Recombinant binding proteins with activatable effector domains

Abstract

The present invention relates to a recombinant binding protein pair and uses thereof, for example for activating an effector domain upon binding to a target cell. In particular, the present invention relates to the recombinant binding protein pair and a pharmaceutical composition comprising the recombinant binding protein pair.

Description

Technical Field

[0001] The present invention relates to a recombinant binding protein pair and its use, for example, for activating an effector domain after binding to a target cell. Specifically, the present invention relates to the recombinant binding protein pair and a pharmaceutical composition comprising the recombinant binding protein pair. Background Art

[0002] Bispecific antibodies, for example, target antigens expressed on cancer cells and T cells via CD3, thereby mediating ADCC against cancer cells. Cancer treatment with such bispecific antibodies poses challenges in terms of administration due to off-target T cell activation, which is undesirable.

[0003] EP3180361 discloses a pair of precursor molecules in which a binding site specifically binding to CD3 is activated on a target cell. Such a precursor molecule comprises a Fab fragment, wherein the C-terminus of the Fab fragment is fused to a CH2 domain and a variable antibody domain (e.g., binding to CD3). After binding of two precursor molecules comprising different variable domains to a target cell, a functional antigen-binding site (e.g., binding to CD3) is formed by the association of the variable domains.

[0004] EP2802607 also discloses a pair of precursor molecules in which a binding site specifically binding to CD3 is activated on a target cell. These precursor molecules comprise a single-chain Fv fragment capable of binding to a target cell and an antibody variable domain that associates with a complementary variable domain contained in another precursor molecule to form a functional CD3-binding site.

[0005] However, alternative solutions are still needed to activate an effector domain at a target site. Summary of the Invention

[0006] The present invention relates to a recombinant binding protein pair comprising (a) a first fusion polypeptide comprising (i) a first antigen-binding domain capable of binding to a target antigen, (ii) a first part of an effector domain, and (iii) a first complementary domain capable of associating with the first part of the effector domain, wherein the first part of the effector domain and the first complementary domain are linked via a peptide linker; and (b) a second fusion polypeptide comprising (i) a second antigen-binding domain capable of binding to the target antigen, (ii) a second part of the effector domain, and (iii) a second complementary domain capable of associating with the second part of the effector domain, wherein the first part of the effector domain and the first complementary domain are linked via a peptide linker; wherein the first part of the effector domain and the second part of the effector domain are capable of associating with each other to form a functional effector domain, characterized in that the effector domain is an antibody Fab fragment.

[0007] One embodiment of the present invention relates to a recombinant binding protein pair, wherein the first part of the effector domain comprises a variable heavy chain domain having a Q39E mutation, and wherein the first complementary domain comprises a variable light chain domain having a Q38E mutation, wherein the second part of the effector domain comprises a variable light chain domain having a Q38K mutation, and wherein the second complementary domain comprises a variable heavy chain domain having a Q39K mutation.

[0008] One embodiment of the present invention relates to a recombinant binding protein pair, wherein the first part of the effector domain and the first complementary domain are contained in a single-chain Fab fragment, and wherein the second part of the effector domain and the second complementary domain are contained in a single-chain Fab fragment, and wherein in the single-chain Fab fragment of the first fusion polypeptide, the VH and VL domains are exchanged with each other, and wherein in the single-chain Fab fragment of the second fusion polypeptide, the CH1 and CL domains are exchanged with each other.

[0009] One embodiment of the present invention relates to a recombinant binding protein pair, wherein the first fusion polypeptide comprises a (heterodimeric) Fc domain, and wherein the second fusion polypeptide comprises a (heterodimeric) Fc domain. In one embodiment, wherein the first fusion polypeptide and the second fusion polypeptide comprise a heterodimeric Fc domain, the heterodimeric Fc domain comprises two CH3 domains, wherein one of the CH3 domains comprises the mutations S354C and T366W, and the other CH3 domain comprises the mutations Y349C, L368A and Y407V.

[0010] One embodiment of the present invention relates to a recombinant binding protein pair, wherein the effector domain is an effector domain that is an anti-CD3 antibody binding domain.

[0011] Another aspect of the present invention is a method for forming a functional effector domain from a recombinant binding protein pair according to one of the preceding claims, wherein the first antigen-binding domain and the second antigen-binding domain of the recombinant binding domain specifically bind to an epitope on the surface of a target cell, the method comprising contacting the recombinant binding protein pair with the target cell under conditions that allow the first fusion polypeptide and the second fusion polypeptide to bind to the target cell.

[0012] Another aspect of the present invention is a pharmaceutical formulation comprising: a recombinant binding protein pair according to any one of the preceding claims; and a pharmaceutically acceptable carrier.

[0013] According to the present invention, a functional effector domain composed of a first part contained in a first fusion polypeptide and a second part contained in a second fusion polypeptide is formed by the association of the first part and the second part after the first fusion polypeptide and the second fusion polypeptide bind to a target cell. The therapeutic application of the multiple pairs of recombinant binding proteins of the present invention allows the formation of the effector domain only at the target site, such as the anti-CD3 binding site, thus reducing unwanted off-target toxicity. The methods and multiple pairs of recombinant binding proteins of the present invention can be advantageously used to provide antigen-binding proteins for therapeutic use; for example, for the treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : Design and modular composition of an inactive precursor module, where a prodrug entity is covalently fused in a single-chain-Fab-like manner to produce a Fab.

[0015] Figure 2 : Generation of bispecific antibodies from monospecific precursors

[0016] Figure 3 : Generation of trispecific antibodies from monospecific precursors

[0017] Figure 4 : A) Expression profiles of the tumor antigens EGFR and HER2 on SK-BR-3 cells. B) Activation of CD3 binding function by a previously inactive precursor molecule targeting HER2 on SK-BR-3 cells detected by T cell reporter gene assay. C) Expression of EGFR and HER2 on A431 cells. D) Activation of CD3 binding function by a previously inactive precursor molecule targeting EGFR on A-431 cells detected by T cell reporter gene assay.

[0018] Figure 5 : Activation of CD3 binding function by previously inactive precursor molecules targeting (i) HER2 and (ii) EGFR on double-positive SK-BR-3 cells detected by T cell reporter gene assay.

[0019] Figure 6 : Generation of bispecific antibodies from one monospecific precursor and one non-specific precursor with a covalently linked exchange unit (as a measure of exchange in solution)

[0020] Figure 7: A) Activation of CD3 binding function on HER2-positive SK-BR-3 cells detected by T cell reporter gene assay by (i) a previously inactive precursor molecule targeting HER2 and (ii) a non-targeting one carrying a non-covalently linked exchange unit (as a measure of rearrangement in solution). B) Activation of CD3 binding function on HER2-positive SK-BR-3 cells detected by T cell reporter gene assay by (i) a previously inactive precursor molecule targeting HER2 and (ii) a non-targeting one carrying a covalently linked exchange unit (as a measure of rearrangement in solution).

[0021] Figure 8 : Generation of trispecific antibodies from monospecific precursors

[0022] Figure 9 : Activation of CD3 binding function on dual-positive SK-BR-3 cells detected by T cell reporter gene assay by a previously inactive HER2-targeting precursor molecule containing a combination of CD3 VH / VL with Dig or non-binding "Nada" VH / VL in internal and external positions. Nomenclature of the molecule: <target>(internal conjugate)(external conjugate)[prodrug A / B]

[0023] Figure 10 : Activation of Dig binding function on Sk-Br-3 cells detected by flow cytometry by a previously inactive HER2-targeting precursor molecule containing a combination of CD3 VH / VL with Dig or non-binding "Nada" VH / VL in internal and external positions. Precursor molecules containing Dig and CD3 VH / VL pairs in different positions (internal or external positions) were added to Sk-Br-3 cells in 96-well plates alone or in combination. After incubation and washing, digoxigeninylated Cy5 dye (Dig-Cy5)

[0024] Figure 11A CD28 co-stimulation of T cells using HER2-targeting precursor molecules.

[0025] Co-culture of HER2-positive Sk-Br-3 cells (60,000 cells per well) with Jurkat IL-2 promoter cells (100,000 cells per well) and treatment with increasing concentrations of Fab-PACE prodrug combinations. After incubation at 37 °C for 16 h, ONE-Glo TM solution (Promega, catalog number E6120) was added to the plates and luminescence was measured according to the manufacturer's instructions. CD28 co-stimulation of Jurkat T cells was observed using CD28 in both the internal and external positions, as indicated by an increase in luminescence for precursor molecule combinations containing CD28 compared to those containing Nada.

[0026] Figure 11B As Figure 11A shown, in the presence of a separate HER2-targeting precursor molecule, CD28 co-stimulation of Jurkat IL-2 promoter cells was monitored. Even at high concentrations, no T cell activation or co-stimulation was observed with either of the individual prodrugs. The nomenclature for the Fab-PACE molecules in Figure 11 is: <target>(internal conjugate)(external conjugate)[prodrug A / B].

[0027] Figure 12A Generation of tetra-specific antibodies from monospecific precursors

[0028] Figure 12B Schematic of the assay setup for the HEK-Blue IL-2 transactivation assay as described in Example 8

[0029] Figure 13 IL-2 transactivation as described in Example 8. (A) After accumulation on PD-1-expressing cells, the inactive PD-1-targeting precursor molecule is converted into an active IL-2 receptor agonist. (B) Control experiments on PD-1-negative cells demonstrate that the activation of the active IL2 receptor agonist is target-specific. Detailed Description

[0030] 1. Definition

[0031] Unless otherwise defined herein, scientific and technical terms associated with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. The methods and techniques of the present disclosure are generally carried out according to conventional methods well known in the art. Generally, the terms and techniques related to biochemistry, enzymology, molecular and cell biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art.

[0032] Unless the context clearly indicates otherwise, the terms "a", "an", "the" generally include plural referents.

[0033] Unless otherwise defined herein, the term "comprising" shall include the term "consisting of".

[0034] Unless the context clearly indicates otherwise, the two alternatives provided by the term "either...or" represent mutually exclusive alternatives.

[0035] As used herein, the term "antigen-binding domain" refers to a domain that specifically binds to a target antigen. The term includes antibody-binding sites and other natural (e.g., receptors, ligands) or synthetic (e.g., DARPin) molecules capable of specifically binding to a target antigen.

[0036] The term "antibody" is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0037] As used herein, the term "binding site" or "antigen-binding site" refers to one or more regions of the antigen-binding portion where the antigen actually binds. In the case where the antigen-binding portion is an antibody, the antigen-binding site includes the variable domain of the antibody heavy chain (VH) and / or the variable domain of the antibody light chain (VL), or a VH / VL pair. An antigen-binding site derived from an antibody that specifically binds to a target antigen can be derived from: a) a known antibody that specifically binds to the antigen; or b) a new antibody or antibody fragment obtained by methods such as reimmunization using, in particular, the antigen protein or nucleic acid or a fragment thereof, or by phage display methods.

[0038] When derived from an antibody, the antigen-binding site of an antibody according to the present invention can comprise six complementarity-determining regions (CDRs), which contribute to the affinity of the antigen-binding site to varying degrees. There are three heavy-chain variable domain CDRs (CDRH1, CDRH2, and CDRH3) and three light-chain variable domain CDRs (CDRL1, CDRL2, and CDRL3). The boundaries of the CDRs and framework regions (FRs) are determined by comparison with a compilation database of amino acid sequences in which those regions have been defined based on sequence variability. Also included within the scope of the present invention are functional antigen-binding sites composed of fewer CDRs (i.e., the binding specificity is determined by three, four, or five CDRs). For example, fewer than a complete set of 6 CDRs may be sufficient for binding.

[0039] As used herein, the term "valence" refers to the presence of a specified number of binding sites in an antibody molecule. For example, a natural antibody has two binding sites and is bivalent. Thus, the term "trivalent" refers to the presence of three binding sites in an antibody molecule.

[0040] "Antibody fragment" refers to a molecule other than a full antibody that contains a portion of a full antibody that binds to the antigen to which the full antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv, scFab); and multispecific antibodies formed from antibody fragments.

[0041] "Specificity" refers to the selective recognition of a particular epitope of an antigen by an antigen-binding portion (e.g., an antibody). For example, a native antibody is monospecific. As used herein, the term "monospecific antibody" refers to an antibody having one or more binding sites, each of which binds to the same epitope of the same antigen. A "multispecific antibody" binds two or more different epitopes (e.g., two, three, four, or more different epitopes). The epitopes can be on the same or different antigens. An example of a multispecific antibody is a "bispecific antibody" that binds two different epitopes. When an antibody has more than one specificity, the recognized epitopes may associate with a single antigen or more than one antigen.

[0042] An epitope is the region of an antigen that is bound by an antigen-binding portion (e.g., an antibody). The term "epitope" includes any polypeptide determinant capable of specifically binding to an antibody or antigen-binding portion. In certain embodiments, an epitope determinant includes chemical reactive surface groups such as amino acids, carbohydrate side chains, phosphoryl or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural features and / or specific charge characteristics.

[0043] As used herein, the terms "bind" and "specifically bind" refer to the binding of an antibody or antigen-binding portion to an antigen epitope in an in vitro assay, preferably in a surface plasmon resonance assay using purified wild-type antigen ( GE-Healthcare Uppsala, Sweden). In certain embodiments, an antibody or antigen-binding portion is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0044] The affinity of an antibody for an antigen is defined by the terms k a (the association rate constant of the antibody from the antibody / antigen complex), k D (the dissociation constant), and K D (k D / ka). In one embodiment, binding or specifically binding thereto refers to a binding affinity (K -8 ) of 10 D mol / l or less, and in one embodiment, from 10 -8 M to 10-13 mol / l. Thus, the antigen-binding portion, particularly the antibody-binding site, has a binding affinity of 10 -8 mol / l or lower (K D ) and specifically binds each antigen to which it is specific. For example, the binding affinity (K D ) is from 10 -8 mol / l to 10 -13 mol / l. In one embodiment, the binding affinity (K D ) is from 10 -9 mol / l to 10 -13 mol / l.

[0045] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to antigen. The variable domains of the heavy chain and light chain of a native antibody (VH and VL, respectively) generally have similar structures, each domain including four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, antibodies that bind a particular antigen can be isolated using the VH or VL domain from an antibody that binds that antigen to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0046] As used herein, the term "constant domain" or "constant region" refers to the sum total of the antibody domains other than the variable regions. The constant regions do not directly participate in binding of antigen, but exhibit various effector functions.

[0047] Antibodies are classified into the following "classes" according to the amino acid sequence of the constant region of their heavy chains: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subclasses such as IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different immunoglobulin classes are designated α, δ, ε, γ, and μ, respectively. The light chain constant regions (CL) that can be found in all five antibody classes are designated κ (kappa) and λ (lambda).

[0048] As used herein, a "constant domain" is preferably of human origin and is derived from the constant heavy chain region and / or the constant light chain kappa or lambda region of a human antibody of subclass IgG1, IgG2, IgG3 or IgG4. Such constant domains and regions are well known in the art and are described, for example, by Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0049] In a wild-type antibody, the "hinge region" is a flexible amino acid segment in the central part of the heavy chain of the IgG and IgA immunoglobulin classes that links the two heavy chains by disulfide bonds, i.e., "interchain disulfide bonds", since these disulfide bonds are formed between the two heavy chains. The hinge region of human IgG1 is typically defined as extending from about Glu216 or about Cys226 of human IgG1 to about Pro230 (Burton, Molec. Immunol. 22:161-206 (1985)). Formation of disulfide bonds in the hinge region is avoided by deleting cysteine residues in the hinge region or substituting cysteine residues in the hinge region with other amino acids such as serine.

[0050] The "light chain" of an antibody from any vertebrate species can be assigned to one of two different types based on the amino acid sequence of its constant domain, which are called kappa (κ) and lambda (λ), respectively. A wild-type light chain typically contains two immunoglobulin domains, usually a variable domain (VL) that is important for binding to an antigen and a constant domain (CL).

[0051] There are several different types of "heavy chains", which define the class or isotype of an antibody. A wild-type heavy chain contains a series of immunoglobulin domains, usually having a variable domain (VH) that is important for binding to an antigen and several constant domains (CH1, CH2, CH3, etc.).

[0052] The term "Fc region" as used herein is defined as the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0053] The "CH2 domain" of the human IgG Fc region generally extends from the amino acid residue at about position 231 to about position 340. A multispecific antibody does not have a CH2 domain. "Does not have a CH2 domain" means that the antibody according to the present invention does not contain a CH2 domain.

[0054] The "CH3 domain" comprises a segment of residues C-terminal to the CH2 domain in the Fc region (i.e., from the amino acid residue at about position 341 of IgG to about position 447). The "CH3 domain" herein is a variant CH3 domain in which the amino acid sequence of the native CH3 domain has been modified by at least one different amino acid substitution (i.e., a modification of the amino acid sequence of the CH3 domain) to promote heterodimerization of two CH3 domains facing each other in a multispecific antibody.

[0055] Typically, in heterodimerization methods known in the art, the CH3 domain of one heavy chain and the CH3 domain of another heavy chain are both engineered in a complementary manner such that a heavy chain comprising one engineered CH3 domain no longer homodimerizes with a heavy chain of the same structure as the other heavy chain. Thus, a heavy chain comprising one engineered CH3 domain is forced to heterodimerize with another heavy chain comprising a CH3 domain engineered in a complementary manner.

[0056] One hetero - dimerization method known in the art is the so - called "knob - in - hole" technology, which is described in detail with several examples in, for example, WO 96 / 027011; Ridgway, J.B., et al., Protein Eng. 9(1996)617 - 621; Merchant, A.M., et al., Nat. Biotechnol. 16(1998)677 - 681 and WO 98 / 050431, which are hereby incorporated by reference. In the "knob - in - hole" technology, within the interface formed between two CH3 domains in the antibody tertiary structure, specific amino acids on each CH3 domain are engineered to respectively produce a protrusion ("knob") in one CH3 domain and a cavity ("hole") in the other CH3 domain. In the tertiary structure of a multispecific antibody, the protrusion introduced into one CH3 domain can be positioned within the cavity introduced into the other CH3 domain.

[0057] In combination with the substitutions according to the knob - in - hole technology, additional inter - chain disulfide bonds can be introduced into the CH3 domains to further stabilize the heterodimeric polypeptide (Merchant, A.M., et al., Nature Biotech. 16(1998)677 - 681). For example, such inter - chain disulfide bonds are formed by introducing the following amino acid substitutions into the CH3 domains: D399C in one CH3 domain and K392C in the other CH3 domain; Y349C in one CH3 domain and S354C in the other CH3 domain; Y349C in one CH3 domain and E356C in the other CH3 domain; Y349C in one CH3 domain and E357C in the other CH3 domain; L351C in one CH3 domain and S354C in the other CH3 domain; T394C in one CH3 domain and V397C in the other CH3 domain. As used herein, a "cysteine mutation" refers to an amino acid substitution of an amino acid in a CH3 domain by a cysteine, which cysteine is capable of matching with another amino acid substitution of an amino acid in a second CH3 domain by a cysteine to form an inter - chain disulfide bond.

[0058] In addition to the "stub-into-hole" technique mentioned previously, other techniques for modifying the CH3 domain to enhance heterodimerization are known in the art. These techniques, especially those described in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO2011 / 143545, WO2012 / 058768, WO 2013 / 157954 and WO 2013 / 096291, are considered herein as "stub-and-hole structure techniques" for the polypeptides provided by the present invention. All these techniques involve engineering the CH3 domain in a complementary manner, by introducing amino acids with opposite charges or different side-chain volumes, thus supporting heterodimerization.

[0059] As used herein, the term "polypeptide chain" refers to a linear organic polymer comprising a large number of amino acids linked together by peptide bonds. One or more polypeptide chains form a "polypeptide" or "protein", and the two terms are used interchangeably herein. The heterodimeric precursor polypeptides provided in a group according to the present invention comprise at least two polypeptide chains comprising a CH3 domain. Thus, a first polypeptide chain comprising a first CH3 domain "associates" with a second polypeptide chain comprising a second CH3 domain to form a dimeric polypeptide. Since the first CH3 domain and the second CH3 domain include amino acid substitutions according to the stub-into-hole technique, the two polypeptide chains form a "heterodimer", i.e., a dimer formed by two different polypeptides.

[0060] A polypeptide chain may comprise one or more polypeptide domains. When indicating the order of polypeptide domains herein, it is represented in the N-terminal to C-terminal direction.

[0061] Each of the first fusion polypeptide and the second fusion polypeptide comprises at least two polypeptide chains comprising a CH3 domain.

[0062] Heavy chain / light chain and heavy chain polypeptide / light chain polypeptide

[0063] A "purified" polypeptide (such as an antibody) is an antibody that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99% as determined by, for example, electrophoresis (such as SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (such as ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0064] As used herein, the amino acid positions of all constant regions and domains of the heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). In particular, for the variable domains and the light chain constant domain CL of the κ and λ isotypes, the Kabat numbering system of Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) is used (see pages 647 - 660), while for the constant heavy chain domains (CH1, hinge, CH2 and CH3), the Kabat EU index numbering system is used (see pages 661 - 723). The amino acid positions provided herein are generally represented by the following

[0065] An amino acid “substitution” or “replacement” or “mutation” (all terms used interchangeably herein) within a polypeptide chain is prepared by introducing the appropriate nucleotide change into the antibody DNA or by nucleotide synthesis. However, such modifications can only be made within a very limited range. For example, the modifications do not alter the above-described antibody characteristics, such as IgG isotype and antigen binding, but can further improve the yield of recombinant production, protein stability or facilitate purification. In certain embodiments, antibody variants having one or more conservative amino acid substitutions are provided. As referred to herein, a “double mutation” means that two of the indicated amino acid substitutions are present in their respective polypeptide chains.

[0066] The term “amino acid” as used herein refers to an organic molecule having an amino moiety at the α-position of the carboxyl group. Examples of amino acids include: arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline. The amino acids employed in the various cases are optionally L-amino acids. The terms “positively charged” or “negatively charged” amino acids refer to the charge of the amino acid side chain at pH 7.4. Amino acids can be grouped according to common side chain characteristics:

[0067] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile, Trp, Tyr, Phe;

[0068] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0069] (3) Acidic or negatively charged: Asp, Glu;

[0070] (4) Basic or positively charged: His, Lys, Arg;

[0071] (5) Residues affecting chain orientation: Gly, Pro.

[0072] Table - Amino acids with specific properties

[0073]

[0074]

[0075] As used herein, the term "purified" refers to a polypeptide that has been removed from its natural environment or from a recombinant production source, or otherwise separated or isolated, and is at least 60% (e.g., at least 80%) free of other components that are naturally associated with it, such as membranes and microsomes. Antibody purification is carried out by standard techniques (e.g., recovering antibodies from host cell cultures) to eliminate cellular components or other contaminants (e.g., other cellular nucleic acids or proteins), and the standard techniques include alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. See Current Protocols in Molecular Biology, edited by Ausubel, F. et al., Greene Publishing and Wiley Interscience, New York (1987). A variety of different methods for protein purification have been successfully established and widely used, such as affinity chromatography using microbial proteins (e.g., using affinity media for purifying κ or λ isotype constant light chain domains, such as KappaSelect or LambdaSelect), ion exchange chromatography (e.g., cation exchange (carboxymethyl resin), anion exchange (aminoethyl resin), and mixed mode exchange), thiophilic adsorption (e.g., using β-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g., using phenyl-agarose, aza-arenophilic resins, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g., using Ni(II)-affinity materials and Cu(II)-affinity materials), size exclusion chromatography, and electrophoretic methods (such as gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, M.A., Appl. Biochem. Biotech. 75 (1998) 93-102).

[0076] As used herein, the term "peptide linker" refers to a peptide having an amino acid sequence that is preferably of synthetic origin. In the fusion polypeptides used in the present invention, the peptide linker can be used to fuse additional polypeptide domains, such as antibody fragments, to the C-terminus or N-terminus of a single polypeptide chain. In one embodiment, the peptide linker is a peptide having an amino acid sequence with a length of at least 5 amino acids, in another embodiment, with a length of 5 to 100 amino acids, and in yet another embodiment, 10 to 50 amino acids. In one embodiment, the peptide linker is a glycine-serine linker. In one embodiment, the peptide linker is a peptide composed of glycine and serine residues. In one embodiment, the peptide linker is

[0077] (G x S) n or (G x S) n G m

[0078] where G = glycine, S = serine, and

[0079] x = 3, n = 3, 4, 5 or 6, m = 0, 1, 2 or 3; or

[0080] x = 4, n = 2, 3, 4 or 5, m = 0, 1, 2 or 3.

[0081] In one embodiment, x = 4 and n = 2 or 3, in another embodiment, x = 4, n = 2. In one embodiment, the peptide linker is (G4S)2.

[0082] As used herein, the term "valence" refers to the presence of a specified number of antigen-binding sites in an antigen-binding molecule. For example, a natural antibody has two binding sites and is divalent. Thus, the term "trivalent" refers to the presence of three binding sites in an antigen-binding molecule.

[0083] The polypeptides according to the invention are produced recombinantly. Recombinant production methods for polypeptides (such as antibodies) are well known in the art and include expressing the protein in prokaryotic and eukaryotic host cells, followed by isolating the polypeptide and generally purifying it to pharmaceutical purity. To express the above polypeptides in host cells, nucleic acids encoding the corresponding polypeptide chains are inserted into expression vectors by standard methods. Expression is carried out in suitable prokaryotic or eukaryotic host cells such as CHO cells, NS0 cells, SP2 / 0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast or E. coli cells, and the polypeptide is recovered from the cells (the supernatant or the cells after lysis). General methods for the recombinant production of polypeptides (such as antibodies) are well known in the art and are reviewed in the following papers: Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R.J., Mol. Biotechnol. 16 (2000) 151-161; Werner, R.G., Drug Res. 48 (1998) 870-880.

[0084] The polypeptides produced by the host cells can undergo post-translational cleavage of one or more, particularly one or two, amino acids at the C-terminus of the polypeptide chain containing the CH3 domain at the C-terminus. Thus, the polypeptides produced by the host cells by expressing a specific nucleic acid molecule encoding the polypeptide chain can include full-length polypeptide chains containing the full-length CH3 domain, or the polypeptide can include a cleaved variant of the full-length polypeptide chain (also referred to herein as a "cleaved variant polypeptide chain"). This may be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447).

[0085] As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can contain modified nucleotides such as methylated nucleotides and their analogs. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can contain modifications that occur after synthesis, such as, for example, conjugation to a label. Other types of modifications include, for example, "capping" with similar inter-nucleotide modifications such as, for example, those having uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates) and those having charged linkages (e.g., phosphorothioates, dithiophosphates, etc.), those containing side group moieties such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those containing intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylating agents, those having modified linkages (e.g., α-anomeric nucleic acids, etc.) and one or more polynucleotides in unmodified form to replace one or more naturally occurring nucleotides. In addition, any hydroxyl group normally present in the sugar can be replaced (e.g., by a phosphate group, a phosphonate group), protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or can be conjugated to a solid or semi-solid support. The OH at the 5' and 3' termini can be phosphorylated or replaced in part by an amine or organic capping group of 1 to 20 carbon atoms. Other hydroxyl groups can also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose that are commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars (such as arabinose, xylose or lyxose, pyranoses, furanoses, sedoheptuloses), acyclic analogs and basic nucleoside analogs such as methyl ribosides. One or more phosphodiester bonds can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, those in which the phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO or CH2 ("acetal"), where each R or R' is independently H or a substituted or unsubstituted alkyl (1-20C) optionally containing an ether (-O-) bond, aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl (araldyl). Not all linkages in the polynucleotide need be the same. The previous description applies to all polynucleotides referred to herein, including RNA and DNA.

[0086] "Isolated" nucleic acid refers to a nucleic acid molecule that has been separated from the components of its natural environment. Isolated nucleic acids include nucleic acid molecules that are contained in a cell that normally contains nucleic acid molecules, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

[0087] "Isolated nucleic acid encoding a heterodimeric polypeptide" refers to one or more nucleic acid molecules encoding one or more polypeptide chains (or fragments thereof) of the heterodimeric polypeptide, including such nucleic acid molecules in a single vector or separate multiple vectors, and such nucleic acid molecules present at one or more locations in a host cell.

[0088] As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of the host cell into which they have been introduced. The term includes vectors used primarily for inserting DNA or RNA into a cell (e.g., chromosomal integration), vectors used primarily for the replication of DNA or RNA, and expression vectors used for the transcription and / or translation of DNA or RNA. Also included are vectors that provide more than one of the above functions.

[0089] "Expression vector" is a vector capable of directing the expression of a nucleic acid operably linked thereto. When an expression vector is introduced into a suitable host cell, it can be transcribed and translated into a polypeptide. When transforming a host cell in the methods according to the invention, an "expression vector" is used; thus, as described herein, the term "vector" in connection with host cell transformation refers to an "expression vector". An "expression system" generally refers to a suitable host cell comprising an expression vector capable of producing the desired expression product.

[0090] As used herein, "expression" refers to the process by which a nucleic acid is transcribed into mRNA and / or the process by which the transcribed mRNA (also referred to as a transcript) is subsequently translated into a peptide or polypeptide. The transcript and the encoded polypeptide are individually or collectively referred to as gene products. If the nucleic acid is derived from genomic DNA, expression in eukaryotic cells may include splicing of the corresponding mRNA.

[0091] As used herein, the term "transformation" refers to the process of transferring a vector or nucleic acid into a host cell. If cells without a strong cell wall barrier are used as host cells, transfection is carried out, for example, by the calcium phosphate precipitation method described by Graham and Van der Eh, Virology 52 (1978) 546ff. However, other methods of introducing DNA into cells can also be used, such as by nuclear injection or by protoplast fusion. If prokaryotic cells or cells containing a substantial cell wall structure are used, for example, one transfection method is carried out using calcium treatment with calcium chloride, as described by Cohen, F. N. et al., PNAS 69 (1972) 7110 et seq.

[0092] As used in this application, the term "host cell" refers to any type of cell system that can be engineered to produce the polypeptides provided by the present invention.

[0093] As used herein, the terms "cell", "cell line", and "cell culture" are used interchangeably and all such names include progeny. Thus, the words "transformant" and "transformed cell" include the primary subject cells and cultures derived therefrom without regard to the number of transfers. It should also be understood that due to deliberate or inadvertent mutations, all progeny may not be precisely identical in DNA content. Variant progeny having the same function or biological activity as screened in the original transformed cells are included. If different names are intended, it will be clear from the context.

[0094] Transient expression is described, for example, in the following references: Durocher, Y. et al., Nucl. Acids Res. 30 (2002) E9. Cloning of variable domains is described, for example, in the following references: Orlandi, R. et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833 - 3837; Carter, P., et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285 - 4289; and Norderhaug, L., et al., J. Immunol. Methods 204 (1997) 77 - 87. A preferred transient expression system (HEK293) is described, for example, in the following references: Schlaeger, E.-J. and Christensen, K., Cytotechnology 30 (1999) 71 - 83; and Schlaeger, E.-J., J. Immunol. Methods 194 (1996) 191 - 199.

[0095] The term "pharmaceutical composition" refers to a preparation that is in a form such that the bioactivity of the active ingredient contained therein is effective and that the preparation contains no additional components that are unacceptably toxic to the subject to which the composition is to be administered. The pharmaceutical compositions of the present invention can be administered by a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired result. In order to administer an antibody according to the present invention by certain routes of administration, it may be necessary to coat the antibody with a material or co-administer the antibody with a material to prevent its inactivation. For example, the heterodimeric polypeptide can be administered to a subject in a suitable carrier such as a liposome or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.

[0096] The pharmaceutical composition contains an effective amount of the fusion polypeptide provided by the present invention. An "effective amount" of an agent (e.g., a fusion polypeptide) is an amount that is effective to achieve the desired therapeutic or prophylactic result at the required dosage and for the required period of time. In particular, the expression "effective amount" refers to the amount of the heterodimeric polypeptide of the present invention which, when administered to a subject, (i) treats or prevents a specific disease, disorder or condition, (ii) alleviates, ameliorates or eliminates one or more symptoms of a specific disease, disorder or condition, or (iii) prevents or delays the onset of one or more symptoms of a specific disease, disorder or condition described herein. The therapeutically effective amount will vary depending on the heterodimeric polypeptide molecule used, the disease state being treated, the severity of the disease being treated, the age and relative health of the subject, the route and form of administration, the judgment of the attending physician or veterinarian and other factors.

[0097] A "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation that is non-toxic to the subject in addition to the active ingredient. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents that are physiologically compatible, etc. In a preferred embodiment, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).

[0098] The pharmaceutical composition according to the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Sterilization can be carried out by the above procedures and by adding various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.) to ensure the absence of microorganisms. It may also be desirable to include isotonic agents such as sugars, sodium chloride, etc. in the composition. In addition, prolonged absorption of injectable pharmaceutical forms can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin).

[0099] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration (usually administered by injection), and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0100] Regardless of the chosen route of administration, the compounds of the invention and / or the pharmaceutical compositions of the invention that can be used in a suitable hydrated form are formulated into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art.

[0101] The actual dosage level of the active ingredient in the pharmaceutical compositions of the invention can vary to obtain an amount of the active ingredient that effectively achieves the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the invention employed, the route of administration, the time of administration, the excretion rate of the particular compound employed, the duration of the treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0102] The composition must be sterile and fluid to the extent that it can be delivered by syringe. In addition to water, in one embodiment, the carrier is an isotonic buffered saline solution.

[0103] For example, fluidity can be maintained by using coatings such as lecithin in the case of dispersion by maintaining the desired particle size and by using surfactants. In many cases, it is preferred to include in the composition an isotonic agent, such as sugars, polyols such as mannitol or sorbitol, and sodium chloride.

[0104] As used herein, "treatment" (and its grammatical variants, such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of the individual being treated, and can be for prophylaxis or during the course of a clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and relieving or improving the prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

[0105] "Individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (such as cows, sheep, cats, dogs, and horses), primates (such as humans and non-human primates, such as monkeys), rabbits, and rodents (such as mice and rats). In certain embodiments, the individual or subject is a human. 2. Detailed Description

[0107] The present invention provides applicable fusion polypeptide pairs, which are, for example, used for the in vivo generation of functional effector domains. One application is to generate antigen-binding sites on cells by associating newly formed antigen-binding sites (e.g., anti-CD3 binding sites). The recombinant fusion polypeptide pair can also be referred to as a precursor polypeptide pair because once the portions of the effector domains contained in the two fusion polypeptides associate with each other and form a functional effector domain, they are capable of forming a polypeptide complex with each other.

[0108] The recombinant fusion protein pair comprises a first fusion polypeptide, which comprises a portion of an effector domain that is complementary to a complementary domain. When associated with each other, the portion of the effector domain and the complementary domain form a non-functional antigen-binding domain. The second fusion polypeptide comprises a second portion of the effector domain that is complementary to a second complementary domain. When associated with each other, the second portion of the effector domain and the complementary domain also form a non-functional antigen-binding domain. According to the present invention, the effector domain is an antibody Fab fragment, wherein the first portion of the effector domain is an antibody light chain polypeptide, and the second portion of the effector domain is an antibody heavy chain polypeptide (CH1-VH).

[0109] When both the first fusion polypeptide and the second fusion polypeptide are in close proximity to each other, the complementary portions of the effector domain dissociate from their respective complementary domains and associate with each other, thereby forming a functional antigen-binding site. This is supported by mutations of charged amino acids in the interfaces of the respective portions of the effector domain and the complementary domain. While in the first fusion polypeptide, the interface between the first portion of the effector domain and the first complementary domain contains at least one pair of amino acid mutations with a positive charge, in the second fusion polypeptide, the interface between the second portion of the effector domain and the second complementary domain contains at least one pair of amino acid mutations with a negative charge.

[0110] Polypeptide pair

[0111] In one aspect, the present invention relates to a recombinant binding protein pair comprising (a) a first fusion polypeptide comprising (i) a first antigen-binding domain capable of binding to a target antigen, (ii) a first portion of an effector domain, and (iii) a first complementary domain capable of associating with the first portion of the effector domain, wherein the first portion of the effector domain and the first complementary domain are linked via a peptide linker; and (b) a second fusion polypeptide comprising (i) a second antigen-binding domain capable of binding to the target antigen, (ii) a second portion of the effector domain, and (iii) a second complementary domain capable of associating with the second portion of the effector domain, wherein the first portion of the effector domain and the first complementary domain are linked via a peptide linker; wherein the first portion of the effector domain and the second portion of the effector domain are capable of associating with each other to form a functional effector domain, characterized in that the effector domain is an antibody Fab fragment.

[0112] (A) Antigen-binding domain

[0113] The first fusion polypeptide comprises a first antigen-binding domain. The second fusion polypeptide comprises a second antigen-binding domain. In one embodiment, the antigen-binding domain is an antibody-derived antigen-binding domain. In one embodiment, the antibody-derived antigen-binding domain is an antibody fragment. In one embodiment, the antibody fragment is a Fab fragment. In one embodiment, the first fusion polypeptide and the second fusion polypeptide comprise antibody-derived antigen-binding domains.

[0114] In one embodiment, the antigen-binding domain comprised in the first fusion polypeptide and / or the second fusion polypeptide binds specifically to a target cell. In one embodiment, the antigen-binding domain binds specifically to an epitope on the surface of the target cell. In one embodiment, the antigen-binding domains of the first fusion polypeptide and the second fusion polypeptide bind to the same epitope on the surface of the target cell. In one embodiment, the antigen-binding domains of the first fusion polypeptide and the second fusion polypeptide bind to different epitopes on the surface of the target cell. In one embodiment, the antigen-binding domains of the first fusion polypeptide and the second fusion polypeptide bind to different epitopes of the same antigen on the surface of the target cell.

[0115] (B) Effector domain

[0116] The first fusion polypeptide comprises a first portion of an effector domain. The second fusion polypeptide comprises a second portion of the effector domain. The first portion of the effector domain and the second portion of the effector domain are capable of associating with each other to form a functional effector domain.

[0117] In one embodiment, the effector domain is a dimer.

[0118] In one embodiment, the effector domain is an antigen-binding domain. In one embodiment, the effector domain is an antibody-derived antigen-binding domain. In one embodiment, the effector domain is an antibody fragment. In one embodiment, the effector domain is a Fab fragment. In one embodiment, the effector domain is an antibody Fab fragment, wherein the first part of the effector domain is an antibody light chain polypeptide and the second part of the effector domain is an antibody heavy chain polypeptide (CH1-VH).

[0119] In one embodiment, the first part of the effector domain is an antibody heavy chain polypeptide and the first complementary domain is an antibody light chain polypeptide, and the second part of the effector domain is an antibody light chain polypeptide and the second complementary domain is an antibody heavy chain polypeptide.

[0120] In one embodiment, the effector domain specifically binds to an antigen. In one embodiment, the effector domain specifically binds to a T cell antigen, particularly an activated T cell antigen. In one embodiment, the effector domain specifically binds to CD3, particularly CD3ε. In one embodiment, the effector domain specifically binds to human CD3.

[0121] In one embodiment, the effector domain specifically binds to human CD28.

[0122] In one embodiment, the effector domain specifically binds to a cell surface receptor. In one embodiment, the effector domain specifically binds to a domain of a cytokine receptor. In one embodiment, the effector domain specifically binds to IL2Rγ. In one embodiment, the effector domain specifically binds to IL2Rβ.

[0123] The first fusion polypeptide comprises the first part of the first effector domain and the first part of the second effector domain. The second fusion polypeptide comprises the second part of the first effector domain and the second part of the second effector domain. The first part of the effector domain and the second part of the effector domain are capable of associating with each other to form the corresponding functional first and second effector domains.

[0124] In one embodiment, the second effector domain specifically binds to a factor, preferably a cell surface factor, which further activates the immune response at the tumor site.

[0125] (C) Complementary domain

[0126] The first fusion polypeptide comprises a first complementary domain. The first complementary domain is capable of associating with the first part of the effector domain. The second fusion polypeptide comprises a second complementary domain. The second complementary domain is capable of associating with the second part of the effector domain.

[0127] In one embodiment, the first complementary domain is capable of associating with the second complementary domain to form a dimer of the first complementary domain and the second complementary domain (also referred to herein as a "complementary domain dimer").

[0128] In one embodiment, when the first complementary domain and the second complementary domain associate with each other, the first complementary domain and the second complementary domain form a non-functional antigen-binding domain. In one embodiment, when the first complementary domain and the second complementary domain associate with each other, the first complementary domain and the second complementary domain form a functional antigen-binding domain.

[0129] In one embodiment, the complementary domain dimer is an antigen-binding domain. In one embodiment, the complementary domain dimer is an antibody-derived antigen-binding domain. In one embodiment, the complementary domain dimer is an antibody fragment. In one embodiment, the effector domain is a Fab fragment.

[0130] (D) Domain arrangements and amino acid substitutions

[0131] The first fusion polypeptide and the second fusion polypeptide are precursor polypeptides that are capable of forming a complex with each other once the first part of the effector domain and the second part of the effector domain associate with each other. To support this dimerization, the first fusion polypeptide and the second fusion polypeptide are arranged such that the association of the two parts of the effector domain is preferred over the association of each part of the effector domain with its respective complementary domain.

[0132] In one embodiment, the first part of the effector domain comprises an antibody variable domain and the first complementary domain comprises a complementary antibody variable domain. In one embodiment, the first part of the effector domain comprises a VL domain and the first complementary domain comprises a VH domain. In one embodiment, the first part of the effector domain comprises a VH domain and the first complementary domain comprises a VL domain. In one embodiment, the VH and VL domains comprise an interface having at least one amino acid substitution that introduces a positively charged amino acid in each domain. In one embodiment, the VH and VL domains comprise an interface having at least one amino acid substitution that introduces a negatively charged amino acid in each domain. In one embodiment, the VH domain comprises a Q39E mutation and the VL domain comprises a Q38E mutation.

[0133] In one embodiment, the second portion of the effector domain comprises an antibody variable domain and the second complementary domain comprises a complementary antibody variable domain. In one embodiment, the second portion of the effector domain comprises a VL domain and the second complementary domain comprises a VH domain. In one embodiment, the second portion of the effector domain comprises a VH domain and the second complementary domain comprises a VL domain. In one embodiment, the VH and VL domains comprise an interface having at least one amino acid substitution introducing a positively charged amino acid in each domain. In one embodiment, the VH and VL domains comprise an interface having at least one amino acid substitution introducing a negatively charged amino acid in each domain. In one embodiment, the VH domain comprises a Q39K mutation and the VL domain comprises a Q38K mutation.

[0134] In one embodiment, the first fusion polypeptide comprises a first portion of the effector domain containing a VL domain and VH domain pair and a first complementary domain, wherein the VH domain comprises a Q39E mutation and the VL domain comprises a Q38E mutation. In one embodiment, the first fusion polypeptide comprises a first portion of the effector domain comprising a VH domain containing a Q39E mutation; and a first complementary domain comprising a VL domain containing a Q38E mutation.

[0135] In one embodiment, the second fusion polypeptide comprises a second portion of the effector domain containing a VL domain and VH domain pair and a second complementary domain, wherein the VH domain comprises a Q39K mutation and the VL domain comprises a Q38K mutation. In one embodiment, the second fusion polypeptide comprises a second portion of the effector domain comprising a VL domain containing a Q38K mutation; and a second complementary domain comprising a VH domain containing a Q39K mutation.

[0136] In one embodiment, the first fusion polypeptide comprises a first portion of the effector domain comprising a VH domain containing a Q39E mutation; and a first complementary domain comprising a VL domain containing a Q38E mutation; and the second fusion polypeptide comprises a second portion of the effector domain comprising a VL domain containing a Q38K mutation; and a second complementary domain comprising a VH domain containing a Q39K mutation. Due to the amino acid mutations with charged amino acids, the interaction between the multiple portions of the effector domain and their respective complementary domains is weaker (because the interface contains amino acids of the same charge), while the interaction between the two portions of the effector domain and optionally the two complementary domains is stronger because the interface contains amino acids of opposite charges.

[0137] In one embodiment, the first portion of the effector domain is an antibody light chain polypeptide. In one embodiment, the first complementary domain is an antibody heavy chain polypeptide.

[0138] In one embodiment, the second part of the effector domain is an antibody heavy chain polypeptide. In one embodiment, the second complementary domain is an antibody light chain polypeptide.

[0139] In one embodiment, the first part of the effector domain is an antibody light chain polypeptide, the first complementary domain is an antibody heavy chain polypeptide, the second part of the effector domain is an antibody heavy chain polypeptide, and the second complementary domain is an antibody light chain polypeptide.

[0140] In one embodiment, the first fusion polypeptide is a recombinant antibody consisting of the following three polypeptides: (i) an antibody light chain, and (ii) an antibody heavy chain, wherein the variable domains of the antibody light chain and the antibody heavy chain form a first antigen-binding domain, and (iii) an antibody heavy chain / light chain fusion polypeptide that comprises an antibody light chain comprising the first part of the effector domain fused via a peptide linker to an antibody heavy chain comprising the first complementary domain, wherein the C-terminus of the antibody light chain is fused to the N-terminus of the antibody heavy chain; and wherein the second fusion polypeptide is a recombinant antibody consisting of the following three polypeptides: (i) an antibody light chain, and (ii) an antibody heavy chain, wherein the variable domains of the antibody light chain and the antibody heavy chain form a second antigen-binding domain, and (iii) an antibody heavy chain / light chain fusion polypeptide that comprises an antibody light chain comprising the second part of the effector domain fused via a peptide linker to an antibody heavy chain comprising the second complementary domain, wherein the C-terminus of the antibody light chain is fused to the N-terminus of the antibody heavy chain.

[0141] In one embodiment, the first part of the effector domain and the first complementary domain are included in a single-chain Fab fragment. In one embodiment, the second part of the effector domain and the second complementary domain are included in a single-chain Fab fragment. It has been observed that the use of single-chain Fab fragments significantly reduces the remaining solution-phase activation of the effector domain ( Figure 7 ).

[0142] In one embodiment, the C-terminus of the first part of the effector domain is fused to the N-terminus of the first complementary domain via a peptide linker.

[0143] In a preferred embodiment of the present invention, the linker is a peptide of at least 20 amino acids. In a preferred embodiment of the present invention, the linker is a peptide of at least 25 amino acids. In another embodiment of the present invention, the linker is a peptide of 25 to 70 amino acids. In another embodiment of the present invention, the linker is a peptide of 25 to 35 amino acids. In one embodiment of the present invention, the linker is a glycine-serine linker. In one embodiment of the present invention, the linker is a peptide composed of glycine and serine residues. In one embodiment of the present invention, the glycine-serine linker contains at least more than 5, preferably more than six (Gly-Gly-Gly-Gly-Ser) repeat sequences.

[0144] In one embodiment, the first and second fusion polypeptides comprise multiple parts containing effector domains and single-chain Fab fragments of corresponding complementary domains, the single-chain Fab fragment containing a domain crossover such that a first part of the effector domain can associate with a second part of the effector domain. Domain crossovers in multispecific antibodies are known in the art, such as et al. (Schaefer W, Regula JT, M, Schanzer J, Croasdale R, Dürr H, Gassner C, Georges G, Kettenberger H, Imhof-Jung S, Schwaiger M, Stubenrauch KG, Sustmann C, Thomas M, Scheuer W, Klein C. Proc Natl Acad Sci U SA. July 5, 2011; 108(27):11187-92, Klein C, Sustmann C, Thomas M, Stubenrauch K, Croasdale R, Schanzer J, Brinkmann U, Kettenberger H, Regula JT, SchaeferW. MAbs. November-December 2012; 4(6):653-63.doi:10.4161 / mabs.21379.Epub2012 August 27) in the disclosed CrossMab technology.

[0145] This results in an improved by-product profile for the recombinant expression of the first and second fusion polypeptides. In addition, the use of different domain crossovers in the first and second fusion polypeptides allows the two parts of the effector domain to associate with each other.

[0146] In one embodiment, the single-chain Fab fragment of the first fusion polypeptide comprises a domain crossover of the VH and VL domains, i.e., the VH and VL domains are exchanged with each other, and the single-chain Fab fragment of the second fusion polypeptide comprises a domain crossover of the CH1 and CL domains, i.e., the CH1 and CL domains are exchanged with each other.

[0147] In one embodiment, the first fusion polypeptide comprises a first part of the effector domain, which comprises a VH domain and a CL domain in the N-terminal to C-terminal direction; and a first complementary domain, which comprises a VL domain and a CH1 domain in the N-terminal to C-terminal direction; and the second fusion polypeptide comprises a second part of the effector domain, which comprises a VL domain and a CH1 domain in the N-terminal to C-terminal direction, and a second complementary domain, which comprises a VH domain and a CL domain in the N-terminal to C-terminal direction.

[0148] In one embodiment, the single-chain Fab fragment of the first fusion polypeptide comprises a VH domain, a CL domain, a peptide linker, a VL domain, and a CH1 domain in the N-terminal to C-terminal direction, and wherein the single-chain Fab fragment of the second fusion polypeptide comprises a VL domain, a CH1 domain, a peptide linker, a VH domain, and a CL domain in the N-terminal to C-terminal direction.

[0149] In one embodiment, the first fusion polypeptide comprises a first part of the effector domain, which comprises a VH domain with a Q39E mutation and a CL domain in the N-terminal to C-terminal direction; and a first complementary domain, which comprises a VL domain with a Q38E mutation and a CH1 domain in the N-terminal to C-terminal direction; and the second fusion polypeptide comprises a second part of the effector domain, which comprises a VL domain with a Q38K mutation and a CH1 domain in the N-terminal to C-terminal direction; and a second complementary domain, which comprises a VH domain with a Q39K mutation and a CL domain in the N-terminal to C-terminal direction.

[0150] In one embodiment, the single-chain Fab fragment of the first fusion polypeptide comprises a VH domain with a Q39E mutation, a CL domain, a peptide linker, a VL domain with a Q38E, and a CH1 domain in the N-terminal to C-terminal direction, and wherein the single-chain Fab fragment of the second fusion polypeptide comprises a VL domain with a Q38K mutation, a CH1 domain, a peptide linker, a VH domain with a Q39K mutation, and a CL domain in the N-terminal to C-terminal direction.

[0151] (E) Fc domain

[0152] In one embodiment, the first fusion polypeptide comprises an Fc domain, particularly a heterodimeric Fc domain. In one embodiment, the second fusion polypeptide comprises an Fc domain, particularly a heterodimeric Fc domain. In one embodiment, the first fusion polypeptide and the second fusion polypeptide comprise an Fc domain, preferably a heterodimeric Fc domain.

[0153] In one embodiment, the Fc domain comprises a knobs-into-holes mutation. In one embodiment, the first fusion polypeptide and the second fusion polypeptide comprise a heterodimeric Fc domain that comprises two CH3 domains, wherein one of the CH3 domains comprises the mutations S354C and T366W, and the other CH3 domain comprises the mutations Y349C, L368A, and Y407V. This results in an improved by-product profile upon recombinant expression of the first fusion polypeptide and the second fusion polypeptide.

[0154] (F) Antibody isotype

[0155] In one embodiment of the invention, the first fusion polypeptide and the second fusion polypeptide comprise immunoglobulin constant regions of one or more immunoglobulin classes. Immunoglobulin classes include IgG, IgM, IgA, IgD, and IgE isotypes, and in the case of IgG and IgA, also their subtypes. In one embodiment of the invention, the precursor polypeptide has the constant domain structure of an IgG-type antibody.

[0156] In one embodiment of the invention, the CH3 domains comprised in the first fusion polypeptide and the second fusion polypeptide belong to mammalian IgG classes. In one embodiment of the invention, the CH3 domains comprised in the first fusion polypeptide and the second fusion polypeptide belong to mammalian IgG1 subclass. In one embodiment of the invention, the CH3 domains comprised in the first fusion polypeptide and the second fusion polypeptide belong to mammalian IgG4 subclass.

[0157] In one embodiment of the invention, the CH3 domains comprised in the first fusion polypeptide and the second fusion polypeptide belong to human IgG classes. In one embodiment of the invention, the CH3 domains comprised in the first fusion polypeptide and the second fusion polypeptide belong to human IgG1 subclass. In one embodiment of the invention, the CH3 domains comprised in the first fusion polypeptide and the second fusion polypeptide belong to human IgG4 subclass.

[0158] In one embodiment, the constant domains of the first fusion polypeptide and the second fusion polypeptide according to the invention belong to human IgG classes. In one embodiment, the constant domains of the first fusion polypeptide and the second fusion polypeptide according to the invention belong to human IgG1 subclass. In one embodiment, the constant domains of the first fusion polypeptide and the second fusion polypeptide according to the invention belong to human IgG4 subclass.

[0159] In one embodiment, the first fusion polypeptide and the second fusion polypeptide do not have a CH4 domain.

[0160] In one embodiment of the present invention, the constant domains of the first fusion polypeptide and the second fusion polypeptide according to the present invention belong to the same immunoglobulin subclass. In one embodiment of the present invention, the variable domains and the constant domains of the first fusion polypeptide and the second fusion polypeptide according to the present invention belong to the same immunoglobulin subclass.

[0161] In one embodiment of the present invention, the first fusion polypeptide and the second fusion polypeptide are isolated precursor polypeptides.

[0162] In one embodiment, the heterodimeric fusion polypeptide comprising a polypeptide chain including a CH3 domain comprises a full-length CH3 domain or a CH3 domain in which one or both C-terminal amino acid residues (i.e., G446 and / or K447) are absent.

[0163] Methods for forming a functional effector domain and recombinant methods

[0164] In another aspect, the present invention relates to a method for forming a functional effector domain from a pair of recombinant binding proteins according to the present invention, wherein the first antigen-binding domain and the second antigen-binding domain of the recombinant binding protein specifically bind to an epitope on the surface of a target cell, the method comprising contacting the pair of recombinant binding proteins with the target cell under conditions that permit the first fusion polypeptide and the second fusion polypeptide to bind to the target cell.

[0165] The fusion polypeptide according to the present invention is prepared by a recombinant method. Accordingly, the present invention also relates to a method for preparing a fusion polypeptide according to the present invention, comprising culturing a host cell comprising a nucleic acid encoding the fusion polypeptide under conditions suitable for the expression of the fusion polypeptide.

[0166] In one aspect, there is provided a method for preparing a fusion polypeptide of the present invention, wherein the method comprises culturing a host cell as provided above comprising a nucleic acid encoding the fusion polypeptide under conditions suitable for the expression of the fusion polypeptide, and optionally recovering the fusion polypeptide from the host cell (or the host cell culture medium).

[0167] In one embodiment, the method comprises the steps of: transforming a host cell with an expression vector comprising a nucleic acid encoding the fusion polypeptide, culturing the host cell under conditions permitting the synthesis of the fusion polypeptide, and recovering the fusion polypeptide from the host cell culture.

[0168] For recombinant production of a fusion polypeptide, a nucleic acid encoding the fusion polypeptide, such as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to the gene encoding the polypeptide chain of the fusion polypeptide), or produced by recombinant methods or obtained by chemical synthesis.

[0169] Suitable host cells for vectors encoding antibodies for cloning or expression include the prokaryotic or eukaryotic cells described herein. For example, the fusion polypeptide can be produced in bacteria. For expression of polypeptides in bacteria, see, e.g., US 5,648,237, US 5,789,199, and US 5,840,523. (See also Charlton, K.A., in: Methods in Molecular Biology, Vol. 248, Lo, B.K.C., ed., Humana Press, Totowa, NJ (2003), pp. 245 - 254, describing the expression of antibody fragments in E. coli.) The fusion polypeptide can be isolated from the bacterial cell paste in the soluble fraction after expression and can be further purified.

[0170] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding the fusion polypeptides of the invention, including such fungal and yeast strains in which the glycosylation pathways have been "humanized" such that polypeptides with a partially or fully human glycosylation pattern are produced. See Gerngross, T.U., Nat. Biotech. 22 (2004) 1409 - 1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210 - 215.

[0171] Suitable host cells for expression of (glycosylated) fusion polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. A number of baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0172] Plant cell cultures can also be used as hosts. See, e.g., US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (describing the PLANTIBODIES™ technology for production of antibodies in transgenic plants).

[0173] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are the monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney cell lines (such as the 293 or 293T cells described, for example, in Graham, F. L. et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (such as the TM4 cells described in Mather, J. P., Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); dog kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (such as described, for example, in Mather, J. P. et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines, such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, A. M., Methods in Molecular Biology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0174] In one aspect, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphocyte (such as Y0, NS0, Sp20 cells).

[0175] In one aspect, the invention provides an isolated nucleic acid encoding the fusion polypeptide of the invention. In one aspect, the invention provides an expression vector comprising the nucleic acid according to the invention. In another aspect, the invention provides a host cell comprising the nucleic acid of the invention.

[0176] In one embodiment, the method of the present invention includes providing a recombinant binding protein pair according to the present invention. Thus, the first fusion polypeptide and the second fusion polypeptide are expressed recombinantly. In one embodiment, the first fusion polypeptide and the second fusion polypeptide are expressed in eukaryotic cells, preferably HEK293 cells or CHO cells. In one embodiment, the first fusion polypeptide and the second fusion polypeptide are purified after recombinant expression.

[0177] In one embodiment, the first fusion protein and the second fusion protein are contacted with a target cell, in one embodiment in an aqueous solution. The first antigen-binding domain and the second antigen-binding domain contact a target antigen on the target cell. Due to the binding of the two precursor proteins bringing multiple parts of the effector domain into close proximity, the first part of the effector domain and the second part of the effector domain associate with each other to form a functional effector domain. Optionally, the first complementary domain and the second complementary domain also associate with each other, optionally forming another functional domain, such as an antigen-binding domain.

[0178] Therapeutic applications

[0179] The recombinant binding protein pair can be used in therapies, such as cancer therapy.

[0180] Thus, one aspect of the present invention is a recombinant binding protein pair for use as a medicament. In one embodiment, the effector domain is CD3 that specifically binds to CD3, and the recombinant binding protein pair is used as a medicament for treating cancer.

[0181] Another aspect is a method of treating an individual suffering from a disease, the method comprising administering to the individual an effective amount of the recombinant binding protein pair of the present invention or the pharmaceutical composition of the present invention.

[0182] 3. Specific Embodiments of the Present Invention

[0183] Specific embodiments of the present invention are listed below.

[0184] 1. A recombinant binding protein pair, comprising

[0185] (a) A first fusion polypeptide comprising (i) a first antigen-binding domain capable of binding to a target antigen, (ii) a first part of an effector domain, and (iii) a first complementary domain capable of associating with the first part of the effector domain, wherein the first part of the effector domain and the first complementary domain are connected via a peptide linker; and

[0186] (b) A second fusion polypeptide comprising (i) a second antigen-binding domain capable of binding to a target antigen, (ii) a second portion of an effector domain, and (iii) a second complementary domain capable of associating with the second portion of the effector domain, wherein the first portion of the effector domain and the first complementary domain are linked via a peptide linker;

[0187] wherein the first portion of the effector domain and the second portion of the effector domain are capable of associating with each other to form a functional effector domain, characterized in that the effector domain is a Fab fragment.

[0188] 2. The recombinant binding protein pair according to embodiment 1, wherein the antigen-binding domain specifically binds to an epitope on the surface of a target cell.

[0189] 3. The recombinant binding protein pair according to embodiment 2, wherein the antigen-binding domain of the first fusion polypeptide and the antigen-binding domain of the second fusion polypeptide bind to the same epitope on the surface of a target cell.

[0190] 4. The recombinant binding protein pair according to embodiment 2, wherein the antigen-binding domain of the first fusion polypeptide and the antigen-binding domain of the second fusion polypeptide bind to different epitopes on the surface of a target cell.

[0191] 5. The recombinant binding protein pair according to any one of the preceding embodiments, wherein the effector domain is an antigen-binding domain.

[0192] 6. The recombinant binding protein pair according to any one of the preceding embodiments, wherein the effector domain is an antibody heavy chain polypeptide and the first complementary domain is an antibody light chain polypeptide, and the second portion of the effector domain is an antibody light chain polypeptide and the second complementary domain is an antibody heavy chain polypeptide.

[0193] 7. The recombinant binding protein pair according to any one of the preceding embodiments, wherein the effector domain specifically binds to a T cell antigen, particularly an activated T cell antigen.

[0194] 8. The recombinant binding protein pair according to any one of the preceding embodiments, wherein the effector domain specifically binds to CD3.

[0195] 9. The recombinant binding protein pair according to any one of the preceding embodiments, wherein the effector domain specifically binds to CD28.

[0196] 10. The recombinant binding protein pair according to one of the foregoing embodiments, wherein the effector domain is an antibody Fab fragment, wherein the first part of the effector domain is an antibody light chain polypeptide, and wherein the second part of the effector domain is an antibody heavy chain polypeptide (CH1-VH).

[0197] 11. The recombinant binding protein pair according to one of the foregoing embodiments,

[0198] wherein the first part of the effector domain is an antibody heavy chain polypeptide, and the first complementary domain is an antibody light chain polypeptide, and

[0199] wherein the second part of the effector domain is an antibody light chain polypeptide, and the second complementary domain is an antibody heavy chain polypeptide.

[0200] 12. The recombinant binding protein pair according to one of the foregoing embodiments, wherein the complementary domain dimer is an antibody-derived antigen-binding domain.

[0201] 13. The recombinant binding protein pair according to one of the foregoing embodiments, wherein when the first complementary domain and the second complementary domain associate with each other, the first complementary domain and the second complementary domain form a non-functional antigen-binding domain.

[0202] 14. The recombinant binding protein pair according to one of the foregoing embodiments, wherein the Fc domain contains a coiled-coil mutation.

[0203] 15. The recombinant binding protein pair according to one of the foregoing embodiments, wherein the first part of the effector domain contains a variable heavy chain domain with a Q39E mutation, and wherein the first complementary domain contains a variable light chain domain with a Q38E mutation,

[0204] wherein the second part of the effector domain contains a variable light chain domain with a Q38K mutation, and wherein the second complementary domain contains a variable heavy chain domain with a Q39K mutation.

[0205] 16. The recombinant binding protein pair according to one of the foregoing embodiments, wherein the first part of the effector domain and the first complementary domain are contained in a single-chain Fab fragment.

[0206] 17. The recombinant binding protein pair according to one of the foregoing embodiments, wherein the second part of the effector domain and the second complementary domain are contained in a single-chain Fab fragment.

[0207] 18. A recombinant binding protein pair according to one of embodiments 15 or 16, wherein in the single-chain Fab fragment of the first fusion protein, the VH and VL domains are exchanged with each other, and wherein in the single-chain Fab fragment of the second fusion protein, the CH1 and CL domains are exchanged with each other.

[0208] 19. A recombinant binding protein pair according to one of embodiments 15 or 16, wherein the single-chain Fab fragment of the first fusion protein comprises, from the N-terminal to the C-terminal direction, a VH domain, a CL domain, a peptide linker, a VL domain, and a CH1 domain, and wherein the single-chain Fab fragment of the second fusion protein comprises, from the N-terminal to the C-terminal direction, a VL domain, a CH1 domain, a peptide linker, a VH domain, and a CL domain.

[0209] 20. A recombinant binding protein pair according to one of the foregoing embodiments, wherein the first antigen-binding domain is an antibody Fab fragment.

[0210] 21. A recombinant binding protein pair according to one of the foregoing embodiments, wherein the second antigen-binding domain is an antibody Fab fragment.

[0211] 22. A recombinant binding protein pair according to one of the foregoing embodiments, wherein the first fusion polypeptide comprises an (heterodimeric) Fc domain.

[0212] 23. A recombinant binding protein pair according to one of the foregoing embodiments, wherein the second fusion polypeptide comprises an (heterodimeric) Fc domain.

[0213] 24. A recombinant binding protein pair according to one of the foregoing embodiments, wherein the first fusion polypeptide and the second fusion polypeptide comprise a heterodimeric Fc domain, the heterodimeric Fc domain comprising two CH3 domains, wherein one of the CH3 domains comprises the mutations S354C and T366W, and the other CH3 domain comprises the mutations Y349C, L368A, and Y407V.

[0214] 25. A recombinant binding protein pair according to one of the foregoing embodiments, wherein the first fusion polypeptide is a recombinant antibody composed of the following three polypeptides: (i) an antibody light chain, and (ii) an antibody heavy chain, wherein the variable domains of the antibody light chain and the antibody heavy chain form the first antigen-binding domain, and (iii) an antibody heavy chain / light chain fusion polypeptide, the antibody heavy chain / light chain fusion polypeptide comprising an antibody light chain comprising the first part of the effector domain fused to an antibody heavy chain comprising the first complementary domain via a peptide linker, wherein

[0215] The C-terminus of the antibody light chain is fused to the N-terminus of the antibody heavy chain; and

[0216] wherein the second fusion polypeptide is a recombinant antibody composed of the following three polypeptides: (i) an antibody light chain, and (ii) an antibody heavy chain, wherein the variable domains of the antibody light chain and the antibody heavy chain form the second antigen-binding domain, and (iii) an antibody heavy chain / light chain fusion polypeptide, the antibody heavy chain / light chain fusion polypeptide comprising an antibody light chain containing the second part of the effector domain fused to the antibody heavy chain containing the second complementary domain via a peptide linker, wherein the C-terminus of the antibody light chain is fused to the N-terminus of the antibody heavy chain.

[0217] 26. A recombinant binding protein pair, wherein the effector domain is an antibody-binding domain.

[0218] 27. A recombinant binding protein pair, wherein the effector domain is an anti-CD3 antibody-binding domain.

[0219] 28. A method for forming a functional effector domain from a recombinant binding protein pair according to one of the preceding embodiments, wherein the first and second antigen-binding domains of the recombinant binding protein specifically bind to an epitope on the surface of a target cell, the method comprising contacting the recombinant binding protein pair with the target cell under conditions that permit the first and second fusion polypeptides to bind to the target cell.

[0220] 29. A pharmaceutical formulation comprising: a recombinant binding protein pair according to any one of the preceding embodiments; and a pharmaceutically acceptable carrier.

[0221] Description of amino acid sequences

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233] Example

[0234] The following examples are provided to assist in understanding the present invention, the true scope of which is set forth in the appended claims. It should be understood that the procedures described may be modified without departing from the spirit of the present invention.

[0235] Example 1:

[0236] Design and mode of action of an (inactive) fusion polypeptide comprising a single-chain Fab fragment

[0237] Figure 1Shows the general design of the recombinant binding protein pair of the present invention. In this example, an IgG-type molecule composed of three separate polypeptide chains is provided: a light chain (e.g., a full-length light chain containing a light chain variable domain and a light chain constant domain), a heavy chain (e.g., a full-length heavy chain containing a heavy chain variable domain and all heavy chain constant domains including a hinge region), and a heavy chain polypeptide containing a portion of the Fc domain (e.g., a fragment of the heavy chain Fc region containing the hinge-CH2-CH3) fused to the scFab polypeptide at its N-terminus. The variable domain of the light chain and the variable domain of the heavy chain form an antigen-binding domain, here the antigen-binding site. The heavy chain (e.g., derived from the human IgG1 subclass) contains a knobs-into-holes mutation (e.g., mutations T366W and S354C in the CH3 domain of the antibody heavy chain are designated as "knobs", and mutations T366S, L368A, Y407V, Y349C in the CH3 domain of the antibody heavy chain are designated as "holes". In the first fusion polypeptide ("Precursor A"), the scFab-polypeptide contains a domain crossover of the VH and VL domains to ensure correct light chain pairing (N--VH-Ck-linker-dVL-CH1--C). The complementary domain contains the variable light chain domain dVL, the linker consists of 6x(G4S) units, and the first part of the effector domain is the VH domain, e.g., derived from a CD3-binding entity. Repulsive charges are introduced between dVL and VH (dVL: Q38E; VH: Q39E) to create a partially defective interface between the two domains. In the second fusion protein ("Precursor B"), the scFab-polypeptide contains a domain crossover of the CH1 and CL domains to ensure correct light chain pairing (N--VL-CH1-linker-dVH-Ck--C). The complementary domain contains the variable heavy chain domain dVH, the linker consists of 6x(G4S) units, and the second part of the effector domain is VL, e.g., derived from a CD3-binding entity. Repulsive charges are introduced between dVH and VL (VL: Q38K; dVH: Q39K) to create a partially unstable interface between the two domains.

[0238] Figure 2 Shows the principle of activation of the effector domain after the first fusion polypeptide and the second fusion polypeptide bind via their first antigen-binding domain and second antigen-binding domain to the same epitope on the surface of the target cell. The partially unstable interfaces in the first fusion polypeptide and the second fusion polypeptide trigger <cd3>Dissociation of the derived VH / VL chains. In the case driven by attracting charges (VH, Q39E (precursor A); VL, Q38K (precursor B)), the active CD3-binding Fab is generated from precursors in close proximity and mediates T cell engagement and activation.

[0239] Figure 3 The principle of activation of the effector domain after binding of the first fusion polypeptide and the second fusion polypeptide to the target cell via their first antigen-binding domain and second antigen-binding domain that bind to different epitopes on the surface of the target cell is shown. The partially labile interface in the prodrug module triggers <cd3>Dissociation of the derived VH / VL chains. In the case driven by attractive charges (VH, Q39E (precursor A); VL, Q38K (precursor B)), the active CD3-binding Fab is generated from the precursors in close proximity and mediates T cell engagement and activation.

[0240] Example 2:

[0241] Expression of (inactive) fusion polypeptides containing single-chain Fab fragments

[0242] To express the fusion polypeptides used in the following examples, the following method was adopted:

[0243] Expression of the first and second fusion polypeptides was achieved by co-transfecting plasmids encoding the light chain, the heavy chain (with a pestle or mortar mutation), and a matching heavy chain polypeptide containing a part of the effector domain and the complementary domain (mortar or pestle) into mammalian cells (e.g., HEK293) via the prior art.

[0244] More specifically, for example, to produce the first and second fusion polypeptides by transient transfection (e.g., in HEK293 cells), expression plasmids based on cDNA organization with or without the CMV-intron A promoter or on genomic organization with the CMV promoter were applied.

[0245] In addition to the antibody expression cassette, the plasmid also contains:

[0246] - An origin of replication that allows replication of the plasmid in Escherichia coli (E. coli),

[0247] - The β-lactamase gene, which confers ampicillin resistance in E. coli, and

[0248] - The dihydrofolate reductase gene from Mus musculus as a selection marker in eukaryotic cells.

[0249] The transcription unit of each antibody gene consists of the following elements:

[0250] -- A unique restriction site at the 5' end,

[0251] - The immediate early enhancer and promoter from human cytomegalovirus,

[0252] - In the case of cDNA organization, followed by the intron A sequence,

[0253] -- The 5' untranslated region of the human antibody gene,

[0254] - The immunoglobulin heavy chain signal sequence,

[0255] - An antibody chain, which is present as cDNA or in genomic organization (maintaining immunoglobulin exon-intron organization),

[0256] - A 3'-untranslated region having a polyadenylation signal sequence, and

[0257] - A unique restriction site at the 3' end.

[0258] A fusion gene containing the antibody chain is generated by gene synthesis and the fusion gene is assembled by known recombinant methods and techniques, for example, by ligating the corresponding nucleic acid segments using the unique restriction sites in the corresponding plasmid. The nucleic acid sequence of the subclone is verified by DNA sequencing. For transient transfection, a relatively large amount of plasmid is prepared from the transformed Escherichia coli culture by plasmid preparation (Nucleobond AX, Macherey-Nagel).

[0259] Standard cell culture techniques as described in Current Protocols in Cell Biology (2000), Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott-Schwartz, J. and Yamada, K.M. (Eds.), John Wiley & Sons, Inc are used.

[0260] The first fusion polypeptide and the second fusion polypeptide are generated by transient transfection with the corresponding plasmid using the HEK293-Expi system (ThermoFisher) according to the manufacturer's instructions. Since the fusion polypeptides contain the Fc region, they are purified by applying standard protein A affinity chromatography. Using MabSelectSure-Sepharose TM (GE Healthcare, Sweden) and Superdex 200 size exclusion (GE Healthcare, Sweden) chromatography, the antibody is purified from the cell culture supernatant by affinity chromatography.

[0261] Briefly, the sterile-filtered cell culture supernatant was captured on MabSelect SuRe resin equilibrated with PBS buffer (10 mM Na2HPO4, 1 mM KH2PO4, 137 mM NaCl, and 2.7 mM KCl, pH 7.4), washed with the equilibration buffer, and eluted with 25 mM sodium citrate at pH 3.0. The eluted antibody fractions were pooled and neutralized with 2 M Tris, pH 9.0. The antibody pool was further purified by size-exclusion chromatography using a Superdex 200 26 / 60 GL (GE Healthcare, Sweden) column equilibrated with 20 mM histidine, 140 mM NaCl (pH 6.0). The fractions containing 2 / 3-IgG were pooled, concentrated to the desired concentration using a Vivaspin ultrafiltration device (Sartorius Stedim Biotech S.A., France), and stored at -80 °C.

[0262] For both the first and second fusion polypeptides targeting HER2, the product purity > 98%. The samples were stored at 4 °C for > 14 days to monitor stability and potential aggregate formation, and the samples were found to be stable.

[0263] The stability of the non-specific precursor molecule with a covalently linked exchange unit after storage at 4 °C for > 14 days was determined by analytical size-exclusion chromatography.

[0264] Example 3:

[0265] T cell activation mediated by the recombinant binding proteins of the present invention, wherein the first and second fusion polypeptides bind to the same epitope on the target cell

[0266] Having as Figure 1 The multiple pairs of recombinant binding proteins according to the present invention having the domain arrangement shown were expressed as described in Example 2. The two fusion polypeptides included in the recombinant protein pair contain the same antigen-binding domain.

[0267] A first recombinant binding protein pair is provided, wherein the first and second fusion polypeptides contain an antigen-binding domain that specifically binds to Her2 and the effector domain is an anti-CD3 antigen-binding site.

[0268] Light Chain Heavy Chain Heavy Chain - scFab Fusion <her2>pro <cd3>VH SEQ ID NO:01 SEQ ID NO:02 SEQ ID NO:03 <her2>pro <cd3>VL SEQ ID NO:01 SEQ ID NO:02 SEQ ID NO:04

[0269] In addition, a second recombinant binding protein pair is provided, wherein the first and second fusion polypeptides contain an antigen-binding domain that specifically binds to EGFR, and the effector domain is an anti-CD3 antigen-binding site.

[0270] Light Chain Heavy Chain Heavy Chain - scFab Fusion <egfr>pro <cd3>VH < / egfr> SEQ ID NO:05 SEQ ID NO:06 SEQ ID NO:07 <egfr>pro <cd3>VL < / egfr> SEQ ID NO:05 SEQ ID NO:06 SEQ ID NO:04

[0271] Figure 4 A shows the expression profiles of the tumor-associated antigens EGFR and HER2 on SK-BR3, and Figure 4 C shows the expression profiles of the tumor-associated antigens EGFR and HER2 on A431 cells.

[0272] To measure whether a functional effector domain that specifically binds to CD3 is formed after target cell binding, HER2-positive SK-BR3 cells were co-incubated with Jurkat reporter cells (E:T = 1:3) and the corresponding antibody targeting HER2 according to the manufacturer's recommendations (Promega, #J1601). Briefly, on day 1, 30,000 target cells were seeded into a 96-well plate (white clear flat bottom, Corning #3610). On day 2, the medium was removed and 1x10^5 Jurkat cells were added together with the medium and the antibody to reach a final volume of 75 μl. Antibodies targeting HER2 were included individually (to measure inactivation of the CD3 conjugate to a prodrug) or in combination (to measure reconstitution of the CD3 conjugate) at equimolar concentrations. Sixteen hours after treatment, the recommended detection reagent was added and luminescence was measured using a TECAN microplate reader device.

[0273] Figure 4 B indicates activation of the effector domain in the presence of both target cells and both the first and second fusion polypeptides, while no activity was observed when only one of the fusion polypeptides was present. Values in triplicate were fitted using 3-parameter non-linear regression and plotted using GraphPad Prism software.

[0274] To measure whether a functional effector domain that specifically binds to CD3 is formed after binding of the second pair of recombinant binding proteins to target cells, EGFR-positive A431 cells were co-incubated with Jurkat reporter cells (E:T = 1:3) and the corresponding antibody targeting A431 according to the manufacturer's recommendations (Promega, #J1601). Figure 4 D indicates activation of the effector domain in the presence of both target cells and both the first and second fusion polypeptides, while no activity was observed when only one of the fusion polypeptides was present. Values in triplicate were fitted using 3-parameter non-linear regression and plotted using GraphPad Prism software. Values in triplicate were fitted using 3-parameter non-linear regression and plotted using GraphPad Prism software (D).

[0275] Example 4:

[0276] T cell activation mediated by the recombinant binding proteins of the present invention, wherein the first fusion polypeptide and the second fusion polypeptide bind to different epitopes on the target cell

[0277] Pairs of recombinant binding proteins according to the invention having the domain arrangement as shown in Figure 1 are expressed as described in Example 2. The two fusion polypeptides contained in the recombinant protein pair contain the same antigen-binding domain.

[0278] SK-BR-3 cell lines expressing both Her2 and EGFR were used to assess T cell activation mediated by different combinations of the recombinant binding proteins used in Example 3. Briefly, HER2-positive and EGFR-positive SK-BR3 cells were co-incubated with Jurkat reporter cells (E:T = 1:3) and the corresponding fusion polypeptides targeting HER2 or EGFR according to the manufacturer's recommendations (Promega, #J1601). The fusion polypeptides were included individually (control) or in combination (to assess the formation of functional effector domains) at equimolar concentrations. Sixteen hours after treatment, the recommended detection reagent was added and luminescence was measured using a TECAN microplate reader device. The results are shown in Figure 5 which indicates activation of the effector domain in the presence of the target cell and both the first and second fusion polypeptides, while no activity was observed when only one of the fusion polypeptides was present. Values in triplicate were fitted using 3-parameter non-linear regression and plotted using GraphPad Prism software.

[0279] Example 5 (Control):

[0280] T cell activation mediated by the recombinant binding proteins of the present invention, wherein only one of the antigen-binding domains of the first fusion polypeptide and the antigen-binding domain of the second fusion polypeptide binds to an epitope on the target cell

[0281] Pairs of recombinant binding proteins according to the invention having the domain arrangement as shown in Figure 6 are expressed as described in Example 2 as a control.

[0282] Figure 6 Shows the principle of activation of the effector domain after the first and second fusion polypeptides bind via their first and second antigen-binding domains to the target cell, wherein only one of the first and second antigen-binding domains binds to an epitope on the target cell and the other antigen-binding domain does not bind to the target on the surface cell, which describes one form of unwanted activation in solution that needs to be minimized. A partially labile interface in the fusion polypeptide triggers <cd3>Dissociation of the derived VH / VL chains. In the case driven by attractive charges (VH, Q39E (precursor A); VL, Q38K (precursor B)), the active CD3-binding Fab is generated from the closely approximated precursors and mediates T cell engagement and activation.

[0283] Use of a first fusion polypeptide and a second fusion polypeptide comprising antigen-binding domains that specifically bind to Her2 to provide multiple pairs of recombinant binding proteins, and the effector domain is an anti-CD3 antigen-binding site, as used in Example 3. Another set of fusion polypeptides is provided that comprises antigen-binding domains that do not specifically bind to epitopes on target cells. As a control, a recombinant binding protein in the shape of regular IgG is provided, i.e., having regular Fab fragments instead of single-chain Fab fragments.

[0284]

[0285]

[0286] The SK-BR-3 cell line expressing both Her2 and EGFR was used to assess T cell activation mediated by different combinations of these recombinant binding proteins. Briefly, according to the manufacturer's recommendations (Promega, #J1601), HER2-positive and EGFR-positive SK-BR3 cells were co-incubated with Jurkat reporter cells (E:T = 1:3) and the corresponding fusion polypeptides targeting Her2. The fusion polypeptides were included individually (control) or in combination (to assess the formation of functional effector domains) at equimolar concentrations. Sixteen hours after treatment, the recommended detection reagent was added, and luminescence was measured using a TECAN microplate reader device.

[0287] The results using the linkerless control recombinant binding protein are shown in Figure 7 A, indicating activation in the solution of the effector domain.

[0288] The results using the recombinant binding proteins of the present invention are shown in Figure 7 B, which indicates activation of the effector domain in the presence of both the target cells and the first and second fusion polypeptides bound to the target cells, while little activity was observed when only one of the fusion polypeptides was present. The values in triplicate were fitted using 3-parameter non-linear regression and plotted using GraphPad Prism software.

[0289] Example 6:

[0290] Activation of the first CD3 Fab and the second DIG Fab mediated by the recombinant binding proteins of the present invention, wherein the first and second fusion polypeptides bind to the same epitope on the target cell

[0291] The multiple pairs of recombinant binding proteins according to the invention having the domain arrangement as shown in Figure 1 are expressed as described in Example 2.

[0292] Figure 8 The principle of activating two effector domains after the first fusion polypeptide and the second fusion polypeptide bind via their first antigen-binding domain and second antigen-binding domain to the same epitope on the surface of the target cell is shown. The dissociation of the VH / VL chains of the scFab fragment of the precursor molecule is triggered by a partially labile interface in the first fusion polypeptide and the second fusion polypeptide. Two active antigen-binding Fab fragments are generated from precursors that are very close in the case of being driven by attracting charges (conjugate 1: VH, Q39E (precursor A); VL, Q38K (precursor B)) or (conjugate 2: VL, Q38E (precursor A); VH, Q39K (precursor B)).

[0293] Light Chain Heavy Chain Heavy Chain - scFab Fusion <her2>(Dig)(CD3)[A] SEQ ID NO:01 SEQ ID NO:02 SEQ ID NO:13 <her2>(Dig)(CD3)[B] SEQ ID NO:01 SEQ ID NO:02 SEQ ID NO:14 <her2>(CD3)(Dig)[A] SEQ ID NO:01 SEQ ID NO:02 SEQ ID NO:15 <her2>(CD3)(Dig)[B] SEQ ID NO:01 SEQ ID NO:02 SEQ ID NO:16 <her2>(Nada)(CD3)[A] SEQ ID NO:01 SEQ ID NO:02 SEQ ID NO:03 <her2>(Nada)(CD3)[B] SEQ ID NO:01 SEQ ID NO:02 SEQ ID NO:04 <her2>(CD3)(Nada)[A] SEQ ID NO:01 SEQ ID NO:02 SEQ ID NO:17 <her2>(CD3)(Nada)[B] SEQ ID NO:01 SEQ ID NO:02 SEQ ID NO:18

[0294] To demonstrate that two functional effector domains can be formed using the concept of the present invention, a first fusion polypeptide and a second fusion polypeptide comprising an antigen-binding domain that specifically binds to Her2 are used to provide multiple pairs of recombinant binding proteins, and the first effector domain is an anti-CD3 antigen-binding moiety (see Figure 8 "conjugate 1"), while the second effector domain is a digoxin (Dig) binding entity (see Figure 8 "conjugate 2"). Combining the VH or VL of the CD3 conjugate in the first fusion polypeptide and the second fusion polypeptide with the VL or VH of the Dig conjugate renders the two binding entities non-functional. Targeted strand exchange on SK-BR-3 cells expressing Her2 activates the two binding entities, thereby generating a CD3 binding entity and a Dig binding entity on the surface of the target cell. Depending on the position of the CD3 and Dig variable regions, the CD3 and Dig binding functions of the cell-binding prodrug are proximal to the IgG hinge region (defined herein as 'internal conjugate') or distal, i.e., at the N-terminus of the product (defined herein as 'external conjugate'). Figure 9 and 10 The nomenclature for the middle molecules is as follows: <target>(internal conjugate)(external conjugate)[prodrug A / B]. The function of the CD3 conjugate was demonstrated in T cell activation assays of different combinations of these recombinant binding proteins evaluated using the SK-BR-3 cell line expressing Her2, as described in the examples above. The formation of a functional Dig binding conjugate was evaluated by detecting the Dig binding function on the cells with fluorescently labeled digoxin. The results of these experiments confirmed that the prodrug assembled on the target cell into a product that binds CD3 as well as Dig ( Figure 9 and 10 ). This result also demonstrates that active CD3 binders and active Dig binders can be generated at both internal and external locations.

[0295] Example 7:

[0296] Activation of a first CD3 Fab and a second CD28 Fab mediated by the recombinant binding protein of the present invention, wherein the first fusion polypeptide and the second fusion polypeptide bind to the same epitope on the target cell

[0297] To assess CD28-mediated T cell co-stimulation, the expression of the inventive polypeptides with Figure 8 Four sets of recombinant binding protein pairs targeting HER2 with the domain arrangement shown. Co-stimulation of T cells via CD28 signaling (also known as signal 2 in the T cell activation pathway) is dependent on initial T cell receptor activation, such as by CD3 cross-linking (PMID 23470321).

[0298] In the first two pairs of recombinant binding proteins prepared as controls, the CD3 VH / VL domains were paired with non-binding VH / VL "nada" dummy domains, allowing chain exchange to generate efficient CD3 binders in either internal or external positions.

[0299] In the other two pairs of recombinant binding proteins, the CD3 VH / VL domains were paired with the CD28 VH / VL domains so that chain exchange resulted in effective CD3 and CD28 binders at either internal or external positions. The CD28 binder used in this example was derived from TGN1412, clone 5.11A (PMID12707299, US20040092718A1). The CD3 binder used in this example was 40G5c (US10174124B2).

[0300] Use the following molecules:

[0301] In one set of recombinant binding proteins, the first fusion polypeptide comprises an antigen binding domain that specifically binds to HER2, and the second fusion polypeptide comprises an effector domain consisting of half of an anti-CD3 antigen binding site in the "outer position" paired with either half of a Nada or CD28 binding site in the "inner position".

[0302] Light Chain Heavy Chain Heavy Chain - scFab Fusion <her2>(NADA)(CD3)[A] SEQ ID NO:01 SEQ ID NO:02 SEQ ID NO:03 <her2>(NADA)(CD3)[B] SEQ ID NO:01 SEQ ID NO:02 SEQ ID NO:04 <her2>(CD28)(CD3)[A] SEQ ID NO:01 SEQ ID NO:02 SEQ ID NO:19 <her2>(CD28)(CD3)[B] SEQ ID NO:01 SEQ ID NO:02 SEQ ID NO:20

[0303] In another group of recombinant binding proteins, the first fusion polypeptide comprises an antigen binding domain that specifically binds to HER2, and the second fusion polypeptide comprises an effector domain consisting of half of an anti-CD3 antigen binding site in the "inner position" paired with either half of a NADA or CD28 binding site in the "outer position".

[0304] Light Chain Heavy Chain Heavy Chain - scFab Fusion <her2>(CD3)(NADA)[A] SEQ ID NO:01 SEQ ID NO:02 SEQ ID NO:21 <her2>(CD3)(NADA)[B] SEQ ID NO:01 SEQ ID NO:02 SEQ ID NO:22 <her2>(CD3)(CD28)[A] SEQ ID NO:01 SEQ ID NO:02 SEQ ID NO:23 <her2>(CD3)(CD28)[B] SEQ ID NO:01 SEQ ID NO:02 SEQ ID NO:24

[0305] To assess CD28 signaling, HER2-positive SK-BR-3 cells were co-cultured with Jurkat IL-2 promoter cells (Promega, catalog number J1631). These engineered Jurkat T cells express low levels of luciferase in response to T cell activation through CD3-mediated TCR clustering. After CD28 co-stimulation, they express higher levels of luciferase. Thus, these reporter cells integrate CD3 and CD28 signals into an overall luminescence readout. CD28 signaling can be evaluated by comparing the signals obtained from the control CD3 / nada construct with those obtained from the dual-activated CD3 / CD28 construct.

[0306] Figure 11A Examples of T cell co-stimulation by simultaneous CD28 receptor activation and CD3 cross-linking are shown. CD28-mediated T cell co-stimulation was observed using two recombinant binding pairs according to the invention (both having CD28 in internal and external positions). Co-stimulation depends on the presence of two fusion polypeptides and thus on strand exchange, as individual fusion proteins alone do not cause any T cell activation in the absence of their respective counterparts ( Figure 11B ).

[0307] These results indicate that the recombinant binding proteins according to the invention can be used to activate two antigen-binding functions on target cells.

[0308] Example 8:

[0309] Activation of the first IL2-RγFab and the second IL-2RβFab mediated by the recombinant binding proteins of the invention, wherein the first fusion polypeptide and the second fusion polypeptide bind to the same antigen on the target cell

[0310] Additional precursor molecules were prepared to demonstrate that the domain arrangement applied to the recombinant binding proteins of the invention is generally applicable and independent of antigen-binding specificity.

[0311] Another application of the prodrug approach that activates two functions is the antibody-mediated activation of cell surface receptors. For certain receptors, ligand-induced activation of intracellular pathways is conferred by structural changes and / or heterodimerization of receptor subunits on the cell surface. Such events naturally triggered by the corresponding receptor ligands can also be achieved in some cases by antibodies that link and / or modulate receptor subunits. To mimic ligand function, receptor-binding antibodies or bispecific antibodies must bind to the appropriate positions in the appropriate receptor subunits in a suitable form. Due to this complexity, it is very challenging to identify molecules that meet all these parameters and thus trigger receptor signaling.

[0312] Thus, in another example, recombinant binding proteins were generated that are capable of forming two effector domains that separately target IL2-Rγ and IL-2Rβ, respectively. The overall arrangement of the recombinant binding proteins is as Figure 8 and Figure 12A shown.

[0313] Figure 12A A diagram of the recombinant binding protein used in this example is shown in , which undergoes strand exchange to activate the β and γ subunits of IL-2R. The PD1 conjugate was used as the antigen-binding domain to achieve targeted cell surface accumulation of the precursor molecule.

[0314] For these molecules, the applied IL-2Rβ conjugates are Mik-β1 (J Immunol. July 15, 1993; 151(2):1075-85) and Cl.4 (internal), and the IL-2Rγ conjugates are Cl.3 and Cl.1 (both internal). The VH-VL prodrug combinations derived from these conjugates cover Mik-β1 (VH)-Cl.3 (VL), Cl.4 (VH)-Cl.1 (VL), Cl.4 (VH)-Cl.3 (VL), and, as complementary counterparts, Mik-β1 (VL)-Cl.3 (VH), Cl.4 (VL)-Cl.1 (VH), Cl.4 (VL)-Cl.3 (VH). The targeting arms of each complementary group of prodrugs contain the PD-1 conjugate 0376 (heavy chain of SEQ ID NO:25, light chain of SEQ ID NO:26) or the PD-1 conjugate 1040 (heavy chain of SEQ ID NO:30, light chain of SEQ ID NO:31), which bind to two different epitopes on PD-1.

[0315] Pairs of recombinant binding proteins according to the invention having the domain arrangement as Figure 12A shown were expressed as described in Example 2. Each club-containing heavy chain contained in the pair of recombinant proteins contains a different PD-1 antigen-binding domain.

[0316] Provide a first set of recombinant binding proteins (prodrug A), wherein the first fusion polypeptide and the second fusion polypeptide comprise antigen-binding domains that specifically bind to different epitopes on human PD-1, and the effector domain is a split anti-IL-2Rβ binder at an external position and a split anti-IL-2Rγ binder at an internal position.

[0317] Use the following molecules:

[0318] Name Light Chain Heavy Chain Heavy Chain - scFab Fusion XVV142 SEQ ID NO:25 SEQ ID NO:26 SEQ ID NO:27 XVV143 SEQ ID NO:25 SEQ ID NO:26 SEQ ID NO:28 XVV144 SEQ ID NO:25 SEQ ID NO:26 SEQ ID NO:29

[0319] In addition, provide a second set of recombinant binding proteins (prodrug B), wherein the first fusion polypeptide and the second fusion polypeptide comprise antigen-binding domains that specifically bind to a second different epitope on human PD-1, and the effector domain is a complementary split anti-IL-2Rβ binder at an external position and a complementary split anti-IL-2Rγ binder at an internal position.

[0320] Use the following molecules:

[0321] Name Light chain Heavy chain Heavy chain - scFab fusion XVV146 SEQ ID NO:30 SEQ ID NO:31 SEQ ID NO:32 XVV147 SEQ ID NO:30 SEQ ID NO:31 SEQ ID NO:33 XVV148 SEQ ID NO:30 SEQ ID NO:31 SEQ ID NO:34

[0322] To assess the formation after accumulation of the functional IL-2R agonist on cells expressing PD-1, a HEK-Blue IL-2 transactivation assay was performed after co-incubation of the precursor molecule with CHO-K1 cells expressing PD-1 or with PD-1-negative CHO-K1 control cells (for the assay schematic, see Figure 12B ).

[0323] Briefly, 2.5x10 4 CHO-K1 cells overexpressing human PD-1 or 2.5x10 4 parental CHO-K1 cells were seeded into flat-bottom 96-well plates. The next day, 5x10 4 HEK-Blue IL-2 reporter cells (InvivoGen) were added to the plates, and then the corresponding precursor molecules were serially diluted. The plates were incubated at 37 °C, 5% CO2, and 80% relative humidity for 22 hours. After incubation, 20 μl of the supernatant was added to 180 μl of QuantiBlue solution (InvivoGen, rep-qbs2), and the reaction was incubated at 37 °C for 45 minutes. IL-2R activation was quantified by measuring the absorbance at 640 nm using a Tecan Infinite F200 Pro microplate reader. The data were analyzed using GraphPad Prism software and fitted with a three-parameter non-linear regression.

[0324] The results of these analyses are shown in Figure 13 Shown in. The inactive PD-1-targeting precursor molecule (with respect to IL-2R activation) can be converted into an active IL-2 receptor agonist after accumulating on cells expressing PD-1 ( Figure 13 A). Figure 13 B shows that prodrug activation is target-specific because co-incubation with parental CHO-K1 cells that do not express PD-1 generates little IL-2R-dependent signal under otherwise identical conditions.

Claims

1. A recombinant binding protein pair, comprising (a) A first fusion polypeptide comprising (i) a first antigen-binding domain capable of binding to a target antigen, (ii) a first part of an effector domain, and (iii) a first complementary domain capable of associating with the first part of the effector domain, wherein the first part of the effector domain and the first complementary domain are linked via a peptide linker; and (b) A second fusion polypeptide comprising (i) a second antigen-binding domain capable of binding to a target antigen, (ii) a second part of the effector domain, and (iii) a second complementary domain capable of associating with the second part of the effector domain, wherein the first part of the effector domain and the first complementary domain are linked via a peptide linker; wherein the first part of the effector domain and the second part of the effector domain are capable of associating with each other to form a functional effector domain, characterized in that The effector domain is an antibody Fab fragment.

2. The recombinant binding protein pair according to claim 1, wherein the first part of the effector domain is an antibody heavy chain polypeptide, and the first complementary domain is an antibody light chain polypeptide, and wherein the second part of the effector domain is an antibody light chain polypeptide, and the second complementary domain is an antibody heavy chain polypeptide.

3. The recombinant binding protein pair according to one of the preceding claims, wherein the first part of the effector domain comprises a variable heavy chain domain having a Q39E mutation, and wherein the first complementary domain comprises a variable light chain domain having a Q38E mutation, wherein the second part of the effector domain comprises a variable light chain domain having a Q38K mutation, and wherein the second complementary domain comprises a variable heavy chain domain having a Q39K mutation.

4. The recombinant binding protein pair according to one of the preceding claims, wherein the first part of the effector domain and the first complementary domain are contained in a single-chain Fab fragment, and wherein the second part of the effector domain and the second complementary domain are contained in a single-chain Fab fragment.

5. The recombinant binding protein pair according to one of claims 5 or 6, wherein in the single-chain Fab fragment of the first fusion protein, the VH and VL domains are exchanged with each other, and wherein in the single-chain Fab fragment of the second fusion protein, the CH1 and CL domains are exchanged with each other.

6. The recombinant binding protein pair according to one of claims 5 or 6, wherein the single-chain Fab fragment of the first fusion protein comprises a VH domain, a CL domain, a peptide linker, a VL domain, and a CH1 domain in the N-terminal to C-terminal direction, and wherein the single-chain Fab fragment of the second fusion protein comprises a VL domain, a CH1 domain, a peptide linker, a VH domain, and a CL domain in the N-terminal to C-terminal direction.

7. The recombinant binding protein pair according to one of the preceding claims, wherein the first antigen-binding domain is an antibody Fab fragment.

8. The recombinant binding protein pair according to one of the preceding claims, wherein the second antigen-binding domain is an antibody Fab fragment.

9. The recombinant binding protein pair according to one of the preceding claims, wherein the first fusion polypeptide and the second fusion polypeptide comprise a (heterodimeric) Fc domain.

10. The recombinant binding protein pair according to one of the preceding claims, wherein the first fusion polypeptide and the second fusion polypeptide comprise a heterodimeric Fc domain, the heterodimeric Fc domain comprising two CH3 domains, wherein one of the CH3 domains comprises the mutations S354C and T366W, and the other CH3 domain comprises the mutations Y349C, L368A, and Y407V.

11. The recombinant binding protein pair according to one of the preceding claims, wherein the first fusion polypeptide is a recombinant antibody composed of the following three polypeptides: (i) an antibody light chain, and (ii) an antibody heavy chain, wherein the variable domains of the antibody light chain and the antibody heavy chain form the first antigen-binding domain, and (iii) an antibody heavy chain / light chain fusion polypeptide, the antibody heavy chain / light chain fusion polypeptide comprising an antibody light chain comprising the first part of the effector domain fused via a peptide linker to an antibody heavy chain comprising the first complementary domain, wherein the C-terminus of the antibody light chain is fused to the N-terminus of the antibody heavy chain; and wherein the second fusion polypeptide is a recombinant antibody composed of the following three polypeptides: (i) an antibody light chain, and (ii) an antibody heavy chain, wherein the variable domains of the antibody light chain and the antibody heavy chain form the second antigen-binding domain, and (iii) an antibody heavy chain / light chain fusion polypeptide, the antibody heavy chain / light chain fusion polypeptide comprising an antibody light chain comprising the second part of the effector domain fused via a peptide linker to an antibody heavy chain comprising the second complementary domain, wherein the C-terminus of the antibody light chain is fused to the N-terminus of the antibody heavy chain.

12. The recombinant binding protein pair according to one of the preceding claims, wherein the effector domain is an antibody-binding domain.

13. A recombinant binding protein pair according to one of the preceding claims, which comprises two effector domains.

14. A recombinant binding protein pair according to one of the preceding claims, wherein the effector domain specifically binds to an activated T cell antigen.

15. A recombinant binding protein pair according to claim 13 or 14, wherein the recombinant binding protein comprises two effector domains, and the two effector domains specifically bind to two different T cell antigens.

16. A method for forming a functional effector domain from a recombinant binding protein pair according to one of the preceding claims, wherein the first antigen-binding domain and the second antigen-binding domain of the recombinant binding protein specifically bind to an epitope on the surface of a target cell, the method comprising contacting the recombinant binding protein pair with the target cell under conditions that permit the first fusion polypeptide and the second fusion polypeptide to bind to the target cell.

17. A pharmaceutical preparation, which comprises: a recombinant binding protein pair according to any one of the preceding claims; and a pharmaceutically acceptable carrier.

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