two independent protein chains, respectively. Background Art

[0003] In the past four decades since the development of the first monoclonal antibody [R17], antibodies have become an increasingly important class of biomolecules for research, diagnostic, and therapeutic purposes.

[0004] Antibodies as therapeutic agents are evolving towards more rationally designed functions to improve and expand their inherent properties. Examples include optimizing effector functions through glycoengineering [R18], specific targeting such as crossing the blood-brain barrier [R19], or modulating the half-life by, for example, increasing the binding to FcRn.

[0005] A complementary approach to antibody functionalization is to combine different target specificities in one molecule to generate bispecific or multispecific antibodies or antibody fragments, thereby enabling alternative mechanisms of action, such as redirecting T cells, for example, the bispecific antibody Blinatumomab or the trispecific antibody Catumaxomab.

[0006] Although a large number of different multispecific antibody forms [R21] have been developed so far, the current repertoire of bispecific and multispecific antibody forms still poses considerable technical challenges and little flexibility for industrialization. Only a few forms allow trispecific and multispecific binding, and even fewer forms support the formation of heterodimeric proteins.

[0007] Different multispecific forms have been proposed in the past. Conceptually, these forms can be divided into three categories: a) single-chain multispecific forms, in which different target-binding domains are all located on a single protein chain and are expressed from a single gene, b) homodimeric and homotrimeric forms, in which different target-binding domains are located on the same protein chain, which are assembled by using a multimerization domain to form bivalent / multivalent and optionally also multispecific complexes, and c) heterodimeric forms, in which target-binding domains are located on different protein chains, and the assembly of the two protein chains is driven by a heterodimerization domain.

[0008] In principle, heterodimeric multispecific formats offer the following advantages: By simple replacement of two heterodimerizing protein chains, binding domains with different specificities and affinities can be easily tested in various combinations, enabling direct screening of the best combinations of specificity and affinity in the final format without the need for cumbersome cloning.

[0009] Such screening in the final product format is required when the binding properties and / or potencies of the various domains need to be carefully matched to each other to obtain optimal potency of the bispecific protein while simultaneously minimizing the risk of non-specific effects. In a clinical setting, this would translate into optimal efficacy at the lowest risk of adverse effects. Situations where such an optimal combination is needed can be, for example, the simultaneous blockade of two disease-driving cytokines produced at different concentrations during the course of a disease. In such a case, the therapeutic bispecific protein should be able to effectively block both cytokines at one and the same therapeutic dose.

[0010] Another example where the characteristics of the target-binding domains of a multispecific molecule must be coordinated is the treatment of cancer by targeting two cell surface targets on tumor cells with a cytotoxic antibody. Although in this case the two cell surface targets of the antibody may only be co-expressed on cancer cells, they can be expressed individually in various healthy tissues. To obtain the best efficacy in cancer treatment at the lowest risk of adverse side effects, the cytotoxic antibody should preferentially bind to cells where the two targets are co-expressed and should not bind to tissues where only one of the two targets is expressed. To achieve this, the affinities of the two target-binding domains need to be adjusted such that, on the one hand, the individual domains have too weak an affinity for their targets to cause cell lysis and, on the other hand, the cooperative affinity resulting from the simultaneous binding of the bispecific molecule to the two targets on cancer cells is sufficient to induce cell lysis. Due to the geometric constraints arising from the simultaneous binding to different macromolecules immobilized on the cell surface, the domain combination that achieves maximum cooperative binding is not only an affinity function but also an epitope function and can only be identified by testing different domain combinations in the actual product format.

[0011] Native IgG-type antibodies can be considered to be in a homodimeric format.

[0012] To increase the number of specificities of homodimeric antibody formats using the conventional IgG structure as a scaffold, additional binding moieties such as single-chain Fvs [R15], Fvs [R16], single domains [such as Nanobodies: Huang et al., Expert Rev Mol Diagn. 10 (2010): 777-85] or alternative scaffolds [such as Fynomers: Schlatter et al., MAbs. 4 (2012) 497-508] can be appended to the amino or carboxyl termini of the heavy and light chains. An advantage of this approach is that bispecific to trispecific constructs can be generated with a conventional IgG as the core domain, which enables the use of most of the manufacturing and modification techniques that have already been established for conventional IgG. However, due to the homodimeric nature of the conventional Fc region, this approach will always generate at least two identical binding domains in each molecule and thus result in bivalent binding to certain targets. This is not always desirable, especially (a) when the desired effect can only be achieved by cooperative binding to two targets, or (b) when the molecular weight should not be increased further. In addition, this approach often encounters poor domain stability of the appended binding moieties, making them unsuitable for drug development.

[0013] The concept of fusing additional binding domains to increase specificity can also be applied to Fab fragments [R14] or other antigen-binding fragments of IgG [R23]. Due to the heterodimeric nature of the Fab, which consists of a heavy and a light chain, the Fab fragment can be used as a heterodimerization domain. For example, Fab fragments have been used to engineer so-called Tribodies. In this format, an scFv fragment is fused to the carboxyl termini of the light and heavy chains of the Fab, resulting in a truly heterodimeric trispecific molecule. The light-chain - heavy-chain crosslinking of the Fab is mainly driven by the interaction between CL-CH1, which is additionally linked by a covalent disulfide bond [R2]. The challenges of this format are: (a) the stability limitation of the most unstable component, which will most likely be the appended scFv, and (b) the limitation to a maximum of three target specificities.

[0014] As a method to address the limitations of the homodimeric bispecific format, heterodimeric IgG[R31] has been introduced. Simple co-expression of two different mAbs from a single cell results in a very low likelihood of assembling heterodimeric bispecific IgG, in which two different heavy chains will pair with each other and two different light chains will pair with their corresponding heavy chains[R24]. However, it will also result in A) mismatching of heavy and light chains with different specificities, and B) a mixture of different heavy chain combinations forming monospecific and bispecific variants. To address these difficulties, several approaches have been taken, which create artificial asymmetry in the molecule. The "knob-into-hole" concept[R3, R4] uses engineering of the heavy chain / heavy chain or heavy chain / light chain interface to drive co-expressed chains to crosslink into the desired configuration. In another approach, the CrossMab method[R5] allows selective pairing of engineered light chain / heavy chain pairs. The drawback of these methods is that it is difficult to separate any residual fraction of mismatched molecules from the product. Therefore, other techniques focus on separation issues by altering the differential binding properties of monospecific and bispecific conjugates[R22], while also tolerating yield losses caused by the random distribution of variants.

[0015] Another limitation of IgG-based heterodimeric formats is that they must all contain the Fc effector domain. In a format where heterodimerization is driven by binding domains directed to arbitrarily chosen targets, it will be possible to increase the number of specificities / functionalities at the same or lower molecular weight. Molecules with lower molecular weight can penetrate target tissues (such as solid cancers) more effectively, and thus hold promise for improved efficacy at the same or lower doses.

[0016] By simply adding a binding domain that interacts with serum albumin, for example, such small formats can be engineered to have a serum half-life comparable to that of IgG.

[0017] Another approach uses non-antibody fusion proteins to confer, for example, the desired multispecificity to an scFv moiety. Examples of such fusion proteins are Dock-and-Lock[R25], barnase-barstar[R26], jun-fos[R27], TNF[R28], or HSA[R29]. What these concepts have in common is the addition of at least one pair of domains that interact in a heterodimeric manner to link together bispecific or multispecific binding domains. These heterodimerization domains do not directly participate in target binding, but they increase the molecular weight of the protein - similar to constant region 1 (C1) in the trimeric format. Additionally, by incorporating non-human epitopes and sequences, they may have an increased risk of immunogenicity.

[0018] Compared with the interaction between CL and CH1 discussed above, the cross-linking of the VL-VH domains that form the antibody binding site is generally considered weak. However, there are several concepts of heterodimeric antibody fragments consisting only of antibody variable domains. Methods such as diabody [R6], DART [R10], and tandab [R7, R8] provide elegant and parsimonious ways to generate homologous and heterodimeric bispecific and bivalent to tetravalent assemblies. The most important limitations of these format strategies are (a) adding additional specificities by fusing, for example, an scFv to the amino or carboxyl terminus of either chain of a diabody or DART can lead to in-chain pairing of the variable light and variable heavy domains, making heterodimerization of the two protein chains very challenging, and (b) due to the weak domain-binding points between the variable light and variable heavy chains often observed in the past, the monomers of these formats have poor stability and productivity, so further engineering, such as introducing inter-domain disulfide bonds [R12] to stabilize the VL / VH binding points, is considered necessary.

[0019] To construct multi-specific single-chain tandem Fv antibodies, Kipriyanov et al. [R30] proposed a design consisting of two protein chains, each protein chain composed of two separate Fv domains arranged in the order of VL-(linker 1)-VH-(linker 2)-VL-(linker 3)-VH. To construct a heterodimeric tetra-specific protein, the heterodimer will consist of two protein chains with the following structures. Chain 1: VLA-(linker 1)-VHA-(linker 12)-VLB-(linker 13)-VHC, and Chain B: VLD-(linker 1)-VHD-(linker 2)-VLC-(linker 3)-VHB, where the assembly of FvB and FvC will drive the heterodimerization of the two chains (see Figure 10A). To prevent in-chain assembly to form tandem single-chain Fv (scFv2)-like forms and to promote heterodimerization of two monomeric protein chains, it has been proposed to shorten the linker at position 3 of the linker, up to 10 amino acids (EP1293514A1). However, the organization of the two separate variable domains of the linker 2 with at least 15 amino acids proposed leads to the second variable domain possibly folding back onto the N-terminal domain, resulting in a single-chain diabody (scDb)-like form composed of mismatched VH / VL pairs and thus potentially unable to bind to its target. Additionally, there is also a possibility of forming a heterodimer in which all variable heavy and light chains on protein chain 1 will pair with the variable light and heavy chains of protein chain 2 respectively, thereby preventing the formation of the terminal scFv (scFvA and scFvD) and forming pairings of non-homologous variable domains. Tandem scFv (scFv2)-type or scDb-type by-products may be the reason for observing a very high protein fraction at the apparent molecular weight of the non-polymerized protein chains [R30].

[0020] Theoretically, the formation of scDb-like structures in the above method can be further reduced by also shortening the second linker (linker 2) between the two separate variable domains. However, this will limit the flexibility of the construct, which will have a negative impact on the range of available antigenic epitopes that allow simultaneous binding of two targets in many cases. These geometric constraints are particularly restrictive when two membrane proteins bind simultaneously.

[0021] Additionally, however, most importantly, both monomers may form homodimeric fragments (see Figure 10 B), so statistically up to two-thirds of the dimer products consist of two homodimers, while only one-third consists of the desired heterodimer.

[0022] In summary, there is a strong industry demand for heterodimeric multispecific forms that allow simple substitution of different binding domains and subsequent characterization in the final form. The main challenges for this form are (a) the relatively poor efficiency of specific heterodimerization, resulting in unsatisfactory production yields, and (b) the necessity to use non-target-binding proteins as heterodimerization domains or engineered heterodimeric Fc effector domains, which have poor flexibility in regulating serum half-life and limit the flexibility of adding new functions without increasing molecular weight.

[0023] Thus, the optimal heterodimeric multispecific forms will consist only of target-binding domains and will be able to adjust the geometry of the molecule to fit the geometric constraints defined by the interacting partners (targets), for example, by freely varying the linker length between different binding domains. So far, neither a solution to the problem of modifying the order of the variable domains on the monomeric chains has been given nor suggested in the prior art. SUMMARY OF THE INVENTION

[0024] The present invention relates to novel heterodimeric multispecific forms of multiple antibody variable domains, which comprise a core of two separate pairs of variable domains, wherein all two variable light domains and two homologous variable heavy domains are located in series on two independent protein chains respectively, thereby driving the homodimerization of the two protein chains. Up to two additional binding domains, particularly antibody-based binding domains such as scFv fragments, are fused to the amino or carboxyl terminus of either protein chain to form up to a hexaspecific heterodimeric protein.

[0025] Thus, in a first aspect, the present invention relates to a heterodimeric protein comprising a first and a second single-chain protein,

[0026] wherein the first single-chain protein comprises a first amino acid sequence consisting of (from the N-terminus to the C-terminus):

[0027] (ia) a first VL domain;

[0028] (iia) a first polypeptide linker, and

[0029] (iiia) a second VL domain, and

[0030] wherein the second single-chain protein comprises a second amino acid sequence consisting of (from the N-terminus to the C-terminus):

[0031] (ib) a first VH domain;

[0032] (iib) a second polypeptide linker, and

[0033] (iiib) a second VH domain, and

[0034] wherein the first VL domain forms a first cognate pair of variable domains with the first or the second VH domain, which is specific for a first target antigen, and the second VL domain forms a second cognate pair of variable domains with the other VH domain, which is specific for a second target antigen, and wherein at least one of the first or the second single-chain proteins further comprises

[0035] (iv) At least one additional domain as a third functional domain, which is fused to the first or the second amino acid sequence via a third polypeptide linker,

[0036] wherein, optionally, the heterodimeric protein does not comprise a homologous pair of the first and second immunoglobulin constant domains, wherein the first immunoglobulin constant domain is comprised in the first single-chain protein, and wherein the second immunoglobulin constant domain is comprised in the second single-chain protein.

[0037] In a second aspect, the invention relates to one or two nucleic acid sequences encoding the first and second single-chain proteins.

[0038] In a third aspect, the invention relates to one or two vectors comprising the one or two nucleic acid sequences.

[0039] In a fourth aspect, the invention relates to one host cell or a plurality of host cells comprising the one or two vectors.

[0040] In a fourth aspect, the invention relates to a method for producing the first and second single-chain proteins or the heterodimeric protein of the invention, which comprises (i) providing one or more nucleic acids according to the invention, or one or more vectors according to the invention, expressing the one or more nucleic acids or the one or more vectors, and collecting the first and second single-chain proteins, or the heterodimeric protein, from the expression system, or (ii) providing one host cell or a plurality of host cells of the invention, culturing the one host cell or the plurality of host cells, and collecting the first and second single-chain proteins, or the heterodimeric protein, from the cell culture.

[0041] In a fifth aspect, the invention relates to a pharmaceutical composition comprising the heterodimeric protein of the invention and a pharmaceutically acceptable carrier.

[0042] In a sixth aspect, the invention relates to the heterodimeric protein of the invention for treating a disease, particularly a human disease, more particularly a human disease selected from cancer, inflammation and autoimmune diseases, wherein at least one of the homologous pairs of the VL and VH domains or at least one of the third, fourth, fifth or sixth functional domains is capable of specifically interacting with a therapeutically relevant target in the corresponding disease.

[0043] Seventh aspect, the present invention relates to a method for treating a patient suffering from a disease, particularly a human disease, more particularly a human disease selected from cancer, inflammation and autoimmune diseases, the method comprising administering to a subject an effective amount of the heterodimeric protein of the present invention, wherein at least one of the pairs of homologous VL and VH domains or at least one of the third, fourth, fifth or sixth functional domains is capable of specifically interacting with a therapeutically relevant target in the corresponding disease.

[0044] Specific embodiments of the present invention are set forth in the appended dependent claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 A schematic diagram of Assembly 1 is shown (see Example 1).

[0046] Figure 2 A schematic diagram of Assembly 3 is shown (see Example 1).

[0047] Figure 3 A schematic diagram of Assembly 5 is shown (see Example 1).

[0048] Figure 4 A schematic diagram of Assembly 7 is shown (see Example 1).

[0049] Figure 5 The size exclusion chromatogram after 1-step purification is shown. (A) Assembly 1; (B) Assembly 3; (C) Assembly 5; (D) Assembly 7.

[0050] Figure 6 The SDS-PAGE analysis after 1-step purification is shown: Figure A: PRO356 (Assembly 1): Reducing conditions: Lane 4; Non-reducing conditions: Lane 10; PRO357 (Assembly 3): Reducing conditions: Lane 5; Non-reducing conditions: Lane 11; PRO358 (Assembly 5) Reducing conditions: Lane 6; Non-reducing conditions: Lane 12; PRO355 (Assembly 7) Reducing conditions: Lane 3; Non-reducing conditions: Lane 9. Figure B: Repeated SDS-PAGE with a lower temperature during sample preparation, showing significant cross-linking under non-reducing conditions for PRO357 (Assembly 3).

[0051] Figure 7 It shows compared with storage at 4°C ( Figure 7 A) the protein content (1 g / L) of PRO356 (Assembly 1); PRO357 (Assembly 3); PRO358 (Assembly 5); PRO355 (Assembly 7) after storage at 37°C for 28 days ( Figure 7 B).

[0052] Figure 8 It shows compared with storage at 4°C (Figure 8 A) Compared with, monomer content (1 g / L) of PRO356 (Assembly 1); PRO357 (Assembly 3); PRO358 (Assembly 5); PRO355 (Assembly 7) after storage at 37 °C for 28 days ( Figure 8 B).

[0053] Figure 9 Shows SDS-PAGE analysis of stability samples after incubation at 37 °C for 4 weeks: PRO356 (Assembly 1): Reducing conditions: Lane 4; Non-reducing conditions: Lane 10; PRO357 (Assembly 3): Reducing conditions: Lane 5; Non-reducing conditions: Lane 11; PRO358 (Assembly 5) Reducing conditions: Lane 6; Non-reducing conditions: Lane 12; PRO355 (Assembly 7) Reducing conditions: Lane 3; Non-reducing conditions: Lane 9.

[0054] Figure 10 Shows a schematic diagram of a multispecific single-chain tandem Fv antibody according to Kipriyanov et al. [R30]: VL domain: Gray background; VH domain: White background; Homologous pairs are represented by the same filling pattern. (A) Schematic diagram of single-chain and heterodimer products. (B) Schematic diagram of potential homodimers.

[0055] Figure 11 Shows the results from an SPR experiment, where MATCH (a multispecific antibody-based therapeutics by cognate hetero-dimerization formed by homologous heterodimerization) molecules were immobilized on a sensor chip and 4 antigens were applied in the sequence shown. The resulting sensorgram shows that the RU changes are consistent with the simultaneous engagement of all four antigens induced by each MATCH form.

[0056] Figure 12 Shows the analysis results of the binding amount of inactive MATCH molecules. MATCH molecules were pre-incubated with an excess of TNF (an antigen that forms one of the dimers of the Fv domain), and the complex was run on SE-HPLC. The resulting chromatogram was analyzed to calculate the fractions of "active" (bound) and "inactive" MATCH molecules. When applying conservative peak fitting, the analysis showed 11.4% to 4.7% of inactive protein. Detailed Description

[0057] Here, we present a novel format that exhibits quantitative heterodimeric assembly of two protein chains containing multiple antibody variable domains. This format consists of a core of two separate variable domain pairs (two Fv fragments), where two variable light domains and two variable heavy domains are each located on an independent protein chain, thereby driving the homodimerization of the two protein chains. Up to two additional variable domains in the scFv format with high intra- and inter-domain stability are fused to the amino and / or carboxyl termini of either peptide chain to form up to a hexaspecific heterodimeric protein.

[0058] Thus, in a first aspect, the present invention relates to a heterodimeric protein comprising a first and a second single-chain protein, wherein the first single-chain protein comprises a first amino acid sequence consisting of, from the N-terminus to the C-terminus:

[0059] (ia) a first VL domain;

[0060] (iia) a first polypeptide linker, and

[0061] (iiia) a second VL domain, and

[0062] wherein the second single-chain protein comprises a second amino acid sequence consisting of, from the N-terminus to the C-terminus:

[0063] (ib) a first VH domain;

[0064] (iib) a second polypeptide linker, and

[0065] (iiib) a second VH domain, and

[0066] wherein the first VL domain forms a first homologous pair of variable domains with the first or the second VH domain, which is specific for a first target antigen, and the second VL domain forms a second homologous pair of variable domains with the other VH domain, which is specific for a second target antigen, and wherein at least one of the first or the second single-chain proteins further comprises

[0067] (iv) at least one additional domain as a third functional domain, which is fused to the first or the second amino acid sequence via a third polypeptide linker.

[0068] Throughout the specification and the following claims, unless the context requires otherwise, the word "comprising" will be understood to imply the inclusion of the stated integer, composition or step or group of integers or steps, and that any additional integer, composition or step or group of integers, compositions or steps may optionally be present, including embodiments in which no additional integer, composition or step or group of integers, compositions or steps is present. In respect of these latter embodiments, the term "comprising" thus includes the narrower term "consisting of".

[0069] Throughout this specification, a number of documents are cited. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank accession number sequence submissions, etc.), whether supra or infra, is incorporated herein by reference in its entirety to the extent permitted by the respective patent laws. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosures by virtue of prior invention.

[0070] In the context of the present invention, the terms "VL domain" and "VH domain" refer to the variable light chain domain and variable heavy chain domain of an antibody, respectively. In the context of the present invention, the term "antibody" refers to an immunoglobulin molecule and the immunologically active portion of an immunoglobulin molecule, i.e., a molecule containing an antigen-binding site that specifically binds an antigen, i.e., including an antibody portion comprising at least one antigen-binding fragment of an antibody.

[0071] In the context of the present invention, an antibody or any binding molecule is generally considered to "specifically bind" to the antigen (in the case of an antibody) or to the cognate binding partner (generally in the case of a binding molecule) if the antibody or any binding molecule has a dissociation constant KD of 100 μM or less, preferably 50 μM or less, preferably 30 μM or less, preferably 20 μM or less, preferably 10 μM or less, preferably 5 μM or less, more preferably 1 μM or less, more preferably 900 nM or less, more preferably 800 nM or less, more preferably 700 nM or less, more preferably 600 nM or less, more preferably 500 nM or less, more preferably 400 nM or less, more preferably 300 nM or less, more preferably 200 nM or less, even more preferably 100 nM or less, further preferably 90 nM or less, further preferably 80 nM or less, further preferably 70 nM or less, further preferably 60 nM or less, further preferably 50 nM or less, further preferably 40 nM or less, even more preferably 30 nM or less, even more preferably 20 nM or less, even more preferably 10 nM or less.

[0072] In the context of the present invention, the term "functional domain" refers to a proteinaceous domain having a predefined function, such as enzymatic activity or specific binding to a cognate ligand, wherein the proteinaceous domain is a domain having at least one secondary structure element. Methods for determining the presence of secondary structure in a polypeptide or protein, such as X-ray crystallography, circular dichroism (CD), vibrational circular dichroism (VCD), NMR or FT-IR, or methods for predicting the presence of secondary structure in a polypeptide, such as PEP-FOLD (Shen et al., J. Chem. Theor. Comput. 10 (2014) 4745-4758) are well known to those skilled in the art. In certain embodiments, the proteinaceous domain is a structured domain having a tertiary structure. In certain embodiments, the proteinaceous domain comprises at least about 20 amino acid residues (see Heitz et al., Biochemistry 38 (1999) 10615-25), particularly at least about 50 amino acid residues, more particularly at least about 100 amino acid residues.

[0073] In the context of the present invention, the term "polypeptide linker" refers to a linker composed of a chain of amino acid residues linked by peptide bonds that connects two domains, each domain being linked to one end of the linker. In certain embodiments, the polypeptide linker has a continuous chain of 2 to 30 amino acid residues (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues). In certain embodiments, the polypeptide linker is an unstructured polypeptide. As described above, methods for determining the presence of secondary structure in a polypeptide, such as X-ray crystallography, circular dichroism (CD), vibrational circular dichroism (VCD), NMR or FT-IR, or methods for predicting the presence of secondary structure in a polypeptide, such as PEP-FOLD (Shen et al., J. Chem. Theor. Comput. 10 (2014) 4745-4758) are well known to those skilled in the art.

[0074] The features of the present invention are as follows:

[0075] · Using antibody variable domains to produce a heterodimeric form, wherein both V Ls are located on one protein chain and the corresponding V Hs are located on a second protein chain.

[0076] · The heterodimeric core domain allows the addition of additional functional domains, such as binding domains, to produce tri-, tetra-, penta- or hexaspecific entities.

[0077] · Examples of efficient pairing of multiple heterodimeric core assemblies.

[0078] · The solution for combinatorially screening mixtures of multiple binding domains sharing a common heterodimeric core domain is simple.

[0079] In a particular embodiment, the invention relates to a heterodimeric protein, wherein the first or the second single-chain protein further comprises

[0080] (v) a fourth functional domain, which is fused to the first or the second amino acid sequence via a fourth polypeptide linker.

[0081] In a particular embodiment, the invention relates to a heterodimeric protein, wherein the first or the second single-chain protein further comprises

[0082] (vi) a fifth functional domain, which is fused to the first or the second amino acid sequence via a fifth polypeptide linker.

[0083] In a particular embodiment, the invention relates to a heterodimeric protein, wherein the first or the second single-chain protein further comprises

[0084] (vii) a sixth functional domain, which is fused to the first or the second amino acid sequence via a sixth polypeptide linker.

[0085] In certain embodiments, the heterodimeric protein comprises the third and the fourth functional domains. In such embodiments, the heterodimeric protein is tetravalent, and in certain embodiments, the heterodimeric protein is tetra-specific.

[0086] In certain embodiments, the heterodimeric protein comprises the third, the fourth, the fifth and the sixth functional domains. In such embodiments, the heterodimeric protein is hexavalent, and in certain embodiments, the heterodimeric protein is hex-specific.

[0087] In certain embodiments, the heterodimeric protein does not comprise a pair of homologous immunoglobulin constant domains, wherein the first immunoglobulin constant domain is included in the first single-chain protein and wherein the second immunoglobulin constant domain is included in the second single-chain protein. In certain embodiments, at least one of the first and the second single-chain proteins does not comprise an immunoglobulin constant domain. In certain embodiments, neither the first nor the second single-chain protein comprises an immunoglobulin constant domain.

[0088] In certain embodiments, in addition to (i) the homologous pair of the first VL domain and the first VH domain and (ii) the homologous pair of the second VL domain and the second VH domain, the heterodimeric protein does not contain a homologous pair of the first proteinaceous interaction domain in the first single-chain protein and the second proteinaceous interaction domain in the second single-chain protein.

[0089] In certain embodiments, the first polypeptide linker consists of 5 to 20 amino acid residues, particularly 6 to 15 amino acid residues. In certain embodiments, the polypeptide linker has the sequence (GmS)n; where m is independently selected from 2, 3, and 4; and n is selected from 1, 2, 3, 4, and 5.

[0090] In certain other embodiments, the first polypeptide linker consists of 11 to 20 amino acid residues, particularly 11 to 15 amino acid residues. In certain embodiments, the polypeptide linker has the sequence (GmS)n; where m is independently selected from 2, 3, and 4; and n is selected from 3, 4, and 5.

[0091] In certain embodiments, the second polypeptide linker consists of 5 to 20 amino acid residues, particularly 6 to 15 amino acid residues. In certain embodiments, the polypeptide linker has the sequence (GmS)n; where m is independently selected from 2, 3, and 4; and n is selected from 1, 2, 3, 4, and 5.

[0092] In certain other embodiments, the second polypeptide linker consists of 11 to 20 amino acid residues, particularly 11 to 15 amino acid residues. In certain embodiments, the polypeptide linker has the sequence (GmS)n; where m is independently selected from 2, 3, and 4; and n is selected from 3, 4, and 5.

[0093] In certain embodiments, the third, fourth, fifth, and / or sixth polypeptide linker consists of 8 to 20 amino acid residues, particularly 10 to 15 amino acid residues. In certain embodiments, the polypeptide linker independently has the sequence (GmS)n; where m is independently selected from 2, 3, and 4, particularly 4; and n is selected from 1, 2, 3, 4, and 5, particularly selected from 2 and 3.

[0094] In certain embodiments, the first VL domain (ia) and the first VH domain (ib) form a first homologous pair of variable domains that is specific for a first target antigen, and the second VL domain (iia) and the second VH domain (iib) form a second homologous pair of variable domains that is specific for a second target antigen. In such embodiments, the first and the second single-chain proteins form the heterodimeric protein in a parallel arrangement of the single-chain proteins.

[0095] In certain such embodiments, the first polypeptide linker consists of 10 to 20 amino acid residues, particularly 12 to 17 amino acid residues, particularly 15 amino acid residues. In certain embodiments, the polypeptide linker has the sequence (GmS)n; where m is independently selected from 2, 3, and 4, particularly 4; and n is selected from 1, 2, 3, 4, and 5, particularly 3.

[0096] In certain such embodiments, the second polypeptide linker consists of 10 to 20 amino acid residues, particularly 12 to 17 amino acid residues, particularly 15 amino acid residues. In certain embodiments, the polypeptide linker has the sequence (GmS)n; where m is independently selected from 2, 3, and 4, particularly 4; and n is selected from 1, 2, 3, 4, and 5, particularly 3.

[0097] In certain such embodiments, the third, fourth, fifth, and / or sixth polypeptide linkers consist of 10 to 20 amino acid residues, particularly 12 to 17 amino acid residues, particularly 15 amino acid residues. In certain embodiments, the polypeptide linker has the sequence (GmS)n; where m is independently selected from 2, 3, and 4, particularly 4; and n is selected from 1, 2, 3, 4, and 5, particularly 3.

[0098] In certain other embodiments, the first VL domain (ia) and the second VH domain (iib) form a first pair of variable domains that is specific for a first target antigen, and the second VL domain (iia) and the first VH domain (ib) form a second pair of variable domains that is specific for a second target antigen. In such embodiments, the first and the second single-chain proteins form the heterodimeric protein in an antiparallel arrangement of the single-chain proteins.

[0099] In certain such embodiments, the first polypeptide linker consists of 5 to 12 amino acid residues, particularly 5 to 10 amino acid residues, particularly 6 amino acid residues. In certain embodiments, the polypeptide linker has the sequence (GmS)n; where m is independently selected from 2, 3, and 4, particularly 2; and n is selected from 1, 2, 3, 4, and 5, particularly 2.

[0100] In certain such embodiments, the second polypeptide linker consists of 5 to 12 amino acid residues, particularly 6 to 10 amino acid residues, particularly 8 amino acid residues. In certain embodiments, the polypeptide linker has the sequence (GmS)n; where m is independently selected from 2, 3, and 4, particularly 3; and n is selected from 1, 2, 3, 4, and 5, particularly 2.

[0101] In certain such embodiments, the third, fourth, fifth, and / or sixth polypeptide linkers consist of 10 to 20 amino acid residues, particularly 8 to 12 amino acid residues, particularly 10 amino acid residues. In certain embodiments, the polypeptide linker has the sequence (GmS)n; where m is independently selected from 2, 3, and 4, particularly 4; and n is selected from 1, 2, 3, 4, and 5, particularly 2.

[0102] In certain embodiments, the third, fourth, fifth, and / or sixth functional domains are independently selected from the following: binding domains, toxins, enzymes, hormones, signaling proteins, and albumins.

[0103] In certain embodiments, the third, fourth, fifth, and / or sixth functional domains are independently selected from binding domains.

[0104] In certain such embodiments, the binding domains are independently selected from the following: antibody-based binding domains, including but not limited to scFv, Fab, and single antibody variable domains, single-domain antibodies based on the VNAR structure from sharks, and binding domains based on alternative scaffolds, including but not limited to ankyrin-based domains, oligomers, avimers, anticalins, fibronectins, and binding sites engineered into the constant region of an antibody (e.g., f-star technology).

[0105] In certain such embodiments, the binding domain is an antibody-based binding domain selected from single-chain Fv fragments and single antibody variable domains.

[0106] In certain such embodiments, the order of the variable domains in such single-chain Fv fragments is selected from (from the N-terminus to the C-terminus) VL-(linker)-VH and VH-(linker)-VL. In certain embodiments, for all single-chain Fv fragments included in a heterodimeric protein, the order of the variable domains is the same. In certain embodiments, the three VL domains are each connected to one another by the first polypeptide linker and one of the third, fourth, and fifth polypeptide linkers, e.g., where the single-chain Fv fragment in the VL-(linker)-VH order is from the C-terminus of the first amino acid sequence. In certain embodiments, the three VL domains are each connected to one another by the second polypeptide linker and one of the third, fourth, and fifth polypeptide linkers, e.g., where the single-chain Fv fragment in the VL-(linker)-VH order is from the N-terminus of the second amino acid sequence (see Figure 1 and 4 ). Thus, in certain embodiments, at least one of the first and second single-chain proteins comprises an amino acid sequence consisting of three VL domains or three VH domains connected by two polypeptide linkers, respectively.

[0107] In certain other embodiments, the variable domain of any such antibody-based binding domain that is directly linked to the N-terminus and / or C-terminus of the first or second amino acid sequence via a respective linker is (a) a VH domain when fused to the first amino acid sequence, and (b) a VL domain when fused to the second amino acid sequence. Thus, the VH domain is fused to the N-terminus and / or C-terminus of the VL-linker-VL core region, and the VL domain is fused to the N-terminus and / or C-terminus of the VH-linker-VH core region (see, for example, Figure 3 ).

[0108] In certain embodiments, the third, fourth, fifth, and / or sixth binding domains are single-chain Fv fragments.

[0109] In certain such embodiments, the polypeptide linker that connects the variable domains of the single-chain Fv fragment consists of 15 to 25 amino acid residues, particularly 20 amino acid residues. In certain embodiments, the polypeptide linker has the sequence (GGGGS)n, where n is selected from 3, 5, and 4, particularly 4.

[0110] In certain embodiments, at least one of the antibody variable domains comprises CDR regions derived from a parental rabbit antibody.

[0111] In certain embodiments, at least one of the antibody variable domains comprises a human framework region.

[0112] In certain such embodiments, at least one of the VL domains comprises (i) human V K framework regions I to III; (ii) CDR domains CDR1, CDR2, and CDR3; and (iii) a framework region IV selected from

[0113] a. Human Vλ germline sequences for framework region IV, particularly Vλ germline sequences selected from the following list: SEQ ID NOs. 16 to 22 according to WO2014 / 206561;

[0114] b. Vλ-based sequences that are (bi) a consensus Vλ sequence from a human Vλ germline sequence for framework region IV, particularly SEQ ID NO. 17 according to WO 2014 / 206561; or (bii) a consensus Vλ sequence from a rearranged human Vλ sequence for framework region IV, particularly Vλ consensus sequences selected from the following list: SEQ ID NOs. 16 and 17 according to WO 2014 / 206561

[0115] c. A sequence based on Vλ which has one or two mutations, particularly one mutation, compared to the closest human Vλ germline sequence for framework region IV.

[0116] In certain embodiments, one homologous pair of the first and the second VL and VH domains is specific for an antigen selected from the list consisting of: cancer targets; and targets present on immune effector cells such as CD3.

[0117] In certain of said embodiments, the third, fourth, fifth, and / or sixth binding domains are single-chain Fv fragments specific for targets selected from: cancer targets, and targets present on immune effector cells such as CD3.

[0118] In the context of the present application, the term "target" refers to the homologous binding partner of a binding domain, such as the antigen of an antibody specifically bound by such a binding domain.

[0119] In certain embodiments, the target is a cancer target, particularly an antigen or epitope that is present on the surface of one or more tumor cell types or tumor-associated cells at an increased concentration and / or in a different spatial configuration compared to non-tumor cells. In particular, the cancer target is present on the surface of one or more tumor or tumor stromal cell types but not on the surface of non-tumor cells.

[0120] In other certain embodiments, the target is an antigen or epitope that is preferentially expressed on cells involved in autoimmune diseases. In other embodiments, the antigen or epitope is preferentially expressed on cells involved in inflammatory diseases.

[0121] In certain embodiments, the target is a target present on immune effector cells. In certain embodiments, the target is CD3.

[0122] In certain embodiments, the first and the second single-chain proteins are selected from the following, wherein VLA, VLB, VHA, and VHB correspond to the first and second VL and VH domains, and VLC, VLD, VLE, VLF, VHC, VHD, VHE, and VHF are part of single-chain fragments with linkers, corresponding to the third, fourth, fifth, and / or sixth functional domains respectively connected to the core domain (in bold letters) via the third, fourth, fifth, and / or sixth linkers (LINKER3, LINKER4, LINKER5, and LINKER6); all constructs are written in the N-terminal to C-terminal direction:

[0123] A (parallel; 6Fvs):

[0124] Chain 1: VLC-(connector)-VHC-(connector 3)-VLA-(connector 1)-VLB-(connector 4)-VLD-(connector)-VHD

[0125] Chain 2: VLE-(connector)-VHE-(connector 5)-VHA-(connector 2)-VHB-(connector 6)-VLF-(connector)-VHF B (antiparallel 6Fvs):

[0126] Chain 1: VLC-(connector)-VHC-(connector 3)-VLA-(connector 1)-VLB-(connector 4)-VLD-(connector)-VHD

[0127] Chain 2: VLE-(connector)-VHE-(connector 52)-VHB-(connector 2)-VHA-(connector 6)-VLF-(connector)-VHF

[0128] C1 (antiparallel 4Fvs) (see Figure 1 ):

[0129] Chain 1: VLC-(connector)-VHC-(connector 3)-VLA-(connector 1)-VLB

[0130] Chain 2: VLD-(connector)-VHD-(connector 4)-VHB-(connector 2)-VHA

[0131] C2 (antiparallel 4Fvs) (see Figure 3 ):

[0132] Chain 1: VLC-(connector)-VHC-(connector 3)-VLA-(connector 1)-VLB

[0133] Chain 2: VHB-(connector 2)-VHA-(connector 4)-VLD-(connector)-VHD

[0134] C3 (antiparallel 4Fvs):

[0135] Chain 1: VLA-(connector 1)-VLB-(connector 3)-VLC-(connector)-VHC

[0136] Chain 2: VLD-(connector)-VHD-(connector 4)-VHB-(connector 2)-VHA

[0137] C4 (antiparallel 4Fvs):

[0138] Chain 1: VLA-(connector 1)-VLB-(connector 3)-VLC-(connector)-VHC

[0139] Chain 2: VHB-(connector 2)-VHA-(connector 4)-VLD-(connector)-VHD

[0140] D1 (parallel 4Fvs) (see Figure 4 ):

[0141] Chain 1: VLC-(linker)-VHC-(linker 3)-VLA-(linker 1)-VLB

[0142] Chain 2: VLD-(linker)-VHD-(linker 4)-VHA-(linker 2)-VHB

[0143] D2 (parallel 4Fvs):

[0144] Chain 1: VLC-(linker)-VHC-(linker 3)-VLA-(linker 1)-VLB

[0145] Chain 2: VHA-(linker 2)-VHB-(linker 4)-VLD-(linker)-VHD

[0146] D3 (parallel 4Fvs):

[0147] Chain 1: VLA-(linker 1)-VLB-(linker 3)-VLC-(linker)-VHC

[0148] Chain 2: VLD-(linker)-VHD-(linker 4)-VHA-(linker 2)-VHB

[0149] D4 (parallel 4Fvs):

[0150] Chain 1: VLA-(linker 1)-VLB-(linker 3)-VLC-(linker)-VHC

[0151] Chain 2: VHA-(linker 2)-VHB-(linker 4)-VLD-(linker)-VHD

[0152] In this format, the positioning of two separate variable heavy domains VHB and VHC on one protein chain and two corresponding variable light domains VLB and VLC on the other protein chain (VH-VH / VL-VL) prevents the formation of intra-chain domain pairing, which produces an inactive single-chain diabody (scDb)-like structure, as in the case of using the VH-VL / VH-VL orientation of traditional diabodies - similar to the design proposed by Kipriyanov et al. - to drive heterodimerization. In contrast, the VH-VH / VL-VL orientation forces the formation of only heterodimeric bispecific to hexaspecific proteins.

[0153] Inappropriate pairing of VHA with VLB and VHB with VLA can form VHA-VLB and VHB-VLA pairs. Theoretically, the following possibility exists: VH / VL domain pairing of the VHA-VHB / VLA-VLB core domains that bind to targets A and B will result in an inactive core domain. Unexpectedly and surprisingly, no such inactive variant has been observed to date. Without wishing to be bound by theory, dimerization can be driven towards homologous pairing because the assembly of the CDRs of homologous pairs is more efficient due to the potential assembly interference that occurs in mismatched pairings.

[0154] To further drive heterodimerization towards the active pairing in the VH-VH / VL-VL core domain, knob-into-hole or similar techniques can be applied in one or both (if applied to each other) VL / VH pairs of the VH-VH / VL-VL core domain. Thus, in certain embodiments, the active pairing in the VH-VH / VL-VL core domain of the heterodimeric protein is also supported by techniques selected from: knob-into-hole and interchain disulfide bridges.

[0155] In a second aspect, the invention relates to one or two nucleic acid sequences encoding the first and second single-chain proteins.

[0156] In a third aspect, the invention relates to one or two vectors comprising the one or two nucleic acid sequences.

[0157] In a fourth aspect, the invention relates to one host cell or multiple host cells comprising the one or two vectors.

[0158] In a fourth aspect, the invention relates to a method for producing the first and second single-chain proteins or the heterodimeric protein of the invention, which comprises (i) providing one nucleic acid or multiple nucleic acids according to the invention, or one vector or multiple vectors according to the invention, expressing the one nucleic acid or multiple nucleic acids or the one vector or multiple vectors, and collecting the first and second single-chain proteins, or the heterodimeric protein, from the expression system, or (ii) providing one host cell or multiple host cells of the invention, culturing the one host cell or multiple host cells, and collecting the first and second single-chain proteins, or the heterodimeric protein, from the cell culture.

[0159] In a fifth aspect, the invention relates to a pharmaceutical composition comprising the heterodimeric protein of the invention and a pharmaceutically acceptable carrier.

[0160] Sixth aspect, the present invention relates to the heterodimeric protein of the present invention for treating diseases selected from cancer, inflammation and autoimmune diseases, wherein at least one of the homologous pairs of the VL and VH domains or at least one of the third, fourth, fifth or sixth functional domains can specifically interact with a therapeutically relevant target in the corresponding disease.

[0161] Seventh aspect, the present invention relates to a method for treating a patient suffering from a disease selected from cancer, inflammation and autoimmune diseases, the method comprising administering to a subject an effective amount of the heterodimeric protein of the present invention, wherein at least one of the homologous pairs of the VL and VH domains or at least one of the third, fourth, fifth or sixth functional domains can specifically interact with a therapeutically relevant target in the corresponding disease.

[0162] Literature

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[0176] R14. Schoonjans et al., Fab chains as an efficient heterodimerization scaffold for the production of recombinant bispecific and trispecific antibody derivatives. J Immunol. 2000 Dec 15;165(12):7050-7.

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[0193] Example

[0194] Example 1: Construction of Multispecific Forms

[0195] To construct a heterodimeric multispecific format named Multispecific Antibody-based Therapeutic Constructed by Homologous Heterodimerization (MATCH), four well-characterized variable domains were selected that were respectively directed against human tumor necrosis factor α (TNF), human interleukin-5 receptor (IL5R), human CD3ε (CD3), and interleukin-23 receptor (IL23R). Based on the known binding characteristics of the individual variable domains in the scFv format, the activities of the homologous VL / VH pairs were evaluated in the context of the multispecific molecule, and correct crosslinking was thereby evaluated. The individual variable domains at the periphery of the molecule were located at the amino (N)-terminus or carboxy (C)-terminus of each protein chain as single-chain Fv (scFv) fragments, or in the heterodimerization core domain. The homologous variable domains VL and VH of the core domain were located on two different protein chains, as opposed to the peripheral scFv fragments where VL and VH were on the same protein chain. In the examples provided below, the target-binding domains in the two core domains were respectively directed against CD3 or TNF. The variable domains that bind to IL23R or IL5R have been used in peripheral scFv modules that were fused to the N-terminus or C-terminus of the core domain using flexible amino acid linkers of 10 or 15 amino acids.

[0196] To explore different variants of the heterodimeric core assembly presented herein, parallel and antiparallel orientations of the homologous variable domain pairs were generated, each with one or two additional scFv modules appended to the N-terminus or C-terminus of the core domain.

[0197] In the antiparallel arrangement, the core domain was constructed from the N-terminus to the C-terminus of each protein chain (protein chains 1 to 9) with the orientation VHA-VHB / VLB-VLA. In one embodiment, a tetra-specific form was formed by N-terminal fusion of an scFv module to each of the two protein chains (the construct consists of protein chains 1+2). The corresponding tri-specific form contains an scFv module fused to only one of the two protein chains (construct 1+5). To study the stabilizing effect that engineered disulfide bridges might have on the core domain assembly, the above two forms with C-terminal cysteines were also generated, which led to cross-linking of the homologous Fvs in the core domain of each protein chain. For the tetra-specific form, the individual heterodimeric forms consist of protein chains 3+4, and for the tri-specific form, of protein chains 4+6. In a variant of the antiparallel arrangement, the scFv module on the chain containing tandem VH located in the core domain was fused to the C-terminus instead of the N-terminus and bound to the N-terminus of the protein chain containing the assembled scFv module to form a tetra-specific form (protein chains 1+7), or bound to a protein chain containing only one core domain to form a tri-specific form (protein chains 5+7).

[0198] In the parallel arrangement, the core domain was constructed from the N-terminus to the C-terminus of each protein chain arrangement with the orientation VHA-VHB / VLA-VLB. A tetra-specific form with both scFv modules fused to the N-terminal side of the core domain was generated by co-expression of protein chains 9+10. A corresponding tri-specific assembly was generated by co-expression of protein chains 10+11, where the scFv module is only on the chain containing tandem VH.

[0199] To generate the constructs outlined in Table 1, the amino acid sequences of the Fv domains and linkers were back-translated into the corresponding nucleic acid sequences, which were de novo synthesized. The coding sequences were assembled by standard molecular biology techniques (e.g., Sambrook, J. et al., Molecular Cloning: A Laboratory Manual) and cloned into a suitable expression vector (e.g., pcDNA3.1, Invitrogen) for recombinant protein secretion.

[0200] Example 2: Expression and Purification

[0201] By using a transient gene expression protocol (FreeStyle TM MAX system), the constructs were co-transfected into a suspension cell line (e.g., CHO-S Freestyle TM, expression of the multispecific format assemblies was carried out in (Invitrogen). The combinations of co-expression vectors used to generate the multispecific format assemblies are outlined in Table 2. After several days of culture, the supernatants of cells secreting antibody fragments were recovered for purification. The protein was captured on a suitable affinity resin (e.g., Capto L, GE Healthcare), washed thoroughly and eluted by pH shift. The eluted protein was neutralized and buffer exchanged to obtain a purified mixture. The purity of the protein was analyzed by size exclusion high performance liquid chromatography (SE-HPLC) (Table 3 and Figure 5 ) and the protein content was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) ( Figure 6 ). The protein concentration was adjusted to the desired level and stability analysis was performed.

[0202] Using one-step affinity chromatography, all constructs could be eluted in high purity and monomeric fractions ( Figure 5 and 6 ), confirming the effectiveness and correct pairing of the homologous variable domains. In addition, in non-reducing SDS-Page, PRO357 migrated almost quantitatively at the size of a covalently linked heterodimer (-106 kDa), confirming proper MATCH interchain crosslinking and showing high efficiency and near-complete formation of interchain disulfide bonds. Due to structural constraints, it is unlikely that such disulfide bonds can form between mispaired variable domains. Therefore, this result indicates that heterodimerization occurs almost exclusively between pairs of homologous variable domains.

[0203] Example 3: Storage Stability Assessment

[0204] The significant homogeneity of the protein content in samples purified by Protein L further demonstrated efficient MATCH chain dimerization. Over a 4-week period and stored at 4 °C and 37 °C, the oligomerization of the protein was analyzed by SE-HPLC and the degradation of the protein was analyzed by SDS-PAGE (see Figures 7 to 9)。Before the study, the sample concentration was adjusted to 1 g / L, and the t0 time point was determined. The monomer content was quantified by separating the sample on a Shodex KW-402.5-4F (Showa Denko) and evaluating the resulting chromatogram. To calculate the relative percentage of protein monomers, the area of the monomer peak was divided by the total area of the peaks not attributable to the sample matrix. Protein degradation was evaluated by SDS-PAGE analysis using an Any kD Mini-Protean TGX gel (Bio-Rad Laboratories) and Coomassie Brilliant Blue staining. Protein concentration at different time points was monitored by UV-Vis spectroscopy using an Infinity reader M200 Pro equipped with a Nanoquant plate (Tecan Group Ltd.).

[0205] Example 4: Thermal Unfolding

[0206] Essentially as described by Niesen (Niesen et al., Nat Protoc. 2 (2007) 2212-21), the midpoint of the thermal-induced unfolding transition of the tested constructs was determined by differential scanning fluorimetry (DSF). The DSF measurements were performed in a qPCR instrument (e.g., MX3005p, Agilent Technologies). The samples were diluted in a buffer (citrate-phosphate, pH 6.4, 0.25 M NaCl) with a total volume of 25 μL containing a final concentration of 5× SYPRO Orange. The samples were measured three times, and the temperature was programmed from 25 - 96 °C. Fluorescence signals were obtained, and the raw data were analyzed using GraphPad Prism (GraphPad Software Inc.).

[0207] Example 5: Affinity Determination

[0208] Using a MASS-1SPR instrument (Sierra Sensors), the binding affinities of the individual target-binding domains in single-chain Fv (scFv) form and of the purified heterodimeric tetra-specific construct for the recombinant target proteins human IL-5 receptor (IL5R), human IL-23 receptor ECD (IL-2R), and human CD3γ-ε single-chain (CD3) were measured by surface plasmon resonance (SPR). For affinity measurements (performed in HEPES running buffer: 0.01 M HEPES, 0.15 M NaCl, 0.05% Tween), using the standard amine coupling method and a buffer system optimized for each individual target, target proteins such as human heterodimeric single-chain CD3γδ extracellular domain (produced in-house), human IL5R (R&D Systems), human IL23R (Trenzyme), and human TNF (Peprotech) were immobilized at 100 - 250 RU on a sensor chip (SPR-2 Affinity Sensor High Capacity Amine, Sierra Sensors). For human TNF-α (TNF), a standard amine sensor was used. Serial two-fold dilutions of the purified heterodimeric tetra-specific construct ranging from 90 to 0.703 nM were injected into the flow cell for 3 minutes (20 μl / min) and allowed to dissociate for 720 seconds. After each injection cycle, the surface was regenerated by injecting 10 mM glycine-HCl pH 1.5 for 45 seconds. Affinities were calculated by fitting the sensorgrams for at least six concentrations such that the average Chi 2 was below 10% or R max . For TNF, no serial dilutions were performed and only a single concentration measurement was made at 90 nM. The data were double subtracted (subtracting the reference channel and control cycles).

[0209] The affinity of the heterodimeric tetra-specific construct for each of the four targets was generally very similar to the affinity of the individual binding domains (scFv) used in the tetra-specific form, including those CDRs whose immunoreactivity was presumed to be dependent on proper dimerization (i.e., those presented by Fvs formed by dimers targeting TNFα and CD3ε, respectively). This demonstrated the full functionality of each variable domain in the tetra-specific construct and confirmed the correct assembly of the homologous variable domain pairs.

[0210] Furthermore, as demonstrated by SPR analysis of the immobilized MATCH proteins, each of the three multi-specificities appeared to be able to simultaneously bind all four target antigens regardless of the order of antigen-encounter ( Figure 11 ).

[0211] It should be acknowledged that while these data demonstrate proper MATCH inter-chain assembly, they do not necessarily indicate the absence of non-homologous variable domain cross-linking, particularly "inverted" pairing of MATCH chains that can generate chimeric CDR sets. It has been proposed that the CDR set affects the efficiency of VL-VH pairing, and our SE-HPLC, SDS-PAGE, and SPR data would seem to indicate that homologous pairing of MATCH chains is highly favored. However, to assess the extent of MATCH chain inverted pairing, following incubation of MATCH protein with a molar equivalent of trimeric TNFα (i.e., 3-fold excess of TNFα epitopes), we performed SE-HPLC analysis of the antibody and antibody-antigen complexes. When this analytical method was applied to the parental anti-TNFα scFv (data not shown), the SE-HPLC curve showed discrete peaks consistent with three different antibody-antigen complex populations, reflecting the different sizes of 1-fold, 2-fold, and 3-fold scFv: TNFα complexes. In addition, peaks consistent with the presence of uncomplexed TNFα remaining in solution were observed, while no uncomplexed scFv was present in solution, thus validating the application of this method for identification of "inactive" anti-TNFα antibodies.

[0212] Due to the larger molecular weight of the multispecific molecules, the separation efficiency of MATCH protein and MATCH-antigen complexes was low. However, our results ( Figure 12 ) also clearly showed the presence of three MATCH-TNFα complex populations and residual uncomplexed TNFα. In addition, the "shoulder" of the 1×MATCH: TNFα complex peak indicated the presence of inactive, but dimeric MATCH protein. To estimate the proportion of inactive MATCH protein in solution, the peaks were deconvoluted using PeakFit v.1.2 software, assuming each peak was Gaussian distributed, and plotted to optimize goodness of fit ( Figure 12 ). This analysis estimated that the proportion of inactive MATCH protein was between 4.7 and 11.4% of the total MATCH protein content (PRO357 < PRO356 < PRO355), indicating that proper dimerization of MATCH chains is highly favored, particularly in the antiparallel form.

[0213] Table 1: Constructs

[0214]

[0215] Table 2: Multispecific Form Assembly

[0216] Protein ID (Numab) Assembly Protein Chain 1 Protein Chain 2 PRO356 1(See Figure 1 ). 1 2 PRO469 2 1 5 PRO357 3(See Figure 2 ). 3 4 PRO470 4 4 6 PRO358 5(See Figure 3 ). 1 7 PRO471 6 5 7 PRO355 7(See Figure 4 ). 9 10 PRO468 8 10 11

[0217] Table 3: Size Exclusion Chromatogram after 1-step Purification

[0218]

[0219] Table 4: Median of protein thermally induced unfolding determined by differential scanning calorimetry

[0220]

[0221] Table 5: Affinity of heterodimeric tetra - specific constructs

[0222] Sequence Listing <110> Numa Innovation Co., Ltd. <120> Heterodimeric multispecific antibody form <130> 111473P877PC <150> EP15001758.0 2015 - 06 - 15 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Artificial linker <400> 1 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 2 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Artificial linker <400> 2 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 3 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Artificial linker <400> 3 Gly Gly Ser Gly Gly Ser 1 5 <210> 4 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial Linker <400> 4 Gly Gly Gly Ser Gly Gly Gly Ser 1 5

Claims

1. A heterodimeric protein comprising a first and a second single-chain protein, wherein, the first single-chain protein consists of a first amino acid sequence which consists of, from the N-terminus to the C-terminus: (ia) a first VL domain; (iia) a first polypeptide linker, (iiia) a second VL domain, (iva) optionally, a first scFv fragment fused to the first VL domain via a third polypeptide linker, and (va) optionally, a second scFv fragment fused to the second VL domain via a fourth polypeptide linker, and wherein the second single-chain protein consists of a second amino acid sequence which consists of, from the N-terminus to the C-terminus: (ib) a first VH domain; (iib) a second polypeptide linker, (iiib) a second VH domain, (ivb) optionally, a third scFv fragment fused to the first VH domain via a fifth polypeptide linker, and (vb) optionally, a fourth scFv fragment fused to the second VH domain via a sixth polypeptide linker, provided that at least one of the first, second, third and fourth scFv fragments is present, wherein the first VL domain forms a first pair of variable domains with the first or the second VH domain, which is specific for a first target antigen, and the second VL domain forms a second pair of variable domains with the other VH domain, which is specific for a second target antigen, and wherein at least one of the VL domains comprises (i) human Vκ framework regions I to III; (ii) CDR domains CDR1, CDR2 and CDR3; (iii) a framework region IV which is based on a human Vλ sequence and has one or two mutations compared to the human λ germline sequence closest to the framework region IV.

2. The heterodimeric protein according to claim 1, wherein at least one of the VL domains comprises (i) human Vκ framework regions I to III; (ii) CDR domains CDR1, CDR2 and CDR3; (iii) a framework region IV which is based on a human Vλ sequence and has one mutation compared to the human λ germline sequence closest to the framework region IV.

3. The heterodimeric protein according to claim 1, wherein (a) the second of the first, second, third and fourth scFv fragments is present; (b) the second and the third of the first, second, third and fourth scFv fragments are present; or (c) all of the first, second, third and fourth scFv fragments are present.

4. The heterodimeric protein according to claim 1, wherein the first polypeptide linker consists of 5 to 20 amino acid residues.

5. The heterodimeric protein according to claim 4, wherein the first polypeptide linker consists of 6 to 15 amino acid residues.

6. The heterodimeric protein according to claim 1, wherein (a) the first VL domain (ia) and the first VH domain (ib) form a first homologous pair of variable domains that is specific for a first target antigen, and the second VL domain (iia) and the second VH domain (iib) form a second homologous pair of variable domains that is specific for a second target antigen; or (b) the first VL domain (ia) and the second VH domain (iib) form a first homologous pair of variable domains that is specific for a first target antigen, and the second VL domain (iia) and the first VH domain (ib) form a second homologous pair of variable domains that is specific for a second target antigen.

7. The heterodimeric protein according to claim 1, wherein at least one of the VL, VH, or scFv domains comprises a CDR region derived from a parental rabbit antibody.

8. The heterodimeric protein according to claim 1, wherein, one of the homologous pairs of the first and the second VL and VH domains is specific for an antigen selected from: a cancer target; and a target present on an immune effector cell.

9. The heterodimeric protein according to claim 8, wherein, one of the homologous pairs of the first and the second VL and VH domains is specific for CD3.

10. A nucleic acid or two nucleic acids encoding the first and second single-chain proteins of the heterodimeric protein according to any one of claims 1-9.

11. A vector or two vectors comprising the nucleic acid or two nucleic acids according to claim 10.

12. A host cell or multiple host cells comprising the vector or two vectors according to claim 11.

13. A method for producing the heterodimeric protein according to any one of claims 1 to 9 or the first or second single-chain protein of the heterodimeric protein, which comprises (i) providing the nucleic acid or two nucleic acids according to claim 7 or the vector or two vectors according to claim 11, expressing the nucleic acid or two nucleic acids or the vector or two vectors, and collecting the heterodimeric protein from the expression system, or (ii) providing the host cell or multiple host cells according to claim 12, culturing the host cell or multiple host cells, and collecting the first and second single-chain proteins, or the heterodimeric protein from the cell culture.

14. A pharmaceutical composition comprising the heterodimeric protein according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

15. Use of the heterodimeric protein according to claim 1 in the preparation of a medicament for treating a disease, wherein at least one of the homologous pairs of the VL and VH domains, or, in the case where present, at least one of the first, second, third, or fourth scFv fragments, is capable of specifically interacting with a therapeutically relevant target in the corresponding disease.

16. Use of the heterodimeric protein according to claim 1 in the preparation of a medicament for the treatment of human diseases, wherein at least one of the pairs of homologous VL and VH domains, or, if present, at least one of the first, second, third or fourth scFv fragments, is capable of specifically interacting with a therapeutically relevant target in the corresponding disease.

17. Use of the heterodimeric protein according to claim 1 in the preparation of a medicament for the treatment of human diseases selected from cancer, inflammation and autoimmune diseases, wherein at least one of the pairs of homologous VL and VH domains, or, if present, at least one of the first, second, third or fourth scFv fragments, is capable of specifically interacting with a therapeutically relevant target in the corresponding disease.

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