Production of antibody-derived polypeptides by polypeptide chain exchange

Abstract

To provide a method for producing a multispecific antigen-binder by polypeptide chain exchange.SOLUTION: The present invention provides sets of heterodimeric polypeptides and uses thereof, e.g. for the generation of multispecific antigen-binders by exchange of polypeptide chains. One embodiment of the invention relates to a set of heterodimeric polypeptides of the invention, wherein the first and / or second antigen binding moiety is an antibody fragment. Another aspect of the invention is a method for producing a heterodimeric polypeptide comprising the steps of a) contacting a first heterodimeric precursor polypeptide and a second heterodimeric precursor polypeptide according to the invention to form a third heterodimeric polypeptide comprising at least one polypeptide from the first heterodimeric precursor polypeptide and at least one polypeptide from the second heterodimeric polypeptide, and b) recovering the third heterodimeric polypeptide.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a set of heterodimeric polypeptides and their use for generating multispecific antigen binders, for example by exchange of polypeptide chains. [Background technology]

[0002] Background of the Invention Providing the optimal combination of antigen binders in a multispecific antibody from a wide variety of possible antigen binders is a difficult task, as each resulting multispecific antibody must be provided individually by recombinant methods. Alternative methods for screening multiple multispecific antibodies are desirable.

[0003] Labrijn, AF, et al. disclosed the efficient generation of stable bispecific IgG1s by controlled Fab arm exchange (Proc. Natl. Acad. Sci. USA 110 (2013) 5145-5150 (Non-Patent Document 1)). Briefly, two monospecific precursor molecules with IgG-like domain configurations containing point mutations in the CH3 domains are contacted to cause exchange of polypeptide chains and form bispecific product molecules that also have IgG-like domain configurations. As a result, the precursor and product molecules are of the same size, making isolation of the product molecules from unreacted precursor molecules and analysis of the product molecules difficult.

[0004] The unpublished prior art PCT / EP2018 / 078675 (Patent Document 1) and PCT / EP2018 / 079523 (Patent Document 2) disclose methods for generating multispecific antigen binders from two different precursor molecules by exchanging polypeptide chains. Both precursor molecules are heterodimeric polypeptides with asymmetric domain configurations. Both precursor molecules contain CH3 domains modified according to the "knob-into-hole" technique (WO 96 / 027011 (Patent Document 3); Ridgway, JB, et al., Protein Eng. 9 (1996) 617-621 (Non-Patent Document 2); and Merchant, AM, et al., Nat. Biotechnol. 16 (1998) 677-681 (Non-Patent Document 3)), and further contain destabilizing mutations arranged in an asymmetric pattern. In each precursor molecule, only one of the CH3 domains contains such destabilizing mutations. Upon exchange of polypeptide chains, two product molecules are formed, each containing polypeptides from each of the precursor molecules. The precursor and product molecules have different domain compositions. PCT / EP2018 / 078675 (Patent Document 1) and PCT / EP2018 / 079523 (Patent Document 2) disclose the amino acid positions within the CH3 / CH3 interface of the precursor molecules that are replaced.

[0005] However, there remains a need for additional methods for generating multispecific antigen binders by polypeptide chain exchange. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] PCT / EP2018 / 078675 [Patent Document 2] PCT / EP2018 / 079523 [Patent Document 3] International Publication No. 96 / 027011 [Non-patent literature]

[0007] [Non-Patent Document 1] Labrijn,AF,et al.,Proc.Natl.Acad.Sci.USA 110(2013)5145-5150 [Non-patent document 2] Ridgway, JB, et al., Protein Eng. 9(1996)617-621 [Non-patent document 3] Merchant, AM, et al., Nat. Biotechnol. 16(1998)677-681 Summary of the Invention

[0008] The present invention provides - a first heterodimeric precursor polypeptide comprising at least two polypeptide chains comprising CH3 domains, wherein the two polypeptide chains comprising CH3 domains associate with each other via the CH3 domains to form a heterodimer, one of the CH3 domains comprising a knob mutation and the other CH3 domain comprising a hole mutation; wherein the first heterodimeric precursor polypeptide comprises a first antigen-binding portion, and at least a portion of the first antigen-binding portion is located on one of the two polypeptide chains comprising a CH3 domain; and - a second heterodimeric precursor polypeptide comprising at least two polypeptide chains comprising CH3 domains, wherein the two polypeptide chains comprising CH3 domains associate with each other via the CH3 domains to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; wherein the second heterodimeric precursor polypeptide comprises a second antigen-binding portion, and at least a portion of the second antigen-binding portion is located on one of the two polypeptide chains comprising the CH3 domain; and and a set of heterodimeric precursor polypeptides comprising: where: A) i) in the first heterodimeric precursor polypeptide, a polypeptide chain comprising a CH3 domain that includes a knob mutation comprises at least a portion of a first antigen-binding portion, and in the second heterodimeric precursor polypeptide, a polypeptide chain comprising a CH3 domain that includes a hole mutation comprises at least a portion of a second antigen-binding portion; or ii) in the first heterodimeric precursor polypeptide, the polypeptide chain comprising the CH3 domain comprising the hole mutation comprises at least a portion of a first antigen-binding portion, and in the second heterodimeric precursor polypeptide, the polypeptide chain comprising the CH3 domain comprising the knob mutation comprises at least a portion of a second antigen-binding portion; and B) i) a CH3 domain of a first heterodimeric precursor polypeptide comprising a knob mutation and a CH3 domain of a second heterodimeric precursor polypeptide comprising a hole mutation; or ii) a CH3 domain of a first heterodimeric precursor polypeptide comprising a hole mutation, and a CH3 domain of a second heterodimeric precursor polypeptide comprising a knob mutation. contains the following amino acid substitutions, numbered according to the Kabat numbering system: - CH3 domain with hole mutations · Replacement of S354 with a hydrophobic amino acid; · Replacement of D356 with a positively charged amino acid; Replacement of E357 with a positively charged or hydrophobic amino acid; replacement of D356 with a positively charged amino acid and replacement of E357 with a positively charged or hydrophobic amino acid; · Replacement of S364 with a hydrophobic amino acid; ·Replacement of A368 with a hydrophobic amino acid; Replacement of E392 with a negatively charged amino acid; · Replacement of T394 with a hydrophobic amino acid; · Replacement of D399 with a hydrophobic amino acid and replacement of S400 with a positively charged amino acid; · Replacement of D399 with a hydrophobic amino acid and replacement of F405 with a positively charged amino acid; replacement of V407 with a hydrophobic amino acid; and Replacement of K409 with a negatively charged amino acid; and Replacement of K439 with a negatively charged amino acid and comprising at least one amino acid substitution selected from the group - CH3 domain with knob mutations · Replacement of Q347 with a positively charged amino acid and replacement of K360 with a negatively charged amino acid; · Replacement of Y349 with a negatively charged amino acid; replacement of L351 with a hydrophobic amino acid and replacement of E357 with a hydrophobic amino acid; · Replacement of S364 with a hydrophobic amino acid; · Replacement of W366 with a hydrophobic amino acid and replacement of K409 with a negatively charged amino acid; ·Replacement of L368 with a hydrophobic amino acid; · Replacement of K370 with a negatively charged amino acid; · Replacement of K370 with a negatively charged amino acid and replacement of K439 with a negatively charged amino acid; · Replacement of K392 with a negatively charged amino acid; · Replacement of T394 with a hydrophobic amino acid; · Replacement of V397 with a hydrophobic amino acid; Replacement of D399 with a positively charged amino acid and replacement of K409 with a negatively charged amino acid; · Replacement of S400 with a positively charged amino acid; ·F405W; Y407W; and Replacement of K439 with a negatively charged amino acid The amino acid sequence of the present invention comprises at least one amino acid substitution selected from the group consisting of:

[0009] One embodiment of the present invention relates to a set of heterodimeric polypeptides of the present invention, wherein i) the CH3 domain comprising the knob mutation of the first heterodimeric precursor polypeptide comprises a cysteine ​​mutation, and the CH3 domain comprising the hole mutation of the second heterodimeric precursor polypeptide comprises a cysteine ​​mutation, or ii) the CH3 domain comprising the hole mutation of the first heterodimeric precursor polypeptide comprises a cysteine ​​mutation, and the CH3 domain comprising the knob mutation of the second heterodimeric precursor polypeptide comprises a cysteine ​​mutation.

[0010] One embodiment of the present invention relates to a set of heterodimeric polypeptides of the invention, wherein the first antigen-binding moiety and / or the second antigen-binding moiety is an antibody fragment.

[0011] Another aspect of the present invention is a method for producing a heterodimeric polypeptide, the method comprising: a) contacting a first heterodimeric precursor polypeptide according to the present invention with a second heterodimeric precursor polypeptide to form a third heterodimeric polypeptide comprising at least one polypeptide from the first heterodimeric precursor polypeptide and at least one polypeptide from the second heterodimeric polypeptide; and b) recovering the third heterodimeric polypeptide Includes.

[0012] One embodiment of the present invention relates to a method of producing the heterodimeric polypeptides of the invention, wherein a second heterodimeric precursor polypeptide comprises an antigen-binding portion that specifically binds to a second antigen, and a third heterodimeric polypeptide comprises an antigen-binding portion that specifically binds to a first antigen and an antigen-binding portion that specifically binds to a second antigen.

[0013] Another aspect of the present invention is a heterodimeric polypeptide obtained by the method according to the present invention.

[0014] Another aspect of the present invention is a method for identifying a multispecific heterodimeric polypeptide, the method comprising: a) A method for producing a heterodimeric polypeptide of the invention, comprising: - a first heterodimeric precursor polypeptide from the plurality of first heterodimeric precursor polypeptides comprising an antigen-binding portion that specifically binds to a first antigen; and - a second heterodimeric precursor polypeptide from the plurality of second heterodimeric precursor polypeptides comprising an antigen-binding portion that specifically binds to a second antigen; and generating a plurality of multispecific heterodimeric polypeptides by providing each combination of b) individually identifying the desired properties of each multispecific heterodimeric polypeptide from the plurality of multispecific heterodimeric polypeptides generated in step a); and c) selecting the multispecific heterodimeric polypeptide Includes.

[0015] Another aspect of the invention is a set of heterodimeric precursor polypeptides according to the invention for use as a medicament.

[0016] The invention disclosed herein provides precursor polypeptides that can undergo polypeptide chain exchange to generate product polypeptides, thereby generating multispecific antigen-binding polypeptides. Multispecific antigen-binding polypeptides can be generated by combining two precursor polypeptides containing antigen-binding portions that specifically bind different antigens, or by activating antigen-binding sites through polypeptide chain exchange that results in the association of antibody variable domains that specifically bind to antigens, or both.

[0017] The present invention provides methods for producing product polypeptides of different sizes from precursor polypeptides, thereby allowing the desired product polypeptide to be easily separated from unreacted precursor polypeptides. The methods and sets of polypeptides according to the present invention are suitable for providing product polypeptides, such as bispecific or multispecific antibodies, using in vitro polypeptide chain exchange and avoiding recombinant expression of the product polypeptides. This makes the methods and sets of polypeptides of the present invention suitable for screening product polypeptides formed from multiple precursor polypeptides, allowing, for example, the identification of advantageous combinations of antigen-binding moieties. The methods and sets of polypeptides of the present invention can be advantageously used to provide antigen-binding polypeptides for therapeutic or diagnostic use.

[0018] Therapeutic application of the precursor polypeptide sets of the present invention may allow for the production of desired product polypeptides at target sites, thus reducing off-target effects of the product polypeptides. [Brief explanation of the drawings]

[0019] [Figure 1]Exemplary structures of heterodimeric precursor polypeptides and corresponding product polypeptides formed upon polypeptide chain exchange. The first heterodimeric precursor polypeptide comprises three polypeptide chains: 1. A first heavy chain polypeptide comprising, from N- to C-terminal, antibody domains VH, CH1, hinge, CH2, and CH3. The CH3 domain comprises a knob mutation and a cysteine ​​mutation, but does not comprise a destabilizing mutation. 2. A second heavy chain polypeptide comprising, from N- to C-terminal, antibody domains hinge, CH2, and CH3. A tagging moiety is fused to the C-terminus of the CH3 domain. The CH3 domain comprises a hole mutation and a destabilizing mutation. 3. A light chain polypeptide comprising, from N- to C-terminal, antibody domains VL and CL. The VH and VL domains form an antigen-binding site that specifically binds to a first antigen. The heavy chain polypeptides of the first heterodimeric precursor polypeptide associate with each other via their CH3 domains. The hinge region comprises an interchain disulfide bond. The second heterodimeric precursor polypeptide comprises three polypeptide chains: 1. A first heavy chain polypeptide comprising, from N- to C-terminal, antibody domains VH, CH1, hinge, CH2, and CH3. The CH3 domain comprises a hole mutation and a cysteine ​​mutation, but does not comprise a destabilizing mutation. 2. A second heavy chain polypeptide comprising, from N- to C-terminal, antibody domains hinge, CH2, and CH3. A tagging moiety is fused to the C-terminus of the CH3 domain. The CH3 domain comprises a knob mutation and a destabilizing mutation. 3. A light chain polypeptide comprising, from N- to C-terminal, antibody domains VL and CL. The VH and VL domains form an antigen-binding site that specifically binds to a second antigen. The heavy chain polypeptides of the second heterodimeric precursor polypeptide associate with each other via their CH3 domains. The hinge region comprises an interchain disulfide bond. In the presence of a reducing agent, the interchain disulfide bond in the hinge region is reduced, thereby destabilizing the heterodimer formed by the first and second heavy chain polypeptides and supporting exchange of polypeptide chains, resulting in the formation of a heterodimeric product polypeptide.The first product polypeptide comprises two antigen-binding sites, one from a first heterodimeric precursor polypeptide and one from a second heterodimeric precursor polypeptide. The first product polypeptide comprises first heavy chain polypeptides from first and second heterodimeric precursor polypeptides associated via their CH3 domains. Both heavy chain polypeptides in the first product polypeptide comprise CH3 domains that do not contain destabilizing mutations. Both CH3 domains contain cysteine ​​mutations that interact to support the formation of heterodimeric product polypeptides. In this example, the second product polypeptide, which does not comprise an antigen-binding site, comprises a second heavy chain polypeptide from the first heterodimeric precursor polypeptide and a second heavy chain polypeptide from the second heterodimeric precursor polypeptide. Both heavy chain polypeptides are associated via their CH3 domains. Both CH3 domains contain destabilizing mutations that interact to support the formation of heterodimeric product polypeptides. The product polypeptides comprise a tagging moiety that allows for purification by tag-specific chromatography. [Figure 2]1 shows exemplary structures of heterodimeric precursor polypeptides and corresponding product polypeptides formed by polypeptide chain exchange, resulting in activation of the antigen-binding site. The first heterodimeric precursor polypeptide comprises three polypeptide chains: 1. A first heavy chain polypeptide comprising, from N- to C-terminal, antibody domains VH, CH1, a peptide connector, a VH domain derived from a first antibody, and CH3. The CH3 domain comprises a knob mutation and no destabilizing mutation. 2. A second heavy chain polypeptide comprising, from N- to C-terminal, the following antibody domains: a VL domain derived from a second antibody and CH3. The CH3 domain comprises a hole mutation and a destabilizing mutation. 3. A light chain polypeptide comprising, from N- to C-terminal, antibody domains VL and CL. The N-terminal VH domain from the first heavy chain polypeptide and the VL domain from the light chain polypeptide form an antigen-binding site that specifically binds to a target antigen. The heavy chain polypeptides of the first heterodimeric precursor polypeptide associate with each other via their CH3 domains. No interchain disulfide bond is formed between the first heavy chain polypeptide and the second heavy chain polypeptide. A VH domain-VL domain pair is formed between the VH domain derived from the first antibody and the VL domain from the second heavy chain polypeptide. Both variable domains associate with each other but do not form an antigen-binding site that specifically binds to the antigen. The second heterodimeric precursor polypeptide comprises three polypeptide chains: 1. A first heavy chain polypeptide comprising, from N- to C-terminal, antibody domains VH, CH1, a peptide connector, a VL domain derived from the first antibody, and CH3. The CH3 domain comprises a hole mutation and no destabilizing mutation. 2. A second heavy chain polypeptide comprising, from N- to C-terminal, the following antibody domains: a VH domain derived from a third antibody and CH3. The CH3 domain comprises a knob mutation and a destabilizing mutation. 3. A light chain polypeptide comprising, from N- to C-terminal, antibody domains VL and CL. The N-terminal VH domain of the first heavy chain polypeptide and the VL domain from the light chain polypeptide form an antigen-binding site that specifically binds to a target antigen, and the heavy chain polypeptides of the second heterodimeric precursor polypeptide associate with each other via their CH3 domains.No interchain disulfide bond is formed between the first heavy chain polypeptide and the second heavy chain polypeptide. A VH and VL domain pair is formed between the VL domain derived from the first antibody and the VH domain from the second heavy chain polypeptide. Both variable domains associate with each other but do not form an antigen-binding site that specifically binds to the antigen. Upon exchange of polypeptide chains, a heterodimeric product polypeptide is formed. The first product polypeptide contains two antigen-binding sites from the precursor polypeptides: one from the first heterodimeric precursor polypeptide and one from the second heterodimeric precursor polypeptide. The first product polypeptide contains first heavy chain polypeptides from the first and second heterodimeric precursor polypeptides associated via their CH3 domains. Both heavy chain polypeptides contained in the first product polypeptide contain CH3 domains that do not contain destabilizing mutations. Association of the first heavy chain polypeptide from the first and second heterodimeric precursor polypeptides results in the formation of a pair of a VH domain derived from the first antibody and a VL domain derived from the first antibody, which forms an antigen-binding site that specifically binds to the first antigen. This antigen-binding site is not present in either precursor polypeptide and is formed (activated) solely by the exchange of polypeptide chains. The second product polypeptide comprises a second heavy chain polypeptide from the first heterodimeric precursor polypeptide and a second heavy chain polypeptide from the second heterodimeric precursor polypeptide. Both heavy chain polypeptides are associated via their CH3 domains. Both CH3 domains contain destabilizing mutations that interact to support the formation of the heterodimeric product polypeptide. Association of the second heavy chain polypeptide from the first and second heterodimeric precursor polypeptides results in the formation of a novel pair of a VH domain and a VL domain. Both variable domains are associated in the second product polypeptide. [Figure 3]1 shows exemplary structures of heterodimeric precursor polypeptides and corresponding product polypeptides formed by polypeptide chain exchange, resulting in activation of the antigen-binding site. The first heterodimeric precursor polypeptide comprises three polypeptide chains: 1. A first heavy chain polypeptide comprising, from N- to C-terminus, antibody domains VH, CH1, a peptide connector, a VH domain derived from a first antibody, CH2, and CH3. The CH3 domain comprises a knob mutation and no destabilizing mutation. 2. A second heavy chain polypeptide comprising, from N- to C-terminus, the following antibody domains: a VL domain derived from a second antibody, CH2, and CH3. The CH3 domain comprises a hole mutation and a destabilizing mutation. 3. A light chain polypeptide comprising, from N- to C-terminus, antibody domains VL and CL. The N-terminal VH domain from the first heavy chain polypeptide and the VL domain from the light chain polypeptide form an antigen-binding site that specifically binds to a target antigen. The heavy chain polypeptides of the first heterodimeric precursor polypeptide associate with each other via their CH3 domains. No interchain disulfide bond is formed between the first heavy chain polypeptide and the second heavy chain polypeptide. A VH domain-VL domain pair is formed between the VH domain derived from the first antibody and the VL domain from the second heavy chain polypeptide. Both variable domains associate with each other but do not form an antigen-binding site that specifically binds to the antigen. The second heterodimeric precursor polypeptide comprises three polypeptide chains: 1. A first heavy chain polypeptide comprising, from N- to C-terminal, antibody domains VH, CH1, a peptide connector, a VL domain derived from the first antibody, CH2, and CH3. The CH3 domain comprises a hole mutation and no destabilizing mutation. 2. A second heavy chain polypeptide comprising, from N- to C-terminal, the following antibody domains: a VH domain derived from a third antibody, CH2, and CH3. The CH3 domain comprises a knob mutation and a destabilizing mutation. 3. A light chain polypeptide comprising, from N- to C-terminal, antibody domains VL and CL. The N-terminal VH domain of the first heavy chain polypeptide and the VL domain from the light chain polypeptide form an antigen-binding site that specifically binds to a target antigen.The heavy chain polypeptides of the second heterodimeric precursor polypeptide are associated with each other via their CH3 domains. No interchain disulfide bond is formed between the first heavy chain polypeptide and the second heavy chain polypeptide. A VH and VL domain pair is formed between the VL domain derived from the first antibody and the VH domain from the second heavy chain polypeptide. Both variable domains associate with each other but do not form an antigen-binding site that specifically binds to the antigen. Upon exchange of polypeptide chains, a heterodimeric product polypeptide is formed. The first product polypeptide contains two antigen-binding sites from the precursor polypeptides: one from the first heterodimeric precursor polypeptide and one from the second heterodimeric precursor polypeptide. The first product polypeptide contains first heavy chain polypeptides from the first and second heterodimeric precursor polypeptides associated with each other via their CH3 domains. Both heavy chain polypeptides contained in the first product polypeptide contain CH3 domains that do not contain destabilizing mutations. Association of the first heavy chain polypeptide from the first and second heterodimeric precursor polypeptides results in the formation of a pair of a VH domain derived from the first antibody and a VL domain derived from the first antibody, which forms an antigen-binding site that specifically binds to the first antigen. This antigen-binding site is not present in either precursor polypeptide and is formed (activated) solely by the exchange of polypeptide chains. The second product polypeptide comprises a second heavy chain polypeptide from the first heterodimeric precursor polypeptide and a second heavy chain polypeptide from the second heterodimeric precursor polypeptide. Both heavy chain polypeptides are associated via their CH3 domains. Both CH3 domains contain destabilizing mutations that interact to support the formation of the heterodimeric product polypeptide. Association of the second heavy chain polypeptide from the first and second heterodimeric precursor polypeptides results in the formation of a novel pair of a VH domain and a VL domain. Both variable domains are associated in the second product polypeptide. [Figure 4]Exemplary domain configurations of a first heterodimeric precursor polypeptide. The knob-into-hole mutations, destabilizing mutations, and cysteine ​​mutations shown are the same as those in Figures 1-3. The precursor polypeptide can contain one or more antigen-binding sites, which can be located at the C-terminus or N-terminus. While the figure shows a precursor polypeptide containing a Fab fragment, it is understood that the precursor polypeptide can contain other suitable antigen-binding moieties. The hinge region disulfide bond shown is exemplary, and the hinge region disulfide bond may or may not be present depending on the desired application. The cysteine ​​mutation shown is exemplary and is not required for the precursor polypeptides of the invention. A) A precursor polypeptide containing an N-terminal Fab fragment and a CH2 domain. B) A precursor polypeptide containing C- and N-terminal Fab fragments and a CH2 domain. C) A precursor polypeptide containing a C-terminal Fab fragment and a CH2 domain. D) A precursor polypeptide with an activatable binding site containing an N-terminal Fab fragment and a VH domain. E) A precursor polypeptide with an activatable binding site containing C- and N-terminal Fab fragments and a VH domain. F) A precursor polypeptide having an activatable binding site comprising a C-terminal Fab fragment and a VH domain. G) A precursor polypeptide having an activatable binding site comprising a VH domain and a CH2 domain comprising an N-terminal Fab fragment. H) A precursor polypeptide having an activatable binding site comprising a VH domain and a CH2 domain comprising N- and C-terminal Fab fragments. I) A precursor polypeptide having an activatable binding site comprising a VH domain and a CH2 domain comprising a C-terminal Fab fragment. J) A precursor polypeptide having an activatable binding site at its C-terminus comprising a VH domain and a CH2 domain comprising an N-terminal Fab fragment. K) A precursor polypeptide having an activatable binding site comprising an N-terminal Fab fragment and a VL domain. [Figure 5]Exemplary domain configurations of a second heterodimeric precursor polypeptide. The knob-into-hole mutations, destabilizing mutations, and cysteine ​​mutations shown are the same as those in Figures 1-3. The precursor polypeptide can contain one or more antigen-binding sites, which can be located at the C-terminus or the N-terminus. While the figure shows a precursor polypeptide containing a Fab fragment, it is understood that the precursor polypeptide can contain other suitable antigen-binding moieties. The hinge region disulfide bond shown is exemplary, and the hinge region disulfide bond may or may not be present depending on the desired application. The cysteine ​​mutation shown is exemplary and is not required for the precursor polypeptides of the invention. A) A precursor polypeptide containing an N-terminal Fab fragment and a CH2 domain. B) A precursor polypeptide containing C- and N-terminal Fab fragments and a CH2 domain. C) A precursor polypeptide containing a C-terminal Fab fragment and a CH2 domain. D) A precursor polypeptide with an activatable binding site containing an N-terminal Fab fragment and a VL domain. E) A precursor polypeptide with an activatable binding site containing C- and N-terminal Fab fragments and a VL domain. F) A precursor polypeptide having an activatable binding site comprising a C-terminal Fab fragment and a VL domain. G) A precursor polypeptide having an activatable binding site comprising a CH2 domain and a VL domain comprising an N-terminal Fab fragment. H) A precursor polypeptide having an activatable binding site comprising a CH2 domain and a VL domain comprising N- and C-terminal Fab fragments. I) A precursor polypeptide having an activatable binding site comprising a CH2 domain and a VL domain comprising a C-terminal Fab fragment. J) A precursor polypeptide having an activatable binding site at its C-terminus comprising a CH2 domain and a VL domain comprising an N-terminal Fab fragment. K) A precursor polypeptide having an activatable binding site comprising an N-terminal Fab fragment and a VH domain. DETAILED DESCRIPTION OF THE INVENTION

[0020] 1.Definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless the context otherwise requires, the singular shall include the plural and the plural shall include the singular. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art. Generally, the nomenclature and techniques used in connection with biochemistry, enzymology, molecular biology, cell biology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art.

[0021] The terms "a," "an," and "the" generally include plural referents unless otherwise specified.

[0022] Unless otherwise defined herein, the term "consisting of" is intended to include the term "consisting of."

[0023] Providing an alternative using "or" designates mutually exclusive alternatives unless otherwise specified.

[0024] As used herein, the term "antigen-binding moiety" refers to a moiety that specifically binds to a target antigen. This term includes antibodies as well as other natural (e.g., receptors, ligands) or synthetic (e.g., DARPins) molecules that can specifically bind to a target antigen.

[0025] The term "antibody" is used in the broadest sense and encompasses a variety of 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.

[0026] As used herein, the term "binding site" or "antigen-binding site" refers to the region or regions of an antigen-binding portion to which an antigen actually binds. When the antigen-binding portion is an antibody, the antigen-binding site comprises the antibody heavy chain variable domain (VH) and / or the antibody light chain variable domain (VL), or a VH / VL pair. An antigen-binding site derived from an antibody that specifically binds to a target antigen can be obtained from a) a known antibody that specifically binds to the antigen, or b) a novel antibody or antibody fragment obtained by a novel immunization method using, inter alia, an antigen protein or nucleic acid or fragment thereof, or by phage display methods.

[0027] When derived from an antibody, the antigen-binding site of an antibody of the present invention can contain six complementarity-determining regions (CDRs), which contribute to varying degrees to the affinity of the binding site for the antigen. There are three heavy chain variable domain CDRs (CDRH1, CDRH2, and CDRH3) and three light chain variable domain CDRs (CDRL1, CDRL2, and CDRL3). The extent of the CDRs and framework regions (FRs) is determined by comparison with a compiled database of amino acid sequences in which these regions are defined according to variability in the sequences. The scope of the present invention also includes functional antigen-binding sites composed of fewer CDRs (i.e., binding specificity determined by three, four, or five CDRs). For example, less than the complete set of six CDRs may be sufficient for binding.

[0028] As used herein, the term "valency" refers to the presence of a specific number of binding sites within an antibody molecule. A natural antibody, for example, has two binding sites and is bivalent. Thus, the term "trivalent" refers to the presence of three binding sites within an antibody molecule.

[0029] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact 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.

[0030] "Specificity" refers to the selective recognition of a particular epitope of an antigen by an antigen-binding moiety, e.g., an antibody. For example, natural antibodies are 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 to two or more different epitopes (e.g., two, three, four, or more different epitopes). The epitopes can be located on the same or different antigens. An example of a multispecific antibody is a "bispecific antibody" that binds to two different epitopes. When an antibody possesses more than one specificity, the recognized epitope can be associated with a single antigen or more than one antigen.

[0031] An epitope is a region of an antigen to which an antigen-binding moiety, e.g., an antibody, binds. The term "epitope" includes any polypeptide determinant capable of specific binding to an antibody or antigen-binding moiety. In some embodiments, epitopic determinants include chemically active surface groupings of molecules, e.g., amino acids, glycan side chains, phosphoryls, or sulfonyls, and in some embodiments may have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0032] As used herein, the terms "binding" and "specific binding" refer to the binding of an antibody or antigen-binding portion thereof to an epitope of an antigen in an in vitro assay, preferably a plasmon resonance assay (BIAcore®, GE-Healthcare Uppsala, Sweden), using purified wild-type antigen. In some embodiments, an antibody or antigen-binding portion thereof is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0033] The binding affinity of an antibody to an antigen is expressed by the term k a (rate constant for association of antibody from the antibody / antigen complex), k D (dissociation constant), and K D (k DIn one embodiment, binding or specifically binding is defined by 10 -8 Binding affinity (K D ) and in one embodiment, 10 -8 ~10 -13 This means that an antigen-binding moiety, particularly an antibody binding site, has a binding affinity of 10 mol / l for each antigen for which it is specific. -8 Binding affinity (K D ), e.g. 10 -8 ~10 -13 Binding affinity (K D ) and in one embodiment, 10 -9 ~10 -13 Binding affinity (K D ) and binds specifically to it.

[0034] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR) and three complementarity-determining regions (CDR). (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by screening a library of complementary VL or VH domains, respectively, using a VH or VL domain from an antibody that binds the antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0035] The term "constant domain" or "constant region" as used within this application refers to the sum of the domains of an antibody other than the variable region. The constant region is not directly involved in antigen binding, but exhibits various effector functions.

[0036] Depending on the amino acid sequence of the constant region of their heavy chains, antibodies are divided into "classes": IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses: IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2. The heavy chain constant regions that correspond to the different classes of antibodies are called α, δ, ε, λ, and μ, respectively. The light chain constant regions (CL), found in all five antibody classes, are called κ (kappa) and λ (lambda).

[0037] As used herein, a "constant domain" is preferably of human origin, 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).

[0038] In wild-type antibodies, the "hinge region" is a flexible stretch of amino acids in the center of the heavy chains of the IgG and IgA immunoglobulin classes, which, when formed between two heavy chains, connects the two heavy chains by a disulfide bond, i.e., an "interchain disulfide bond." The hinge region of human IgG1 is generally defined as extending from about Glu216 or about Cys226 to about Pro230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). Disulfide bond formation in the hinge region can be avoided by deleting cysteine ​​residues in the hinge region or by substituting them with other amino acids, such as serine.

[0039] The "light chains" of antibodies from any vertebrate species can be assigned to one of two different types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domain. Wild-type light chains generally contain two immunoglobulin domains, usually one variable domain (VL) that is important for binding to antigen, and a constant domain (CL).

[0040] There are several different types of "heavy chains" that define the class or isotype of an antibody. Wild-type heavy chains typically contain a series of immunoglobulin domains, along with one variable domain (VH) that is important for antigen binding, and several constant domains (CH1, CH2, CH3, etc.).

[0041] The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to 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.

[0042] The "CH2 domain" of a human IgG Fc region typically extends from approximately amino acid residue 231 to approximately amino acid residue 340. Multispecific antibodies lack a CH2 domain. "Lack of a CH2 domain" means, according to the present invention, that the antibody does not contain a CH2 domain.

[0043] A "CH3 domain" comprises the stretch of residues C-terminal to the CH2 domain in the Fc region (i.e., from about amino acid residue 341 to about amino acid residue 447 of IgG). A "CH3 domain" herein is a variant CH3 domain in which the amino acid sequence of a native CH3 domain has been subjected to at least one distinct amino acid substitution (i.e., modification of the amino acid sequence of the CH3 domain) to promote heterodimerization of two CH3 domains facing each other within a multispecific antibody.

[0044] Typically, in heterodimerization techniques known in the art, the CH3 domains of one heavy chain and the CH3 domains of the other heavy chain are engineered to be complementary to each other, such that a heavy chain containing one engineered CH3 domain can no longer homodimerize with another heavy chain of the same structure, thereby forcing the heavy chain containing one engineered CH3 domain to heterodimerize with another heavy chain containing a complementary engineered CH3 domain.

[0045] One heterodimerization technique known in the art is the so-called "knobs-into-holes" technique, which is described in detail with multiple examples in, for example, WO 96 / 027011; Ridgway, JB, et al., Protein Eng. 9 (1996) 617-621; Merchant, AM, et al., Nat. Biotechnol. 16 (1998) 677-681; and WO 98 / 050431, which are incorporated herein by reference. In the "knobs-into-holes" technique, specific amino acids in each CH3 domain are engineered within the interface formed between the two CH3 domains in the tertiary structure of an antibody to create a protuberance (a "knob") in one of the CH3 domains and a cavity (a "hole") in the other CH3 domain. In the tertiary structure of a multispecific antibody, a protuberance introduced into one CH3 domain can be positioned within a cavity introduced into the other CH3 domain.

[0046] In combination with the knob-into-hole substitution, additional interchain disulfide bonds can be introduced into the CH3 domains to further stabilize the heterodimerized polypeptide (Merchant, AM, et al., Nature Biotech. 16 (1998) 677-681). Such interchain disulfide bonds can be formed, for example, by introducing the following amino acid substitutions into the CH3 domains: D399C in one CH3 domain and K39k2C in the other; Y349C in one CH3 domain and S354C in the other; Y349C in one CH3 domain and E356C in the other; Y349C in one CH3 domain and E357C in the other; L351C in one CH3 domain and S354C in the other; or T394C in one CH3 domain and V397C in the other. As used herein, a "cysteine ​​mutation" refers to the substitution of one amino acid with a cysteine ​​in a CH3 domain, which is capable of forming an interchain disulfide bond with another matching amino acid with a cysteine ​​in a second CH3 domain.

[0047] Beyond the "knobs-into-holes" technique described above, additional techniques for modifying CH3 domains to force heterodimerization are known in the art. These techniques, in particular those described in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954 and WO 2013 / 096291, are considered herein as alternatives to the "knob-into-hole technology" for the polypeptides provided by the present invention. All of these techniques involve complementary engineering of the CH3 domain by the introduction of amino acids of opposite charge or different side chain volume, thereby supporting heterodimerization.

[0048] The precursor polypeptides of the present invention contain, in only one of their CH3 domains, an amino acid substitution that "destabilizes the CH3 / CH3 interface," referred to herein as a "destabilizing mutation." These amino acid substitutions, by definition, are located only in one of the CH3 domains associated in the heterodimeric precursor polypeptide. In the CH3 domain, one or more amino acid positions known to interact within the CH3 / CH3 interface are replaced with amino acids with alternative side chain properties, as disclosed in the prior art related to the CH3-heterodimerization strategy described above. In contrast to heterodimerization strategies in which pairs of interacting amino acids within associated CH3 domains are typically replaced (i.e., one or more amino acid residues in one CH3 domain of the heterodimer and one or more amino acid residues in the other CH3 domain of the heterodimer), the destabilizing mutation is located only in one of the CH3 domains of the heterodimeric precursor polypeptides of the present invention. Exemplary amino acid substitutions that destabilize the CH3 / CH3 interface are listed in the "Destabilizing Mutations" section below. All exemplary amino acid substitutions specifically disclosed herein are positioned such that the substituted amino acid interacts at the CH3 / CH3 interface within the pair of CH3 domains.

[0049] As used herein, the term "polypeptide chain" refers to a linear organic polymer comprising multiple amino acids linked together via peptide bonds. One or more polypeptide chains form a "polypeptide" or a "protein," and the terms are used interchangeably herein. The heterodimeric precursor polypeptides provided as a set by 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. Because the first CH3 domain and the second CH3 domain contain amino acid substitutions via knob-into-hole technology, the two polypeptide chains form a "heterodimer," i.e., a dimer formed by two non-identical polypeptides.

[0050] The polypeptide chains contained in the heterodimeric polypeptides, i.e., the heterodimeric precursor polypeptide and the heterodimeric product polypeptide, contain one or two polypeptide domains. When the order of the polypeptide domains is indicated herein, it is indicated in the N-terminal to C-terminal direction.

[0051] Each heterodimeric precursor polypeptide comprises at least two polypeptide chains that include a CH3 domain.

[0052] When the antigen-binding moieties present in the two heterodimeric precursor polypeptides are antigen-binding sites derived from antibodies, e.g., antibody fragments, the polypeptide chain comprising the CH3 domain is also referred to herein as a "heavy chain polypeptide." In this case, the heterodimeric precursor polypeptide may also comprise a "light chain polypeptide," which typically comprises antibody variable domains and antibody constant domains, e.g., VL and CL.

[0053] The present invention provides a set comprising at least two polypeptides, the set comprising at least two heterodimeric "precursor" polypeptides. The precursor polypeptides are reacted to undergo polypeptide chain exchange with each other to form "product" polypeptides. The present invention also provides a method for producing heterodimeric polypeptides, i.e., heterodimeric product polypeptides, by contacting at least two heterodimeric precursor polypeptides. The contacting step can be carried out in any suitable manner that allows for polypeptide chain exchange, preferably in a suitable buffer. As referred to herein in the context of the present invention, "polypeptide chain exchange" refers to the exchange of polypeptide chains comprising CH3 domains between two heterodimeric (precursor) polypeptides. Polypeptide chain exchange occurs when two initially associated polypeptide chains comprising CH3 domains from precursor polypeptides dissociate, and at least one of the dissociated polypeptide chains forms a new heterodimer by association with an equally dissociated polypeptide chain comprising a CH3 domain derived from another precursor polypeptide. The mechanism of polypeptide chain exchange is also illustrated in Figures 1, 2, and 3.

[0054] An "isolated" heterodimeric polypeptide, e.g., an antibody, is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or greater than 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0055] As used herein, the amino acid positions of all heavy and light chain constant regions and domains 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 light chain constant domain CL and for the variable domains of the kappa and lambda 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) (see pages 647-660) is used, and for the constant heavy chain domains (CH1, hinge, CH2, and CH3), the Kabat EU index numbering system (see pages 661-723) is used. The amino acid positions provided herein are generally designated by:

[0056] Amino acid "substitutions," "replacements," or "mutations" (all terms used interchangeably herein) within the polypeptide chain are prepared by introducing appropriate nucleotide changes into the antibody DNA or by nucleotide synthesis. However, such modifications can only be performed to a very limited extent. For example, modifications may not alter the above-mentioned antibody properties, such as the IgG isotype or antigen binding, but may further improve recombinant production yield, protein stability, or facilitate purification. In some embodiments, antibody variants with one or more conservative amino acid substitutions are provided. A "double mutation," as referred to herein, means that both of the indicated amino acid substitutions are present in each polypeptide chain.

[0057] The term "amino acid" as used herein refers to an organic molecule having an amino moiety located alpha to a carboxyl group. Examples of amino acids include arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, and proline. The amino acids used are optionally in the L-form in each case. The terms "positively charged" and "negatively charged" amino acids refer to the amino acid side chain charge at pH 7.4. Amino acids have the following general side chain characteristics: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, Trp, Tyr, Phe; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic, i.e., negatively charged: Asp, Glu; (4) basic, i.e., positively charged: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro The items can be grouped according to:

[0058] (Table) Amino acids with specific properties TIFF2026004426000002.tif194129

[0059] As used herein, a "tagging moiety" refers to a peptide sequence genetically grafted onto a polypeptide chain for various purposes, e.g., to support purification. In one embodiment, the tagging moiety is an affinity tag. Thus, a polypeptide comprising said affinity tag can be purified by an appropriate affinity technique, e.g., affinity chromatography. Typically, the tagging moiety is fused to the C-terminus of the CH3 domain via a peptide connector. Typically, the peptide connector is composed of flexible amino acid residues such as glycine and serine. Thus, a typical peptide connector used to fuse a tagging moiety to a polypeptide is a glycine-serine linker, i.e., a peptide connector consisting of a pattern of glycine and serine residues.

[0060] As used herein, the term "purified" refers to polypeptides that have been removed from their natural environment or from a recombinant source, or otherwise isolated or separated, and are at least 60%, e.g., at least 80%, free from other components with which they are naturally associated, such as membranes and microsomes. Purification of antibodies (recovery of antibodies from host cell culture) is carried out by standard techniques, including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art, to eliminate cellular components or other contaminants, such as other cellular nucleic acids or proteins. See Ausubel, F., et al., ed., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987). Various methods for protein purification have been established and are widely used, such as affinity chromatography using microbial proteins (e.g., using affinity media such as KappaSelect or LambdaSelect for the purification of kappa or lambda isotype constant light chain domains), ion exchange chromatography (e.g., cation exchange (carboxymethyl resin), ion exchange (aminoethyl resin), and mixed-mode exchange), thiophilic adsorption (e.g., using beta-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g., using phenyl-Sepharose, aza-arenophilic resin, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g., using Ni(II)- and Cu(II)-affinity materials), size exclusion chromatography, and electrophoretic methods (e.g., gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, MA, Appl. Biochem. Biotech. 75 (1998) 93-102).

[0061] Polypeptides containing tagged moieties can be purified by "tag-specific affinity chromatography." Suitable methods for purifying tags are known in the art. Thus, polypeptides containing poly(his) tags can be purified, for example, by metal chelate affinity chromatography, particularly nickel chelate affinity chromatography.

[0062] As used herein, the term "peptide connector" refers to a peptide having an amino acid sequence, preferably of synthetic origin. Within the heterodimeric polypeptides used for the present invention, peptide connectors may be used to fuse additional polypeptide domains, such as antibody fragments, to the C-terminus or N-terminus of individual polypeptide chains. In one embodiment, the peptide connector is a peptide having an amino acid sequence at least 5 amino acids in length, in another embodiment 5 to 100 amino acids in length, and in another embodiment 10 to 50 amino acids in length. In one embodiment, the peptide connector is a glycine-serine linker. In one embodiment, the peptide connector is a peptide consisting of glycine and serine amino acid residues. In one embodiment, the peptide connector is (G x S) n or (G x S) n G m wherein G=glycine, S=serine, and x=3, n=3, 4, 5 or 6, m=0, 1, 2 or 3; or x=4, n=2, 3, 4 or 5, m=0, 1, 2 or 3] is.

[0063] In one embodiment, x = 4 and n = 2 or 3, and in another embodiment, x = 4 and n = 2. In one embodiment, the peptide connector is (G4S)2.

[0064] The term "valency" as used herein refers to the presence of a specific number of binding sites in an antigen-binding molecule. A natural antibody, for example, has two binding sites and is bivalent. Thus, the term "trivalent" refers to the presence of three binding sites in an antigen-binding molecule.

[0065] The polypeptides of the present invention are produced by recombinant means. Methods for the recombinant production of polypeptides, e.g., antibodies, are widely known in the art and include protein expression in prokaryotic and eukaryotic host cells, followed by isolation and purification of the polypeptide, usually to a pharmaceutically acceptable degree of purity. For expression of such proteins in host cells, nucleic acids encoding the respective 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 (supernatant or cells after lysis). General methods for the recombinant production of polypeptides, such as antibodies, are well known in the art and are described, for example, in the reviews of Makrides, SC, Protein Expr. Purif. 17(1999)183-202; Geisse, S., et al., Protein Expr. Purif. 8(1996)271-282; Kaufman, RJ, Mol. Biotechnol. 16(2000)151-161; Werner, RG, Drug Res. 48(1998)870-880.

[0066] Polypeptides produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of a polypeptide chain comprising a C-terminal CH3 domain. Thus, upon expression of a specific nucleic acid molecule encoding such a polypeptide chain, a polypeptide produced by a host cell may comprise a full-length polypeptide chain comprising a full-length CH3 domain, or may comprise a truncated variant of the full-length polypeptide chain (also referred to herein as a truncated variant polypeptide chain). This is also true when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447).

[0067] "Polynucleotide" or "nucleic acid," as used interchangeably herein, refer to a polymer of nucleotides of any length, and include 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 a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can also contain modification(s) performed after synthesis, such as conjugation to a label. Other types of modifications include, for example, "caps," substitutions of one or more analogs of naturally occurring nucleotides, internucleotide modifications such as those with uncharged bonds (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and those with charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those containing intercalators (e.g., acridine, psoralens, etc.), those containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylators, those with modified linkages (e.g., alpha-anomeric nucleic acids, etc.), and unmodified forms of polynucleotide(s). Additionally, any of the hydroxyl groups normally present in the sugar may be replaced, for example, with phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to provide for additional linkages to additional nucleotides, or conjugated to solid or semi-solid supports. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls can be derivatized to standard protecting groups.Polynucleotides may also contain analogous forms of ribose or deoxyribose sugars 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, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and basic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C) (optionally containing an ether (-O-) linkage), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all of the linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0068] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained within a cell that normally contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0069] An "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 said heterodimeric polypeptide, including such nucleic acid molecule(s) in a single vector or separate vectors, and including such nucleic acid molecule(s) present in one or more locations within a host cell.

[0070] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. The term includes vectors that function primarily for the insertion of DNA or RNA into a cell (e.g., chromosomal integration), replicating vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for the transcription and / or translation of DNA or RNA. Additionally, vectors that provide more than one of the recited functions are included.

[0071] An "expression vector" is a vector capable of directing the expression of nucleic acids to which they are covalently linked. Expression vectors, when introduced into an appropriate host cell, 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; thereby, the term "vector" in the context of transforming a host cell as described herein means "expression vector." An "expression system" generally refers to a suitable host cell comprised of an expression vector that can function to produce a desired expression product.

[0072] As used herein, "expression" refers to the process of transcribing a nucleic acid into mRNA and / or the process of subsequently translating the transcribed mRNA (also called a transcript) into a peptide or polypeptide. The transcript and the encoded polypeptide are individually or collectively referred to as the gene product. If the nucleic acid is derived from genomic DNA, expression in a eukaryotic cell may include splicing of the corresponding mRNA.

[0073] The term "transformation" as used herein refers to the process of transferring a vector or nucleic acid into a host cell. When cells without a rigid 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 for introducing DNA into cells, such as by nuclear injection or by protoplast fusion, may also be used. When prokaryotic cells or cells containing substantial cell wall structures are used, one method of transfection, for example, is the calcium treatment using calcium chloride, as described by Cohen, FN, et al., PNAS 69 (1972) 7110 et seq.

[0074] The term "host cell" as used in this application means any type of cell line that can be engineered to produce the polypeptides provided by the present invention.

[0075] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the terms "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where a separate designation is intended, it will be clear from the context.

[0076] Transient expression is described, for example, in Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. Cloning of variable domains is described, for example, in 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 by Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30 (1999) 71-83, and by Schlaeger, E.-J., J. Immunol. Methods 194 (1996) 191-199.

[0077] The term "pharmaceutical composition" refers to a preparation in which the biological activity of the active ingredient contained therein is effective and which does not contain additional components that are unacceptably toxic to the subject to which the composition is administered. The pharmaceutical compositions of the present invention can be administered by a variety of methods known in the art. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. To administer an antibody according to the present invention by a particular route of administration, it may be necessary to coat the antibody with or co-administer the antibody with a material that prevents its inactivation. For example, the heterodimeric polypeptide can be administered to a subject in an appropriate carrier, such as a liposome or diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.

[0078] The pharmaceutical composition comprises an effective amount of a heterodimeric polypeptide provided by the present invention. An "effective amount" of a drug, e.g., a heterodimeric polypeptide, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. In particular, an "effective amount" refers to an amount of a heterodimeric polypeptide of the present invention that, when administered to a subject, (i) treats or prevents a particular disease, condition, or disorder, (ii) reduces, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder, as described herein. A therapeutically effective amount will vary depending on the heterodimeric polypeptide molecule used, the condition being treated, the severity or 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.

[0079] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In a preferred embodiment, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion).

[0080] The pharmaceutical compositions according to the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured both by the above-mentioned sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, etc., in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0081] As used herein, the terms "parenteral administration" and "parenterally administered" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0082] Regardless of the selected route of administration, the compounds of the present invention, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present invention are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0083] The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention may be varied to obtain an amount of the active ingredient that is not toxic to the patient and is effective to achieve the desired therapeutic response for the particular patient, composition, and mode of administration. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, other drugs, compounds, and / or substances used in combination with the particular composition employed, the age, sex, weight, condition, general health, and medical history of the patient being treated, and similar factors well known in the medical arts.

[0084] The composition must be sterile and must be fluid to the extent that the composition is deliverable by syringe. In addition to water, in one embodiment the carrier is an isotonic buffered saline solution.

[0085] The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.

[0086] As used herein, "treatment" (and grammatical variations thereof, e.g., "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed prophylactically or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of disease progression, remission or palliation of disease symptoms, and recovery or improved prognosis. In some embodiments, the antibodies of the invention are used to delay disease onset or to slow disease progression.

[0087] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is human.

[0088] 2. Detailed Description of the Embodiments of the Invention The present invention provides precursor polypeptides applicable to in vitro production of product polypeptides, for example, by polypeptide chain exchange. Each precursor polypeptide contains a pair of CH3 domains, arranged in two separate polypeptide chains that associate with each other via the CH3 domains. The CH3 domains contain multiple amino acid substitutions. As a result, two polypeptide chains containing CH3 domains in a precursor polypeptide form heterodimers. The CH3 domains of the precursor polypeptides provided by the present invention contain at least two patterns of mutations with different functionalities. The first pattern of mutations supports heterodimerization of the two polypeptide chains containing CH3 domains, i.e., knob-into-hole mutations. Thus, the CH3 domain of one precursor polypeptide contains a knob mutation, and the CH3 domain of the other precursor polypeptide contains a hole mutation. The second pattern of mutations is one or more mutations provided only in one of the CH3 domains contained in the precursor polypeptide heterodimer, which destabilizes the interaction of two polypeptides containing CH3 domains. Thus, each precursor polypeptide contains one CH3 domain with a destabilizing mutation selected and positioned to support correct assembly of the product polypeptide upon exchange of polypeptide chains between precursor polypeptides.

[0089] precursor polypeptide In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a first heterodimeric precursor polypeptide comprising at least two polypeptide chains each comprising a CH3 domain, wherein the two polypeptide chains each comprising a CH3 domain associate with each other via the CH3 domain to form a heterodimer, wherein one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; wherein the first heterodimeric precursor polypeptide comprises a first antigen-binding portion, and at least a portion of the first antigen-binding portion is located on one of the two polypeptide chains comprising a CH3 domain; and b) a second heterodimeric precursor polypeptide comprising at least two polypeptide chains comprising CH3 domains, wherein the two polypeptide chains comprising CH3 domains associate with each other via the CH3 domains to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; a second heterodimeric precursor polypeptide, wherein the second heterodimeric precursor polypeptide comprises a second antigen-binding portion, and at least a portion of the second antigen-binding portion is located on one of the two polypeptide chains comprising the CH3 domain; and and providing a set of heterodimeric precursor polypeptides comprising: where: A) i) in the first heterodimeric precursor polypeptide, a polypeptide chain comprising a CH3 domain that includes a knob mutation comprises at least a portion of a first antigen-binding portion, and in the second heterodimeric precursor polypeptide, a polypeptide chain comprising a CH3 domain that includes a hole mutation comprises at least a portion of a second antigen-binding portion; or ii) within the first heterodimeric precursor polypeptide, a polypeptide chain comprising a CH3 domain comprising a hole mutation comprises at least a portion of a first antigen-binding portion, and within the second heterodimeric precursor polypeptide, a polypeptide chain comprising a CH3 domain comprising a knob mutation comprises at least a portion of a second antigen-binding portion; B) i) a CH3 domain of a first heterodimeric precursor polypeptide comprising a knob mutation and a CH3 domain of a second heterodimeric precursor polypeptide comprising a hole mutation; or ii) a CH3 domain of a first heterodimeric precursor polypeptide comprising a hole mutation, and a CH3 domain of a second heterodimeric precursor polypeptide comprising a knob mutation. contains an amino acid substitution that destabilizes the CH3 / CH3 interface, and the amino acid substitution is positioned such that the substituted amino acid interacts at the CH3 / CH3 interface within the pair of CH3 domains.

[0090] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a first heterodimeric precursor polypeptide comprising at least two polypeptide chains each comprising a CH3 domain, wherein the two polypeptide chains each comprising a CH3 domain associate with each other via the CH3 domain to form a heterodimer, wherein one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; wherein the first heterodimeric precursor polypeptide comprises a first antigen-binding portion, and at least a portion of the first antigen-binding portion is located on one of the two polypeptide chains comprising a CH3 domain; and b) a second heterodimeric precursor polypeptide comprising at least two polypeptide chains comprising CH3 domains, wherein the two polypeptide chains comprising CH3 domains associate with each other via the CH3 domains to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; a second heterodimeric precursor polypeptide, wherein the second heterodimeric precursor polypeptide comprises a second antigen-binding portion, and at least a portion of the second antigen-binding portion is located on one of the two polypeptide chains comprising the CH3 domain; and and providing a set of heterodimeric precursor polypeptides comprising: where: A) i) in the first heterodimeric precursor polypeptide, a polypeptide chain comprising a CH3 domain that includes a knob mutation comprises at least a portion of a first antigen-binding portion, and in the second heterodimeric precursor polypeptide, a polypeptide chain comprising a CH3 domain that includes a hole mutation comprises at least a portion of a second antigen-binding portion; or ii) within the first heterodimeric precursor polypeptide, a polypeptide chain comprising a CH3 domain comprising a hole mutation comprises at least a portion of a first antigen-binding portion, and within the second heterodimeric precursor polypeptide, a polypeptide chain comprising a CH3 domain comprising a knob mutation comprises at least a portion of a second antigen-binding portion; B) i) a CH3 domain of a first heterodimeric precursor polypeptide comprising a knob mutation and a CH3 domain of a second heterodimeric precursor polypeptide comprising a hole mutation; or ii) a CH3 domain of a first heterodimeric precursor polypeptide comprising a hole mutation, and a CH3 domain of a second heterodimeric precursor polypeptide comprising a knob mutation. contains the following amino acid substitutions (also referred to herein as "destabilizing mutations"), numbered according to the Kabat numbering system: - CH3 domain with hole mutations · Replacement of S354 with a hydrophobic amino acid; · Replacement of D356 with a positively charged amino acid; Replacement of E357 with a positively charged or hydrophobic amino acid; replacement of D356 with a positively charged amino acid and replacement of E357 with a positively charged or hydrophobic amino acid; · Replacement of S364 with a hydrophobic amino acid; ·Replacement of A368 with a hydrophobic amino acid; Replacement of E392 with a negatively charged amino acid; · Replacement of T394 with a hydrophobic amino acid; · Replacement of D399 with a hydrophobic amino acid and replacement of S400 with a positively charged amino acid; · Replacement of D399 with a hydrophobic amino acid and replacement of F405 with a positively charged amino acid; replacement of V407 with a hydrophobic amino acid; and Replacement of K409 with a negatively charged amino acid; and Replacement of K439 with a negatively charged amino acid at least one amino acid substitution selected from the group - CH3 domain with knob mutations · Replacement of Q347 with a positively charged amino acid and replacement of K360 with a negatively charged amino acid; · Replacement of Y349 with a negatively charged amino acid; replacement of L351 with a hydrophobic amino acid and replacement of E357 with a hydrophobic amino acid; · Replacement of S364 with a hydrophobic amino acid; · Replacement of W366 with a hydrophobic amino acid and replacement of K409 with a negatively charged amino acid; ·Replacement of L368 with a hydrophobic amino acid; · Replacement of K370 with a negatively charged amino acid; · Replacement of K370 with a negatively charged amino acid and replacement of K439 with a negatively charged amino acid; · Replacement of K392 with a negatively charged amino acid; · Replacement of T394 with a hydrophobic amino acid; · Replacement of V397 with a hydrophobic amino acid; · replacement of D399 with a positively charged amino acid and replacement of K409 with a negatively charged amino acid;· replacement of S400 with a positively charged amino acid; ·F405W; ·Y407W; and Replacement of K439 with a negatively charged amino acid The amino acid sequence of the present invention comprises at least one amino acid substitution selected from the group consisting of:

[0091] In another aspect, the present invention provides a first heterodimeric precursor polypeptide comprising at least two polypeptide chains comprising CH3 domains, wherein the two polypeptide chains comprising CH3 domains associate with each other via the CH3 domains to form a heterodimer, wherein one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation, and the first heterodimeric precursor polypeptide comprises a first antigen-binding moiety, at least a portion of which is located in one of the two polypeptide chains comprising the CH3 domains; and one of the CH3 domains (but not the other CH3 domain) comprises the following amino acid substitution (i.e., a destabilizing mutation), numbered according to the Kabat numbering system: - the CH3 domain with the hole mutation comprises at least one amino acid substitution, i.e., a destabilizing mutation, selected from the group consisting of replacement of S354 with a hydrophobic amino acid; replacement of D356 with a positively charged amino acid; replacement of E357 with a positively charged or hydrophobic amino acid; replacement of D356 with a positively charged amino acid and replacement of E357 with a positively charged or hydrophobic amino acid; replacement of S364 with a hydrophobic amino acid; replacement of A368 with a hydrophobic amino acid; replacement of E392 with a negatively charged amino acid; replacement of T394 with a hydrophobic amino acid; replacement of D399 with a hydrophobic amino acid and replacement of S400 with a positively charged amino acid; replacement of D399 with a hydrophobic amino acid and replacement of F405 with a positively charged amino acid; replacement of V407 with a hydrophobic amino acid; and replacement of K409 with a negatively charged amino acid; and replacement of K439 with a negatively charged amino acid; or - CH3 domain with knob mutations: replacement of Q347 with a positively charged amino acid and replacement of K360 with a negatively charged amino acid; replacement of Y349 with a negatively charged amino acid; replacement of L351 with a hydrophobic amino acid and replacement of E357 with a hydrophobic amino acid; replacement of S364 with a hydrophobic amino acid; replacement of W366 with a hydrophobic amino acid and replacement of K409 with a negatively charged amino acid; replacement of L368 with a hydrophobic amino acid; replacement of K370 with a negatively charged amino acid; replacement of replacement of K370 with a negatively charged amino acid and replacement of K439 with a negatively charged amino acid; replacement of K392 with a negatively charged amino acid; replacement of T394 with a hydrophobic amino acid; replacement of V397 with a hydrophobic amino acid; replacement of D399 with a positively charged amino acid and replacement of K409 with a negatively charged amino acid; replacement of S400 with a positively charged amino acid; F405W; Y407W; and replacement of K439 with a negatively charged amino acid.

[0092] In another aspect, the present invention provides a second heterodimeric precursor polypeptide comprising at least two polypeptide chains comprising CH3 domains, wherein the two polypeptide chains comprising CH3 domains associate with each other via the CH3 domains to form a heterodimer, wherein one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation, and the second heterodimeric precursor polypeptide comprises a second antigen-binding moiety, at least a portion of which is located in one of the two polypeptide chains comprising the CH3 domains; and one of the CH3 domains (but not the other CH3 domain) comprises the following amino acid substitution (i.e., a destabilizing mutation), numbered according to the Kabat numbering system: - the CH3 domain with the hole mutation comprises at least one amino acid substitution, i.e., a destabilizing mutation, selected from the group consisting of replacement of S354 with a hydrophobic amino acid; replacement of D356 with a positively charged amino acid; replacement of E357 with a positively charged or hydrophobic amino acid; replacement of D356 with a positively charged amino acid and replacement of E357 with a positively charged or hydrophobic amino acid; replacement of S364 with a hydrophobic amino acid; replacement of A368 with a hydrophobic amino acid; replacement of E392 with a negatively charged amino acid; replacement of T394 with a hydrophobic amino acid; replacement of D399 with a hydrophobic amino acid and replacement of S400 with a positively charged amino acid; replacement of D399 with a hydrophobic amino acid and replacement of F405 with a positively charged amino acid; replacement of V407 with a hydrophobic amino acid; and replacement of K409 with a negatively charged amino acid; and replacement of K439 with a negatively charged amino acid; or - CH3 domain with knob mutations: replacement of Q347 with a positively charged amino acid and replacement of K360 with a negatively charged amino acid; replacement of Y349 with a negatively charged amino acid; replacement of L351 with a hydrophobic amino acid and replacement of E357 with a hydrophobic amino acid; replacement of S364 with a hydrophobic amino acid; replacement of W366 with a hydrophobic amino acid and replacement of K409 with a negatively charged amino acid; replacement of L368 with a hydrophobic amino acid; replacement of K370 with a negatively charged amino acid; replacement of replacement of K370 with a negatively charged amino acid and replacement of K439 with a negatively charged amino acid; replacement of K392 with a negatively charged amino acid; replacement of T394 with a hydrophobic amino acid; replacement of V397 with a hydrophobic amino acid; replacement of D399 with a positively charged amino acid and replacement of K409 with a negatively charged amino acid; replacement of S400 with a positively charged amino acid; F405W; Y407W; and replacement of K439 with a negatively charged amino acid.

[0093] In yet another aspect, the present invention provides the use of a first heterodimeric precursor polypeptide according to the present invention in combination with a second heterodimeric polypeptide according to the present invention for the formation of a heterodimeric product polypeptide, hi one embodiment, the first heterodimeric precursor polypeptide according to the present invention is used in combination with the second heterodimeric polypeptide according to the present invention for the formation of a heterodimeric product polypeptide by exchange of polypeptide chains.

[0094] In yet another aspect, the present invention provides the use of a second heterodimeric precursor polypeptide according to the present invention in combination with a first heterodimeric polypeptide according to the present invention for the formation of a heterodimeric product polypeptide, hi one embodiment, the second heterodimeric precursor polypeptide according to the present invention is used in combination with the first heterodimeric polypeptide according to the present invention to form a heterodimeric product polypeptide by exchange of polypeptide chains.

[0095] In another aspect, the present invention provides the use of a first heterodimeric precursor polypeptide according to the present invention in a set of heterodimeric precursor polypeptides according to the present invention. In another aspect, the present invention provides the use of a second heterodimeric precursor polypeptide according to the present invention in a set of heterodimeric precursor polypeptides according to the present invention.

[0096] Another aspect of the present invention is the use of a first heterodimeric precursor polypeptide according to the present invention in a method for producing a heterodimeric polypeptide according to the present invention.Another aspect of the present invention is the use of a second heterodimeric precursor polypeptide according to the present invention in a method for producing a heterodimeric polypeptide according to the present invention.

[0097] Another aspect of the present invention is the use of a first heterodimeric precursor polypeptide according to the present invention in a method for identifying a multispecific heterodimeric polypeptide according to the present invention.Another aspect of the present invention is the use of a second heterodimeric precursor polypeptide according to the present invention in a method for identifying a multispecific heterodimeric polypeptide according to the present invention.

[0098] Another aspect of the present invention is the use of a set of heterodimeric precursor polypeptides according to the present invention in a method for producing a heterodimeric polypeptide according to the present invention. Another aspect of the present invention is the use of a set of heterodimeric precursor polypeptides according to the present invention in a method for identifying a multispecific heterodimeric polypeptide according to the present invention.

[0099] In one embodiment, the following applies to the first and second heterodimeric precursor polypeptides: - if the CH3 domain with the knob mutation contains the mutation E357K, then the CH3 domain with the hole mutation does not contain the mutation K370E; - If the CH3 domain with the knob mutation contains the mutation D356K, then the CH3 domain with the hole mutation does not contain the mutation K439E.

[0100] In other words, the following applies in accordance with one embodiment of the present invention for the precursor polypeptides provided by the present invention: - if the CH3 domain with the knob mutation contains the amino acid substitution E357K, then the CH3 domain with the hole mutation contains K at position 370; - If the CH3 domain with the knob mutation contains the amino acid substitution D356K, the CH3 domain with the hole mutation contains a K at position 439.

[0101] In one embodiment, the first heterodimeric precursor polypeptide comprises at least two (in one embodiment, exactly two) polypeptide chains comprising a CH3 domain; one of the two polypeptide chains comprising a CH3 domain comprises at least a portion of a (first) antigen-binding portion that specifically binds to an antigen; and the other of the two polypeptide chains comprising a CH3 domain does not comprise an antigen-binding portion that specifically binds to an antigen. In one embodiment, the second heterodimeric precursor polypeptide comprises at least two (in one embodiment, exactly two) polypeptide chains comprising a CH3 domain; one of the two polypeptide chains comprising a CH3 domain comprises at least a portion of a (first) antigen-binding portion that specifically binds to an antigen; and the other of the two polypeptide chains comprising the other CH3 domain does not comprise an antigen-binding portion that specifically binds to an antigen. In one embodiment, the first heterodimeric precursor polypeptide comprises at least two (in one embodiment, exactly two) polypeptide chains comprising a CH3 domain, wherein one of the two polypeptide chains comprising a CH3 domain comprises at least a portion of a (first) antigen-binding moiety that specifically binds to an antigen; and the other of the two polypeptide chains comprising a CH3 domain does not comprise an antigen-binding moiety that specifically binds to an antigen; the second heterodimeric precursor polypeptide comprises at least two (in one embodiment, exactly two) polypeptide chains comprising a CH3 domain, wherein one of the two polypeptide chains comprising a CH3 domain comprises at least a portion of a (first) antigen-binding moiety that specifically binds to an antigen; and the other of the two polypeptide chains comprising a CH3 domain does not comprise an antigen-binding moiety that specifically binds to an antigen. In other words, according to this embodiment of the present invention, one or more functional antigen-binding moieties are located only in one of the two polypeptide chains comprising a CH3 domain, and no functional antigen-binding moiety is located in the other polypeptide chain comprising a CH3 domain. This polypeptide chain is also referred to herein as a "dummy polypeptide." In one embodiment, the dummy polypeptide associates only with another polypeptide chain that comprises a CH3 domain, ie, in a heterodimer, and not with another (eg, third) polypeptide chain.The dummy polypeptide may comprise a portion of the antigen-binding moiety, e.g., an antibody variable domain, that does not participate in a functional antigen-binding site within the heterodimeric precursor polypeptide. One advantage of combining such a construct, e.g., a dummy polypeptide comprising a CH3 domain, with a polypeptide chain comprising a CH3 domain that participates in the formation of one or more functional antigen-binding sites, is that the product polypeptide formed upon exchange of polypeptide chains will differ in size from the heterodimeric precursor molecule, thereby allowing for the improvement of the product polypeptide(s) from unreacted precursor polypeptides.

[0102] As shown, in each of the heterodimeric precursor polypeptides, one of the polypeptide chains comprising a CH3 domain comprises a CH3 domain with a knob mutation, and the other polypeptide chain comprising a CH3 domain comprises a CH3 domain with a hole mutation. Upon exchange of polypeptide chains, the polypeptide chain comprising a CH3 domain with a knob mutation from the first precursor polypeptide forms a heterodimer with the polypeptide chain comprising a CH3 domain with a hole mutation from the second precursor polypeptide (i.e., a first heterodimeric product polypeptide), and the polypeptide chain comprising a CH3 domain with a hole mutation from the first precursor polypeptide forms a heterodimer with the polypeptide chain comprising a CH3 domain with a knob from the second precursor polypeptide (i.e., a second heterodimeric product polypeptide).

[0103] As shown, one CH3 domain of a first heterodimeric precursor polypeptide contains one or more destabilizing mutations as described above, and the other CH3 domain of the first heterodimeric precursor polypeptide does not contain a destabilizing mutation; one CH3 domain of a second heterodimeric polypeptide contains one or more destabilizing mutations as described above, and the other CH3 domain of the second heterodimeric precursor polypeptide does not contain a destabilizing mutation. The destabilizing mutations present in the precursor polypeptides are arranged so that they are present in the same product polypeptide after polypeptide chain exchange. Thus, in one of the precursor polypeptides, one or more destabilizing mutations are arranged in the CH3 domain containing a knob mutation, and in the other precursor polypeptide, one or more destabilizing mutations are arranged in the CH3 domain containing a hole mutation.

[0104] In one embodiment, in a first heterodimeric precursor polypeptide, a polypeptide chain comprising a CH3 domain with a knob mutation comprises at least a portion of a first antigen-binding portion, and in a second heterodimeric precursor polypeptide, a polypeptide chain comprising a CH3 domain with a hole mutation comprises at least a portion of a second antigen-binding portion.

[0105] In one embodiment, in a first heterodimeric precursor polypeptide, a polypeptide chain comprising a CH3 domain with a hole mutation comprises at least a portion of a first antigen-binding portion, and in a second heterodimeric precursor polypeptide, a polypeptide chain comprising a CH3 domain with a knob mutation comprises at least a portion of a second antigen-binding portion.

[0106] In one embodiment, the heterodimeric precursor polypeptide comprises exactly two polypeptide chains that comprise a CH3 domain.

[0107] In one embodiment, the CH3 domain comprising destabilizing mutations comprises one, two or three destabilizing mutations. In one embodiment, the CH3 domain comprising destabilizing mutations comprises one or two destabilizing mutations.

[0108] The present invention includes heterodimeric precursor polypeptides in which the knob mutation is replaced by a destabilizing mutation in the CH3 domain having the knob mutation. For example, the destabilizing mutation may be located at position 366 of the CH3 domain having the knob mutation, e.g., T366W. Thus, the heterodimeric precursor polypeptide contains a destabilizing mutation, i.e., a hydrophobic amino acid but no tryptophan (W) at position 366 of the CH3 domain. Nevertheless, heterodimeric precursor polypeptides having such substitutions are considered to be encompassed by the present invention.

[0109] The present invention also encompasses heterodimeric precursor polypeptides in which one or more of the mutations in the CH3 domain having a hole mutation are replaced with a destabilizing mutation. For example, the destabilizing mutation may be located at position 368 of the CH3 domain having a hole mutation, e.g., T366S L368A Y407V. Thus, the heterodimeric precursor polypeptide contains a destabilizing mutation, i.e., contains another hydrophobic amino acid but not an alanine (A) at position 368 of the CH3 domain. In another example, the destabilizing mutation may be located at position 407 of the CH3 domain having a hole mutation, e.g., T366S L368A Y407V. Thus, the heterodimeric precursor polypeptide contains a destabilizing mutation, i.e., contains another hydrophobic amino acid but not a valine (V) at position 407 of the CH3 domain. Nevertheless, such heterodimeric precursor polypeptides having such one or more substitutions are considered to be encompassed by the present invention.

[0110] In one embodiment of the present invention, no interchain disulfide bond is formed between the two polypeptide chains comprising a CH3 domain of the first heterodimeric polypeptide. In one embodiment of the present invention, no interchain disulfide bond is formed between the two polypeptide chains comprising a CH3 domain of the second heterodimeric polypeptide. In one embodiment of the present invention, no interchain disulfide bond is formed between the two polypeptide chains comprising a CH3 domain of the first heterodimeric polypeptide and the second heterodimeric polypeptide. Heterodimeric precursor polypeptides lacking an interchain disulfide bond between the two polypeptide chains comprising a CH3 domain can undergo polypeptide chain exchange in the absence of a reducing agent. Thus, heterodimeric precursor polypeptides lacking an interchain disulfide bond between the polypeptide chains comprising a CH3 domain are particularly suitable for applications where the presence of a reducing agent is impossible or undesirable, such as therapeutic applications.

[0111] A) Amino acid substitutions in the CH3 domain The precursor polypeptides provided by the present invention contain amino acid substitutions in their CH3 domains.

[0112] Knob-into-Hole Mutation In one embodiment, the knob mutation contained in the first heterodimeric precursor polypeptide is identical to the knob mutation contained in the second heterodimeric precursor polypeptide.

[0113] In one embodiment, the knob mutation is T366W. In one embodiment, the hole mutation is T366S L368A Y407V.

[0114] Destabilizing mutations As noted above, only one CH3 domain of each precursor polypeptide contains one or more destabilizing mutations.

[0115] According to the present invention, either i) the CH3 domain of a first heterodimeric precursor polypeptide comprising a knob mutation and the CH3 domain of a second heterodimeric precursor polypeptide comprising a hole mutation, or ii) the CH3 domain of a first heterodimeric precursor polypeptide comprising a hole mutation and the CH3 domain of a second heterodimeric precursor polypeptide comprising a knob mutation, comprise one or more destabilizing mutations, wherein the one or more destabilizing mutations in the first and second heterodimeric precursor polypeptides are selected such that they interact at the CH3 / CH3 interface of a product polypeptide formed by exchange of polypeptide chains between the precursor polypeptides.

[0116] When a CH3 domain of a heterodimeric precursor polypeptide that includes a knob mutation includes a destabilizing mutation, the CH3 domain of said heterodimeric precursor polypeptide that includes a hole mutation does not include a destabilizing mutation. When a CH3 domain "does not include a destabilizing mutation," it includes a wild-type amino acid residue at a position that interacts with the amino acid residue at the position of the destabilizing mutation in the corresponding CH3 domain of a wild-type immunoglobulin of the same class.

[0117] In one embodiment of the present invention, a CH3 domain having a hole mutation comprises at least one amino acid substitution, i.e., a destabilizing mutation, selected from the group consisting of: replacement of S354 with a hydrophobic amino acid; replacement of D356 with a positively charged amino acid; replacement of E357 with a positively charged or hydrophobic amino acid; replacement of D356 with a positively charged amino acid and replacement of E357 with a positively charged or hydrophobic amino acid; replacement of S364 with a hydrophobic amino acid; replacement of A368 with a hydrophobic amino acid; replacement of E392 with a negatively charged amino acid; replacement of T394 with a hydrophobic amino acid; replacement of D399 with a hydrophobic amino acid and replacement of S400 with a positively charged amino acid; replacement of D399 with a hydrophobic amino acid and replacement of F405 with a positively charged amino acid; replacement of V407 with a hydrophobic amino acid; and replacement of K409 with a negatively charged amino acid; and replacement of K439 with a negatively charged amino acid; a CH3 domain having a knob mutation comprises at least one amino acid substitution, i.e., a destabilizing mutation, selected from the group consisting of: replacement of S354 with a hydrophobic amino acid; replacement of D356 with a positively charged amino acid and replacement of E357 with a positively charged or hydrophobic amino acid; replacement of S364 with a hydrophobic amino acid; replacement of A368 with a hydrophobic amino acid; replacement of E392 with a negatively charged amino acid; replacement of T394 with a hydrophobic amino acid; replacement of D399 with a hydrophobic amino acid and replacement of S400 with a positively charged amino acid; replacement of D399 with a hydrophobic amino acid and replacement of F405 with a positively charged amino acid; replacement of V407 with a hydrophobic amino acid; and replacement of K409 with a negatively charged amino acid; and replacement of K439 with a negatively charged Replacement of Q347 with a charged amino acid and replacement of K360 with a negatively charged amino acid; replacement of Y349 with a negatively charged amino acid; replacement of L351 with a hydrophobic amino acid and replacement of E357 with a hydrophobic amino acid; replacement of S364 with a hydrophobic amino acid; replacement of W366 with a hydrophobic amino acid and replacement of K409 with a negatively charged amino acid; replacement of L368 with a hydrophobic amino acid; replacement of K370 with a negatively charged amino acid; replacement of K370 with a negatively charged amino acid and replacement of K439 with a negatively charged amino acid; replacement of K392 with a negatively charged amino acid; replacement of T394 with a hydrophobic amino acid; replacement of V397 with a hydrophobic amino acid; replacement of D399 with a positively charged amino acid and replacement of K409 with a negatively charged amino acid; replacement of S400 with a positively charged amino acid; F405W; Y407W; and replacement of K439 with a negatively charged amino acid.

[0118] In one embodiment, the hydrophobic amino acid is selected from norleucine, Met, Ala, Val, Leu, Ile, Trp, Tyr, and Phe. In one embodiment, the hydrophobic amino acid is selected from Ala, Val, Leu, Ile, and Tyr. In one embodiment, the hydrophobic amino acid is Val, Leu, or Ile. In one embodiment, the hydrophobic amino acid is Leu or Ile. In one embodiment, the hydrophobic amino acid is Leu. In one embodiment, the hydrophobic amino acid is Tyr. In one embodiment, the hydrophobic amino acid is Phe.

[0119] In one embodiment, the positively charged amino acid is His, Lys, or Arg. In one embodiment, the positively charged amino acid is Lys or Arg. In one embodiment, the positively charged amino acid is Lys.

[0120] In one embodiment, the negatively charged amino acid is Asp or Glu. In one embodiment, the negatively charged amino acid is Asp. In one embodiment, the negatively charged amino acid is Glu.

[0121] Amino acid substitutions with amino acids having the respective side chain properties at the indicated amino acid positions within the CH3 domain were found to support product polypeptide formation from two precursor polypeptides and exchange of polypeptide chains.

[0122] In one embodiment of the present invention, the CH3 domain with hole mutations is S354V, S354I, S354L, D356K, D356R, E357K, E357R, E357F, S364L, S364I, A368F, K392D, K392E, T394L, T394I, V407Y, K409E, K409D, K439D, K439E, as well as double mutations D399A S400K, D399A S400R, D399A and F405W; CH3 domains with knob mutations include at least one amino acid substitution selected from the group consisting of Y349E, Y349D, S364V, S364I, S364L, L368F, K370E, K370D, K392E, K392D, T394L, T394I, V397Y, S400K, S400R, F405W, Y407W, K349E, K439D as well as double mutations Q347K K360E, Q347R K360E, Q347K K360D, Q347R K360D, L351F E357F, W366I K409E, W366L K409E, W366K K409D, W366L K409D, D399K It comprises at least one amino acid substitution selected from the group consisting of K409E, D399R K409E, D399K K409D, and D399K K409E.

[0123] In one embodiment of the present invention, the CH3 domain with hole mutations comprises at least one amino acid substitution selected from the group consisting of S354V, D356K, E357K, E357F, S364L, A368F, K392E, T394I, V407Y, K409E, K439E and the double mutation D399A S400K; the CH3 domain with knob mutations comprises at least one amino acid substitution selected from the group consisting of Y349E, S364V, L368F, K370E, K392D, T394I, V397Y, S400K, F405W, Y407W, K349E and the double mutation Q347K K360E, L351F E357F, W366I K409E and D399K K409E.

[0124] In one embodiment of the present invention, the CH3 domain with hole mutations comprises at least one amino acid substitution selected from the group consisting of D356K, D356R, E357K, E357R, E357F, S364L, S364I, V407Y, K409E, K409D and double mutations D399A S400K, D399A S400R; the CH3 domain with knob mutations comprises at least one amino acid substitution selected from the group consisting of Y349E, Y349D, K370E, K370D, K392E, K392D, T394L, T394I, V397Y, F405W, Y407W, K349E, K439D and double mutations Q347K K360E, Q347R K360E, Q347K K360D, Q347R K360D, W366I. and at least one amino acid substitution selected from the group consisting of K409E, W366L K409E, W366K K409D, W366L K409D, D399K K409E, D399R K409E, D399K K409D, and D399K K409E.

[0125] In one embodiment of the present invention, the CH3 domain having a hole mutation comprises at least one amino acid substitution selected from the group consisting of D356K, E357K, E357F, S364L, V407Y, K409E, and the double mutation D399A S400K; the CH3 domain having a knob mutation comprises at least one amino acid substitution selected from the group consisting of Y349E, K370E, K392D, T394I, V397Y, F405W, Y407W, K349E, and the double mutation Q347K K360E, W366I K409E, and D399K K409E.

[0126] In one embodiment of the invention, the CH3 domain with the hole mutation and the CH3 domain with the knob mutation, including the destabilizing mutation, comprise one of the amino acid substitutions selected from the group shown in the table below: TIFF2026004426000003.tif180139TIFF2026004426000004.tif137139

[0127] For clarity, this table is understood to mean that a CH3 domain containing a hole mutation contains the destabilizing mutation shown in the first column of the table above, and a CH3 domain containing a knob mutation contains the destabilizing mutation listed in the right column of the table above, shown in the same row.

[0128] In one embodiment of the invention, the CH3 domain with the hole mutation and the CH3 domain with the knob mutation, including the destabilizing mutation, comprise one of the amino acid substitutions selected from the group shown in the table below: TIFF2026004426000005.tif87139

[0129] In one embodiment of the invention, the CH3 domain with the hole mutation and the CH3 domain with the knob mutation, including the destabilizing mutation, comprise one of the amino acid substitutions selected from the group shown in the table below: TIFF2026004426000006.tif75139

[0130] In one embodiment of the invention, the CH3 domain with the hole mutation and the CH3 domain with the knob mutation, including the destabilizing mutation, comprise one of the amino acid substitutions selected from the group shown in the table below: TIFF2026004426000007.tif124139

[0131] For clarity, this table is understood to mean that a CH3 domain containing a hole mutation contains the destabilizing mutation shown in the first column of the table, and a CH3 domain containing a knob mutation contains the destabilizing mutation listed in the right column of the table, shown in the same row. Precursor molecules with such combinations of destabilizing mutations exhibit specific beneficial polypeptide chain exchanges.

[0132] In one embodiment of the invention, the CH3 domain with the hole mutation and the CH3 domain with the knob mutation, including the destabilizing mutation, comprise one of the amino acid substitutions selected from the group shown in the table below: TIFF2026004426000008.tif161139

[0133] For clarity, this table will be understood to mean that a CH3 domain containing a hole mutation contains the destabilizing mutation shown in the first column of the table, and a CH3 domain containing a knob mutation contains the destabilizing mutation listed in the right column of the table, shown in the same row. Precursor molecules with such combinations of destabilizing mutations exhibit specific beneficial polypeptide chain exchanges and can be produced in high yields.

[0134] Cysteine ​​mutations In one embodiment of the present invention, the CH3 domain of the heterodimeric precursor polypeptide comprises a third pattern of mutations, i.e., substitution of distinct amino acids within the CH3 / CH3 interface with cysteines, to allow the formation of interchain disulfide bonds between the two CH3 domains with cysteine ​​substitutions at the interacting positions.

[0135] Thus, in one embodiment of the present invention, i) the CH3 domain comprising the knob mutation of the first heterodimeric precursor polypeptide comprises a cysteine ​​mutation, and the CH3 domain comprising the hole mutation of the second heterodimeric precursor polypeptide comprises a cysteine ​​mutation, or ii) the CH3 domain comprising the hole mutation of the first heterodimeric precursor polypeptide comprises a cysteine ​​mutation, and the CH3 domain comprising the knob mutation of the second heterodimeric precursor polypeptide comprises a cysteine ​​mutation. In other words, in one embodiment, i) in the first heterodimeric polypeptide, the CH3 domain comprising the knob mutation comprises a cysteine ​​mutation and the CH3 domain comprising the hole mutation does not comprise a cysteine ​​mutation, and in the second heterodimeric polypeptide, the CH3 domain comprising the knob mutation does not comprise a cysteine ​​mutation and the CH3 domain comprising the hole mutation comprises a cysteine ​​mutation, or ii) in the first heterodimeric polypeptide, the CH3 domain comprising the knob mutation does not comprise a cysteine ​​mutation and the CH3 domain comprising the hole mutation comprises a cysteine ​​mutation, and in the second heterodimeric polypeptide, the CH3 domain comprising the knob mutation comprises a cysteine ​​mutation and the CH3 domain comprising the hole mutation does not comprise a cysteine ​​mutation.

[0136] In one embodiment, i) the CH3 domain comprising the knob mutation of the first heterodimeric precursor polypeptide comprises a first cysteine ​​mutation and the CH3 domain comprising the hole mutation of the second heterodimeric precursor polypeptide comprises a second cysteine ​​mutation, or ii) the CH3 domain comprising the hole mutation of the first heterodimeric precursor polypeptide comprises a first cysteine ​​mutation and the CH3 domain comprising the knob mutation of the second heterodimeric precursor polypeptide comprises a second cysteine ​​mutation, wherein the first and second cysteine ​​mutations are selected from the following pairs: TIFF2026004426000009.tif51139

[0137] In one embodiment, the first cysteine ​​mutation is Y349C and the second cysteine ​​mutation is S354C.

[0138] In one embodiment of the invention, i) the CH3 domain comprising the knob mutation of the first heterodimeric precursor polypeptide comprises the substitution S354C, and the CH3 domain comprising the hole mutation of the second heterodimeric precursor polypeptide comprises the substitution Y349C, or ii) the CH3 domain comprising the hole mutation of the first heterodimeric precursor polypeptide comprises the substitution Y349C, and the CH3 domain comprising the knob mutation of the second heterodimeric precursor polypeptide comprises the substitution S354C.

[0139] In one embodiment of the invention, in the first heterodimeric precursor polypeptide, the CH3 domain comprising the knob mutation comprises a substitution S354C and the CH3 domain comprising the hole mutation comprises a Y at position 349; and in the second heterodimeric precursor polypeptide, the CH3 domain comprising the hole mutation comprises a substitution Y349C and the CH3 domain comprising the knob mutation comprises an S at position 354.

[0140] In one embodiment of the invention, i) the CH3 domain comprising the knob mutation of the first heterodimeric precursor polypeptide comprises the substitution T366W S354C, and the CH3 domain comprising the hole mutation of the second heterodimeric precursor polypeptide comprises the substitution T366S L368A Y407V Y349C, or ii) the CH3 domain comprising the hole mutation of the first heterodimeric precursor polypeptide comprises the substitution T366S L368A Y407V Y349C, and the CH3 domain comprising the knob mutation of the second heterodimeric precursor polypeptide comprises the substitution T366W S354C.

[0141] In one embodiment of the invention, in the first heterodimeric precursor polypeptide, the CH3 domain comprising the knob mutation comprises the substitution T366W S354C, and the CH3 domain comprising the hole mutation comprises a Y at position 349 and the substitution T366S L368A Y407V; and in the second heterodimeric precursor polypeptide, the CH3 domain comprising the hole mutation comprises the substitution T366S L368A Y407V Y349C, and the CH3 domain comprising the knob mutation comprises an S at position 354 and the substitution T366W.

[0142] In one embodiment of the present invention, the CH3 domain of the heterodimeric precursor polypeptide does not comprise an interchain disulfide bond.

[0143] B) Antigen binding portion In one embodiment of the invention, the antigen-binding moiety is a polypeptide that specifically binds to an antigen, hi one embodiment, the antigen-binding moiety is selected from the group of antibodies, receptors, ligands, and DARPins that are capable of specifically binding to an antigen.

[0144] In one embodiment of the invention, the antigen-binding moiety comprised in the (precursor) polypeptide according to the invention is an antibody fragment.

[0145] In one embodiment of the invention, the antigen-binding portion comprises a pair of VH and VL domains that form an antigen-binding site that specifically binds to a target antigen.

[0146] In one embodiment of the present invention, the antibody fragment contained in the (precursor) polypeptide according to the present invention is an antibody fragment selected from the group consisting of Fv, Fab, Fab', Fab'-SH, F(ab')2, diabody, scFv, and scFab. In one embodiment, the antibody fragment contained in the (precursor) polypeptide according to the present invention is Fv or Fab.

[0147] In one embodiment of the invention, the antigen-binding portion is a Fab fragment.

[0148] In one embodiment of the invention, the first antigen-binding moiety is a first Fab fragment and the second antigen-binding moiety is a second Fab fragment. In one embodiment of the invention, the first Fab fragment, the second Fab fragment, or both, the first and second Fab fragments are modified by domain crossover as follows: a) Only the CH1 and CL domains are displaced by each other; b) only the VH and VL domains are replaced by each other; or c) The CH1 and CL domains are replaced by each other, and the VH and VL domains are replaced by each other.

[0149] In one embodiment of the invention, the antigen-binding moieties are Fv fragments. In one embodiment of the invention, the first antigen-binding moiety is a first Fv fragment and the second antigen-binding moiety is a second Fv fragment.

[0150] In one embodiment of the invention, the antigen-binding portion of the first heterodimeric precursor polypeptide and the antigen-binding portion of the second heterodimeric precursor polypeptide bind to the same antigen. In one embodiment of the invention, the antigen-binding portion of the first heterodimeric precursor polypeptide and the antigen-binding portion of the second heterodimeric precursor polypeptide are the same antigen-binding portion.

[0151] In one embodiment of the invention, the antigen-binding portion of the first heterodimeric precursor polypeptide and the antigen-binding portion of the second heterodimeric precursor polypeptide bind to different antigens, and upon exchange of polypeptide chains between the two heterodimeric precursor polypeptides, a multispecific product polypeptide is formed that comprises the antigen-binding portion originating from the first heterodimeric precursor polypeptide and the antigen-binding portion originating from the second heterodimeric precursor polypeptide.

[0152] Additional antigen-binding moieties may be present within the heterodimeric precursor polypeptide, which may be fused to the N-terminus or C-terminus of a polypeptide chain comprised in the heterodimeric precursor polypeptide to provide a product polypeptide of higher valency.

[0153] Such additional antigen-binding moieties are fused to the polypeptide chain via a suitable peptide connector, which in one embodiment is a glycine-serine linker.

[0154] In one embodiment of the present invention, in a heterodimeric precursor polypeptide, only one of the polypeptide chains comprising a CH3 domain comprises at least a portion of an antigen-binding portion. In one embodiment of the present invention, in a heterodimeric precursor polypeptide, one of the polypeptide chains comprising a CH3 domain of an antigen-binding site that specifically binds to a target antigen. In one embodiment of the present invention, in a heterodimeric precursor polypeptide, one of the polypeptide chains comprising a CH3 domain comprises, from N- to C-terminal, a hinge region, an antibody variable domain, and a CH3 domain, and this polypeptide chain is not part of an antigen-binding site that specifically binds to a target antigen. In one embodiment of the present invention, in a heterodimeric precursor polypeptide, one of the polypeptide chains comprising a CH3 domain comprises, from N- to C-terminal, a hinge region, an antibody variable domain, a CH2 domain, and a CH3 domain, and this polypeptide chain is not part of an antigen-binding site that specifically binds to a target antigen.

[0155] C) Domain organization of precursor polypeptide The precursor polypeptides of the present invention are suitable for generating product polypeptides in a variety of formats with different domain configurations. Depending on the choice of domains and the number of antigen-binding moieties provided in the heterodimeric precursor molecule, product polypeptides with different antigen-binding properties (e.g., specificity, valency) and different effector functions can be generated.

[0156] In one embodiment, the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide comprise exactly two polypeptide chains having a CH3 domain. Accordingly, the first and second heterodimeric precursor polypeptides may comprise additional polypeptide chains lacking a CH3 domain.

[0157] Precursor polypeptides containing antibody fragments In one embodiment of the invention, the antigen-binding portion comprises a pair of VH and VL domains that form an antigen-binding site that specifically binds to a target antigen; a) the first heterodimeric precursor polypeptide comprises: - a first heavy chain polypeptide comprising a CH3 domain and a first antibody variable domain; - a second heavy chain polypeptide comprising a CH3 domain, wherein the first heavy chain polypeptide and the second heavy chain polypeptide associate with each other via the CH3 domain to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and - a light chain polypeptide comprising a second antibody variable domain, wherein the first and second antibody variable domains together form a first antigen-binding site that specifically binds to a target antigen. Includes; b) the second heterodimeric precursor polypeptide comprises: - a third heavy chain polypeptide comprising a CH3 domain and a third antibody variable domain; - a fourth heavy chain polypeptide comprising a CH3 domain, wherein the third heavy chain polypeptide and the fourth heavy chain polypeptide associate with each other via the CH3 domain to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and - a light chain polypeptide comprising a fourth antibody variable domain, wherein the third and fourth antibody variable domains together form a second antigen-binding site that specifically binds to a target antigen. where c) i) the first heavy chain polypeptide comprises a CH3 domain comprising a knob mutation and the third heavy chain polypeptide comprises a CH3 domain comprising a hole mutation; or ii) the first heavy chain polypeptide comprises a CH3 domain comprising a hole mutation and the third heavy chain polypeptide comprises a CH3 domain comprising a knob mutation.

[0158] In one embodiment of the invention, the antigen-binding portion comprises a pair of VH and VL domains that form an antigen-binding site that specifically binds to a target antigen; a) the first heterodimeric precursor polypeptide comprises: - a first heavy chain polypeptide comprising, in N-terminal to C-terminal direction, a first VH domain and constant antibody domains CH1, CH2 and CH3; - a second heavy chain polypeptide comprising, from N-terminal to C-terminal, constant antibody domains CH2 and CH3 domains, wherein the first and second heavy chain polypeptides associate with each other via the CH3 domains to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and - a light chain polypeptide comprising, from N-terminal to C-terminal, a first VL domain and a CL domain, wherein the first VH domain and the first VL domain bind to each other to form an antigen-binding site that specifically binds to a target antigen. Includes; b) the second heterodimeric precursor polypeptide comprises: - a third heavy chain polypeptide comprising, in N-terminal to C-terminal direction, a second VH domain and constant antibody domains CH1, CH2 and CH3; - a fourth heavy chain polypeptide comprising, from N-terminal to C-terminal, constant antibody domains CH2 and CH3 domains, wherein the third heavy chain polypeptide and the fourth heavy chain polypeptide associate with each other via the CH3 domains to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and - a light chain polypeptide comprising, from N-terminal to C-terminal, a second VL domain and a CL domain, wherein the second VH domain and the second VL domain associate with each other to form an antigen-binding site that specifically binds to a target antigen. where i) the first heavy chain polypeptide comprises a CH3 domain comprising a knob mutation and the third heavy chain polypeptide comprises a CH3 domain comprising a hole mutation; or ii) the first heavy chain polypeptide comprises a CH3 domain comprising a hole mutation and the third heavy chain polypeptide comprises a CH3 domain comprising a knob mutation.

[0159] In one embodiment of the invention, the antigen-binding portion comprises a pair of VH and VL domains that form an antigen-binding site that specifically binds to a target antigen; a) the first heterodimeric precursor polypeptide comprises: - a first heavy chain polypeptide comprising, in N-terminal to C-terminal direction, a CH2 domain, a CH3 domain, a peptide connector, a first VH domain, and a CH1 domain; - a second heavy chain polypeptide comprising, from N-terminal to C-terminal, constant antibody domains CH2 and CH3 domains, wherein the first and second heavy chain polypeptides associate with each other via the CH3 domains to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and - a light chain polypeptide comprising, from N-terminal to C-terminal, a first VL domain and a CL domain, wherein the first VH domain and the first VL domain bind to each other to form an antigen-binding site that specifically binds to a target antigen. Includes; b) the second heterodimeric precursor polypeptide comprises: - a third heavy chain polypeptide comprising, in N-terminal to C-terminal direction, a CH2 domain, a CH3 domain, a peptide connector, a first VH domain, and a CH1 domain; - a fourth heavy chain polypeptide comprising, from N-terminal to C-terminal, constant antibody domains CH2 and CH3 domains, wherein the third heavy chain polypeptide and the fourth heavy chain polypeptide associate with each other via the CH3 domains to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and - a light chain polypeptide comprising, from N-terminal to C-terminal, a second VL domain and a CL domain, wherein the second VH domain and the second VL domain associate with each other to form an antigen-binding site that specifically binds to a target antigen. where i) the first heavy chain polypeptide comprises a CH3 domain comprising a knob mutation and the third heavy chain polypeptide comprises a CH3 domain comprising a hole mutation; or ii) the first heavy chain polypeptide comprises a CH3 domain comprising a hole mutation and the third heavy chain polypeptide comprises a CH3 domain comprising a knob mutation.

[0160] In one embodiment of the invention, the antigen-binding portion comprises a pair of VH and VL domains that form an antigen-binding site that specifically binds to a target antigen; a) the first heterodimeric precursor polypeptide comprises: - a first heavy chain polypeptide comprising, from N-terminal to C-terminal direction, a first VH domain, and constant antibody domains CH1, CH2, CH3, a peptide connector, the first VH domain, and a CH1 domain; - a second heavy chain polypeptide comprising, from N-terminal to C-terminal, constant antibody domains CH2 and CH3 domains, wherein the first and second heavy chain polypeptides associate with each other via the CH3 domains to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and - a light chain polypeptide comprising, from N-terminal to C-terminal, a first VL domain and a CL domain, wherein the first VH domain and the first VL domain bind to each other to form an antigen-binding site that specifically binds to a target antigen. Includes; b) the second heterodimeric precursor polypeptide comprises: - a third heavy chain polypeptide comprising, in N-terminal to C-terminal direction, a second VH domain, constant antibody domains CH1, CH2 and CH3, a peptide connector, a first VH domain, and a CH1 domain; - a fourth heavy chain polypeptide comprising, from N-terminal to C-terminal, constant antibody domains CH2 and CH3 domains, wherein the third heavy chain polypeptide and the fourth heavy chain polypeptide associate with each other via the CH3 domains to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and - a light chain polypeptide comprising, from N-terminal to C-terminal, a second VL domain and a CL domain, wherein the second VH domain and the second VL domain associate with each other to form an antigen-binding site that specifically binds to a target antigen. where i) the first heavy chain polypeptide comprises a CH3 domain comprising a knob mutation and the third heavy chain polypeptide comprises a CH3 domain comprising a hole mutation; or ii) the first heavy chain polypeptide comprises a CH3 domain comprising a hole mutation and the third heavy chain polypeptide comprises a CH3 domain comprising a knob mutation.

[0161] Precursor polypeptide containing the CH2 domain In one embodiment of the present invention, the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide comprise at least two polypeptide chains comprising a CH2 domain and a CH3 domain. Heterodimeric precursor polypeptides comprising a CH2 domain and a CH3 domain exhibit advantageous properties, such as mediation of Fc-mediated effector functions and long half-life in circulation.

[0162] In one embodiment of the invention, the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide comprise, from the N-terminus to the C-terminus, at least two polypeptide chains comprising a CH2 domain and a CH3 domain.

[0163] In one embodiment of the present invention, i) the first heterodimeric precursor polypeptide comprises a polypeptide chain comprising a VL domain, a CH2 domain, and a CH3 domain, and the second heterodimeric precursor polypeptide comprises a polypeptide chain comprising a VH domain, a CH2 domain, and a CH3 domain, wherein the VL domain and the VH domain specifically bind to an antigen when associated with the VH domain and VL domain pair; or ii) the first heterodimeric precursor polypeptide comprises a polypeptide chain comprising a VH domain, a CH2 domain, and a CH3 domain, and the second heterodimeric precursor polypeptide comprises a polypeptide chain comprising a VL domain, a CH2 domain, and a CH3 domain, wherein the VL domain and the VH domain specifically bind to an antigen when associated with the VH domain and VL domain pair.

[0164] In one embodiment of the invention, the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide lack a CH2 domain. Heterodimeric precursor polypeptides lacking a CH2 domain exhibit advantageous properties, such as rapid clearance from the circulation.

[0165] A precursor polypeptide containing an activatable antigen-binding site In one embodiment, each precursor polypeptide comprises a portion of an antigen-binding moiety that is non-functional in the precursor polypeptide and that is functional in the product polypeptide formed by exchange of polypeptide chains between the precursor polypeptides and specifically binds to a target antigen. Exemplary structures of such precursor polypeptides are shown in Figures 2 and 3.

[0166] In one embodiment of the invention, the antigen-binding portion is an antigen-binding site comprising a pair of antibody variable domains.

[0167] In one embodiment of the present invention, the first heterodimeric precursor polypeptide comprises one polypeptide chain comprising a VL domain and a CH3 domain, and the second heterodimeric precursor polypeptide comprises one polypeptide chain comprising a VH domain and a CH3 domain, and the VL domain and the VH domain specifically bind to an antigen when associated into a VH domain-VL domain pair.

[0168] In one embodiment, the activatable antigen-binding site, e.g., a pair of antibody variable domains, is stabilized by an interchain disulfide bond. In one embodiment, the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide comprise a hinge region, wherein the hinge region does not comprise an interchain disulfide bond, and the activatable antigen-binding site is stabilized by an interchain disulfide bond.

[0169] In one embodiment, the first heterodimeric precursor polypeptide comprises one polypeptide chain comprising a VL domain and a CH3 domain, and one polypeptide chain comprising a VH domain and a CH3 domain, wherein the CH3 domains are associated with each other, and the VH domain and VL domain are associated with each other, and a disulfide bond is formed between the VH domain and the VL domain by cysteine ​​residues introduced at amino acid positions selected from the following pairs: VH position 44 and VL position 100, VH position 105 and VL position 43, and VH position 101 and VL position 100; in one embodiment VH position 44 and VL position 100.

[0170] In one embodiment, the second heterodimeric precursor polypeptide comprises one polypeptide chain comprising a VL domain and a CH3 domain, and one polypeptide chain comprising a VH domain and a CH3 domain, wherein the CH3 domains are associated with each other, and the VH domain and VL domain are associated with each other, and wherein a disulfide bond is formed between the VH domain and the VL domain by cysteine ​​residues introduced at amino acid positions selected from the following pairs: VH position 44 and VL position 100, VH position 105 and VL position 43, and VH position 101 and VL position 100; in one embodiment VH position 44 and VL position 100.

[0171] In one embodiment, the first heterodimeric precursor polypeptide comprises one polypeptide chain comprising a VL domain and a CH3 domain and one polypeptide chain comprising a VH domain and a CH3 domain, wherein the CH3 domains are associated with each other and the VH domain and VL domain are associated with each other, and disulfide bonds are formed between the VH domain and the VL domain by cysteine ​​residues introduced at amino acid positions selected from the following pairs: VH position 44 and VL position 100, VH position 105 and VL position 43, and VH position 101 and VL position 100; the second heterodimeric precursor polypeptide comprises one polypeptide chain comprising a VL domain and a CH3 domain and one polypeptide chain comprising a VH domain and a CH3 domain. and a peptide chain, wherein the CH3 domains are associated with each other, and the VH domain and the VL domain are associated with each other, and a disulfide bond is formed between the VH domain and the VL domain by cysteine ​​residues introduced at amino acid positions selected from the following pairs: VH position 44 and VL position 100, VH position 105 and VL position 43, and VH position 101 and VL position 100; an amino acid substitution by cysteine ​​in the VH domain of the first heterodimeric precursor polypeptide is at the same position in the VH domain of the second heterodimeric precursor polypeptide, and an amino acid substitution by cysteine ​​in the VL domain of the first heterodimeric precursor polypeptide is at the same position in the VL domain of the second heterodimeric precursor polypeptide.

[0172] In one embodiment, the first heterodimeric precursor polypeptide comprises one polypeptide chain comprising a VL domain and a CH3 domain and one polypeptide chain comprising a VH domain and a CH3 domain, wherein the CH3 domains are associated with each other and the VH domain and VL domain are associated with each other, and a disulfide bond is formed between the VH domain and the VL domain by cysteine ​​residues introduced at amino acid positions selected from the following pairs: VH position 44 and VL position 100, VH position 105 and VL position 43, and VH position 101 and VL position 100; and the second heterodimeric precursor polypeptide comprises one polypeptide chain comprising a VL domain and a CH3 domain and one polypeptide chain comprising a VH domain and a CH3 domain. and a polypeptide chain, wherein the CH3 domains are associated with each other, and the VH domain and VL domain are associated with each other, and a disulfide bond is formed between the VH domain and the VL domain by cysteine ​​residues introduced at amino acid positions selected from the following pairs: VH position 44 and VL position 100, VH position 105 and VL position 43, and VH position 101 and VL position 100; an amino acid substitution by cysteine ​​in the VH domain of the first heterodimeric precursor polypeptide is at the same position in the VH domain of the second heterodimeric precursor polypeptide, and an amino acid substitution by cysteine ​​in the VL domain of the first heterodimeric precursor polypeptide is at the same position in the VL domain of the second heterodimeric precursor polypeptide.

[0173] In one embodiment of the present invention, the first heterodimeric precursor polypeptide comprises, from the N-terminus to the C-terminus, a single polypeptide chain comprising a VL domain and a CH3 domain, and the second heterodimeric precursor polypeptide comprises, from the N-terminus to the C-terminus, a single polypeptide chain comprising a VH domain and a CH3 domain, and the VL domain and the VH domain specifically bind to an antigen when associated into a VH domain-VL domain pair.

[0174] In one embodiment of the present invention, a) the first heterodimeric precursor polypeptide comprises: - a first heavy chain polypeptide comprising, from N-terminal to C-terminal, a first VH domain, a CH1 domain, a second antibody variable domain selected from a VH domain and a VL domain, and a CH3 domain; - a second heavy chain polypeptide comprising, from N-terminal to C-terminal, an antibody variable domain capable of associating with a second antibody variable domain of the first heavy chain polypeptide, and a CH3 domain, wherein the first heavy chain polypeptide and the second heavy chain polypeptide associate with each other via the CH3 domain to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and - a light chain polypeptide comprising, from N-terminal to C-terminal, a first VL domain and a CL domain, wherein the first VH domain and the first VL domain bind to each other to form an antigen-binding site that specifically binds to a target antigen. Includes; b) the second heterodimeric precursor polypeptide comprises: - a third heavy chain polypeptide comprising, in N-terminal to C-terminal direction, a second VH domain, a CH1 domain, a third antibody variable domain selected from a VH domain and a VL domain, and a CH3 domain; - a fourth heavy chain polypeptide comprising, from N-terminal to C-terminal, an antibody variable domain capable of associating with a third antibody variable domain of a third heavy chain polypeptide, and a CH3 domain, wherein the third heavy chain polypeptide and the fourth heavy chain polypeptide associate with each other via the CH3 domain to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and - a light chain polypeptide comprising, from N-terminal to C-terminal, a second VL domain and a CL domain, wherein the second VH domain and the second VL domain associate with each other to form an antigen-binding site that specifically binds to a target antigen. where c) i) the first heavy chain polypeptide comprises a CH3 domain comprising a knob mutation and the third heavy chain polypeptide comprises a CH3 domain comprising a hole mutation; or ii) the first heavy chain polypeptide comprises a CH3 domain comprising a hole mutation and the third heavy chain polypeptide comprises a CH3 domain comprising a knob mutation; d) the variable domains of the first heavy chain polypeptide and the third heavy chain polypeptide can form an antigen-binding site that specifically binds to a target antigen.

[0175] In one embodiment, the first heavy chain polypeptide comprises, from N-terminal to C-terminal, a first VH domain, a CH1 domain, a second antibody variable domain selected from a VH domain and a VL domain, a peptide connector, and a CH3 domain; the second heavy chain polypeptide comprises, from N-terminal to C-terminal, an antibody variable domain capable of associating with the second antibody variable domain of the first heavy chain polypeptide, a peptide connector, and a CH3 domain; the first heavy chain polypeptide and the second heavy chain polypeptide associate with each other via the CH3 domains to form a heterodimer, wherein one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation. the third heavy chain polypeptide comprises, from N-terminal to C-terminal, a second VH domain, a CH1 domain, a third antibody variable domain selected from a VH domain and a VL domain, a peptide connector, and a CH3 domain; and the fourth heavy chain polypeptide comprises, from N-terminal to C-terminal, an antibody variable domain capable of associating with the third antibody variable domain of the third heavy chain polypeptide, a peptide connector, and a CH3 domain, wherein the third heavy chain polypeptide and the fourth heavy chain polypeptide associate with each other via the CH3 domains to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation. In one embodiment, the peptide connectors in the first, second, third, and fourth heavy chain polypeptides are identical.

[0176] In one embodiment, in the first heterodimeric precursor polypeptide, the second antibody variable domain contained in the first heavy chain polypeptide is derived from an antibody that specifically binds to a first target antigen, and the antibody variable domain contained in the second heavy chain polypeptide specifically binds to a second target antigen. Both variable domains can associate with each other. Thus, one of the heavy chain polypeptides contains a VH domain, and the other heavy chain polypeptide contains a VL domain. The VH and VL domains can associate with each other; however, a non-functional antigen-binding site is formed. Therefore, in the context of the present invention, the term "variable domains that can associate with each other" means that a pair of VH and VL domains is provided. In this embodiment, within the second heterodimeric precursor polypeptide, the third antibody variable domain contained in the third heavy chain polypeptide is derived from an antibody that specifically binds to a first target antigen (i.e., can form a functional VH / VL pair with the second variable domain contained in the first heavy chain polypeptide of the first heterodimeric precursor polypeptide), and the antibody variable domain contained in the fourth heavy chain polypeptide specifically binds to another, e.g., second, target antigen. The variable domains contained in the first and third heavy chain polypeptides are capable of associating with each other, i.e., one of the variable domains is a VH domain and the other variable domain is a VL domain; and the variable domains contained in the first and third heavy chain polypeptides are capable of forming an antigen-binding site that specifically binds to the target antigen, i.e., both variable domains are derived from the same antibody that specifically binds to the target antigen.

[0177] In one embodiment of the present invention, the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide comprise at least two polypeptide chains comprising, from the N-terminus to the C-terminus, a CH2 domain and a CH3 domain, the first heterodimeric precursor polypeptide comprising one polypeptide chain comprising, from the N-terminus to the C-terminus, a VL domain, a CH2 domain, and a CH3 domain, the second heterodimeric precursor polypeptide comprising one polypeptide chain comprising, from the N-terminus to the C-terminus, a VH domain, a CH2 domain, and a CH3 domain, and the VL domain and VH domain can form an antigen-binding site that specifically binds to a target antigen.

[0178] In one embodiment of the present invention, a) the first heterodimeric precursor polypeptide comprises: - a first heavy chain polypeptide comprising, from N-terminal to C-terminal, a first VH domain, a CH1 domain, a second antibody variable domain selected from a VH domain and a VL domain, a CH2 domain, and a CH3 domain; - a second heavy chain polypeptide comprising, from N-terminal to C-terminal, an antibody variable domain capable of associating with a second antibody variable domain of the first heavy chain polypeptide, a CH2 domain, and a CH3 domain, wherein the first heavy chain polypeptide and the second heavy chain polypeptide associate with each other via the CH3 domain to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and - a light chain polypeptide comprising, from N-terminal to C-terminal, a first VL domain and a CL domain, wherein the first VH domain and the first VL domain bind to each other to form an antigen-binding site that specifically binds to a target antigen. Includes; b) the second heterodimeric precursor polypeptide comprises: - a third heavy chain polypeptide comprising, from N-terminal to C-terminal, a second VH domain, a CH1 domain, a third antibody variable domain selected from a VH domain and a VL domain, a CH2 domain, and a CH3 domain; - a fourth heavy chain polypeptide comprising, from N-terminal to C-terminal, an antibody variable domain capable of associating with a third antibody variable domain of a third heavy chain polypeptide, a CH2 domain, and a CH3 domain, wherein the third heavy chain polypeptide and the fourth heavy chain polypeptide associate with each other via the CH3 domain to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and - a light chain polypeptide comprising, from N-terminal to C-terminal, a second VL domain and a CL domain, wherein the second VH domain and the second VL domain associate with each other to form an antigen-binding site that specifically binds to a target antigen. where c) i) the first heavy chain polypeptide comprises a CH3 domain comprising a knob mutation and the third heavy chain polypeptide comprises a CH3 domain comprising a hole mutation; or ii) the first heavy chain polypeptide comprises a CH3 domain comprising a hole mutation and the third heavy chain polypeptide comprises a CH3 domain comprising a knob mutation; d) the variable domains of the first heavy chain polypeptide and the third heavy chain polypeptide can form an antigen-binding site that specifically binds to a target antigen.

[0179] In one embodiment, the first heavy chain polypeptide comprises, from N-terminal to C-terminal, a first VH domain, a CH1 domain, a second antibody variable domain selected from a VH domain and a VL domain, a peptide connector, a CH2 domain, and a CH3 domain; the second heavy chain polypeptide comprises, from N-terminal to C-terminal, an antibody variable domain capable of associating with the second antibody variable domain of the first heavy chain polypeptide, a peptide connector, a CH2 domain, and a CH3 domain; the first heavy chain polypeptide and the second heavy chain polypeptide associate with each other via the CH3 domain to form a heterodimer, wherein one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation. the third heavy chain polypeptide comprises, from N-terminal to C-terminal, a second VH domain, a CH1 domain, a third antibody variable domain selected from a VH domain and a VL domain, a peptide connector, a CH2 domain, and a CH3 domain; and the fourth heavy chain polypeptide comprises, from N-terminal to C-terminal, an antibody variable domain capable of associating with the third antibody variable domain of the third heavy chain polypeptide, a peptide connector, a CH2 domain, and a CH3 domain, wherein the third and fourth heavy chain polypeptides associate with each other via the CH3 domains to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation. In one embodiment, the peptide connectors in the first, second, third, and fourth heavy chain polypeptides are identical.

[0180] Precursor polypeptide containing the hinge region In one embodiment of the invention, the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide comprise at least two polypeptide chains comprising, from N- to C-terminal, a hinge region and a CH3 domain.

[0181] In one embodiment of the invention, the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide comprise at least two polypeptide chains comprising, from N- to C-terminal, a hinge region, a CH2 domain, and a CH3 domain.

[0182] In one embodiment of the present invention, the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide contain an interchain disulfide bond in the hinge region. Heterodimeric precursor polypeptides having hinge regions containing at least one interchain disulfide bond undergo polypeptide chain exchange in the presence of a reducing agent. Thus, these heterodimeric precursor polypeptides are suitable for applications where the presence of a reducing agent is possible, such as in vitro production of product polypeptides or for screening purposes.

[0183] In one embodiment of the present invention, the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide do not contain interchain disulfide bonds in the hinge region. Heterodimeric precursor polypeptides having hinge regions without interchain disulfide bonds can undergo polypeptide chain exchange in the absence of reducing agents. Therefore, heterodimeric precursor polypeptides having hinge regions without interchain disulfide bonds are particularly suitable for applications where the presence of reducing agents is not possible or desirable. Therefore, these heterodimeric precursor polypeptides may be advantageous in therapeutic methods.

[0184] In one embodiment of the present invention, the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide comprise a native hinge region that does not form interchain disulfides. One example is a hinge region peptide derived from an antibody of the IgG4 isotype.

[0185] Instead of a hinge region that does not contain an interchain disulfide bond, the heterodimeric precursor polypeptide may contain a peptide connector connecting (part of) the antigen-binding portion and the constant antibody domain (i.e., CH2 or CH3). In one embodiment of the present invention, no interchain disulfide bond is formed between the peptide connectors contained in the two polypeptide chains that contain the CH3 domain of the precursor polypeptide. In one embodiment of the present invention, the peptide connectors contained in the two polypeptide chains that contain the CH3 domain are identical to each other.

[0186] In one embodiment of the invention, the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide comprise, from N- to C-terminal, at least two polypeptide chains comprising a peptide connector and a CH3 domain.

[0187] In one embodiment of the invention, the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide comprise at least two polypeptide chains comprising, from N- to C-terminal, a peptide connector, a CH2 domain, and a CH3 domain.

[0188] In one embodiment of the invention, the first heterodimeric precursor polypeptide comprises a first polypeptide chain comprising a first peptide connector, an antibody variable domain, optionally a CH2 domain and a CH3 domain, and a second polypeptide chain comprising a first peptide connector, an antibody variable domain capable of associating with an antibody variable domain from the first polypeptide chain, optionally a CH2 domain and a CH3 domain; and the second heterodimeric precursor polypeptide comprises a first polypeptide chain comprising a first peptide connector, an antibody variable domain, optionally a CH2 domain and a CH3 domain, and a second polypeptide chain comprising a first peptide connector, an antibody variable domain capable of associating with an antibody variable domain from the first polypeptide chain, optionally a CH2 domain and a CH3 domain.

[0189] In one embodiment of the invention, the peptide connector is a peptide of at least 15 amino acids. In another embodiment of the invention, the peptide connector is a peptide of 15-70 amino acids. In another embodiment of the invention, the peptide connector is a peptide of 20-50 amino acids. In another embodiment of the invention, the peptide connector is a peptide of 10-50 amino acids. Shorter (or potentially longer) peptide connectors may also be applicable to heterodimeric precursor polypeptides according to the invention, depending, for example, on the type of antigen to which the activatable binding site binds.

[0190] In yet another embodiment of the invention, the first and second peptide connectors are approximately the length of the natural hinge region (about 15 amino acids for natural antibody molecules of the IgG1 isotype, and about 62 amino acids for the IgG3 isotype). Thus, in one embodiment in which the first and second heterodimeric precursor polypeptides are of the IgG1 isotype, the peptide connector is a peptide of 10 to 20 amino acids, and in a preferred embodiment, 12 to 17 amino acids. In another embodiment, in one embodiment in which the first and second heterodimeric precursor polypeptides are of the IgG3 isotype, the peptide connector is a peptide of 55 to 70 amino acids, and in a preferred embodiment, 60 to 65 amino acids.

[0191] In one embodiment of the invention, the peptide connector is a glycine-serine linker. In one embodiment of the invention, the peptide connector is a peptide consisting of a glycine residue and a serine residue. In one embodiment of the invention, the glycine-serine linker has the structure (GxS)n or (GxS)nGm where G=glycine, S=serine, x=3 or 4, n=2, 3, 4, 5 or 6, and m=0, 1, 2 or 3.

[0192] In one embodiment of the glycine-serine linker defined above, x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3; or x=4, n=2, 3, 4 or 5, and m=0, 1, 2 or 3. In a preferred embodiment, x=4, n=2 or 3, and m=0. In another preferred embodiment, x=4 and n=2. In one embodiment, the peptide connector is (G4S)4 or (G4S)6.

[0193] In one embodiment of the present invention, i) the first heterodimeric precursor polypeptide comprises a polypeptide chain comprising a VL domain, a peptide connector, and a CH3 domain, and the second heterodimeric precursor polypeptide comprises a polypeptide chain comprising a VH domain, a peptide connector, and a CH3 domain, wherein the VL domain and the VH domain specifically bind to an antigen when associated as a VH and VL domain pair; or ii) the first heterodimeric precursor polypeptide comprises a polypeptide chain comprising a VH domain, a peptide connector, and a CH3 domain, and the second heterodimeric precursor polypeptide comprises a polypeptide chain comprising a VL domain, a peptide connector, and a CH3 domain, wherein the VL domain and the VH domain specifically bind to an antigen when associated as a VH and VL domain pair.

[0194] In one embodiment of the present invention, i) the first heterodimeric precursor polypeptide comprises a polypeptide chain comprising a VL domain, a peptide connector, a CH2 domain, and a CH3 domain, and the second heterodimeric precursor polypeptide comprises a polypeptide chain comprising a VH domain, a peptide connector, a CH2 domain, and a CH3 domain, wherein the VL domain and the VH domain specifically bind to an antigen when associated as a VH-VL domain pair; or ii) the first heterodimeric precursor polypeptide comprises a polypeptide chain comprising a VH domain, a peptide connector, a CH2 domain, and a CH3 domain, and the second heterodimeric precursor polypeptide comprises a polypeptide chain comprising a VL domain, a peptide connector, a CH2 domain, and a CH3 domain, wherein the VL domain and the VH domain specifically bind to an antigen when associated as a VH-VL domain pair.

[0195] D) Antibody isotype and valency In one embodiment of the present invention, the precursor polypeptide comprises an immunoglobulin constant region of one or more immunoglobulin classes, including IgG, IgM, IgA, IgD, and IgE isotypes, and in the case of IgG and IgA, their subtypes. In one embodiment of the present invention, the precursor polypeptide has the constant domain structure of an IgG-type antibody.

[0196] In one embodiment of the present invention, the CH3 domain contained in the precursor polypeptide is of the mammalian IgG class. In one embodiment of the present invention, the CH3 domain contained in the precursor polypeptide is of the mammalian IgG1 subclass. In one embodiment of the present invention, the CH3 domain contained in the precursor polypeptide is of the mammalian IgG4 subclass.

[0197] In one embodiment of the present invention, the CH3 domain contained in the precursor polypeptide is of the human IgG class. In one embodiment of the present invention, the CH3 domain contained in the precursor polypeptide is of the human IgG1 subclass. In one embodiment of the present invention, the CH3 domain contained in the precursor polypeptide is of the human IgG4 subclass.

[0198] In one embodiment, the constant domain of the precursor polypeptide according to the invention is of the human IgG1 subclass. In one embodiment, the constant domain of the precursor polypeptide according to the invention is of the human IgG1 subclass. In one embodiment, the constant domain of the precursor polypeptide according to the invention is of the human IgG4 subclass.

[0199] In one embodiment, the precursor polypeptide lacks a CH4 domain.

[0200] In one embodiment of the invention, the constant domains of the precursor polypeptide according to the invention are of the same immunoglobulin subclass. In one embodiment of the invention, the variable domains and the constant domains of the precursor polypeptide according to the invention are of the same immunoglobulin subclass.

[0201] In one embodiment of the invention, the precursor polypeptide is an isolated precursor polypeptide. In one embodiment of the invention, the product polypeptide is an isolated product polypeptide.

[0202] In one embodiment, the heterodimeric precursor polypeptide or heterodimeric product polypeptide comprising a polypeptide chain comprising a CH3 domain comprises a full-length CH3 domain or a CH3 domain in which one or two C-terminal amino acid residues, i.e., G446 and / or K447, are absent.

[0203] In one embodiment, the first heterodimeric precursor polypeptide is monospecific. In one embodiment, the second heterodimeric precursor polypeptide is monospecific. In one embodiment, the heterodimeric product polypeptide is bispecific.

[0204] In one embodiment, the first heterodimeric precursor polypeptide is monospecific and comprises a portion of the second antigen-binding site; and the second heterodimeric precursor polypeptide is monospecific and comprises another portion of the second antigen-binding site. In said embodiment, the heterodimeric product polypeptide is trispecific.

[0205] In one embodiment, the first heterodimeric precursor polypeptide is bispecific. In one embodiment, the second heterodimeric precursor polypeptide is monospecific. In one embodiment, the heterodimeric product polypeptide is trispecific.

[0206] In one embodiment, the first heterodimeric precursor polypeptide is bispecific. In one embodiment, the second heterodimeric precursor polypeptide is bispecific. In one embodiment, the heterodimeric product polypeptide is tetraspecific.

[0207] In one embodiment, the first heterodimeric precursor polypeptide is monovalent. In one embodiment, the second heterodimeric precursor polypeptide is monovalent.

[0208] In one embodiment, the first heterodimeric precursor polypeptide is bivalent. In one embodiment, the second heterodimeric precursor polypeptide is bivalent.

[0209] In one embodiment, the first heterodimeric precursor polypeptide is trivalent. In one embodiment, the second heterodimeric precursor polypeptide is trivalent.

[0210] In one embodiment, the heterodimeric product polypeptide is bivalent. In one embodiment, the heterodimeric product polypeptide is trivalent. In one embodiment, the heterodimeric product polypeptide is tetravalent.

[0211] E) Further Components of the Precursor Polypeptide In one embodiment of the present invention, i) a polypeptide chain in a first heterodimeric precursor polypeptide comprising a CH3 domain that includes a knob mutation, and a polypeptide chain in a second heterodimeric precursor polypeptide comprising a CH3 domain that includes a hole mutation; or ii) a polypeptide chain in the first heterodimeric precursor polypeptide comprising a CH3 domain containing a hole mutation, and a polypeptide chain in the second heterodimeric precursor polypeptide comprising a CH3 domain containing a knob mutation. comprises a tagging moiety. In one embodiment, the tagging moiety is fused to the C-terminus of the CH3 domain. In one embodiment, the tagging moiety is an affinity tag. In one embodiment, the tagging moiety is a poly(his) tag, a C-tag, or a streptococcus-tag. In one embodiment, the tagging moiety is a poly(his) tag or a C-tag.

[0212] F) Methods for Producing Product Polypeptides In one aspect of the invention, a method for producing a heterodimeric product polypeptide is provided, the method comprising: a) contacting a first heterodimeric precursor polypeptide according to the present invention with a second heterodimeric precursor polypeptide to form a third heterodimeric polypeptide comprising at least one polypeptide chain comprising a CH3 domain from the first heterodimeric precursor polypeptide and at least one polypeptide chain comprising a CH3 domain from the second heterodimeric polypeptide; and b) recovering the third heterodimeric polypeptide Includes.

[0213] In one embodiment, a first heterodimeric precursor polypeptide according to the present invention is contacted with a second heterodimeric precursor polypeptide to form a third heterodimeric polypeptide comprising at least one polypeptide chain comprising a CH3 domain from the first heterodimeric precursor polypeptide and at least one polypeptide chain comprising a CH3 domain from the second heterodimeric polypeptide, and a fourth heterodimeric polypeptide comprising another polypeptide comprising a CH3 domain from the first heterodimeric precursor polypeptide and another polypeptide comprising a CH3 domain from the second heterodimeric precursor polypeptide. In one embodiment, the method further comprises recovering the fourth heterodimeric product polypeptide.

[0214] In one embodiment of the invention, the method includes forming a third heterodimeric product polypeptide and a fourth heterodimeric product polypeptide, wherein one of the product polypeptides does not comprise an antigen binding site that specifically binds to an antigen.

[0215] In one embodiment of the invention, the first heterodimeric precursor polypeptide comprises an antigen-binding portion that specifically binds to a first antigen, the second heterodimeric precursor polypeptide comprises an antigen-binding portion that specifically binds to a second antigen, and the third heterodimeric polypeptide comprises an antigen-binding portion that specifically binds to the first antigen and an antigen-binding portion that specifically binds to the second antigen.

[0216] In one embodiment, the contacting step is carried out in a liquid solution, hi one embodiment, the contacting step is carried out in a buffered solution.

[0217] In one embodiment, the recovering step is carried out by chromatography.

[0218] Hinge disulfide reduction In one embodiment of the present invention, the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide comprise a hinge region containing an interchain disulfide bond, and the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide are contacted in the presence of a reducing agent. If the precursor polypeptides comprise a hinge region with an interchain disulfide, exchange of polypeptide chains occurs only after reduction of the interchain disulfide with the reducing agent.

[0219] In one embodiment of the present invention, the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide have hinge regions that do not contain an interchain disulfide bond. In this case, exchange of polypeptide chains can occur in the absence of a reducing agent. Thus, in one embodiment, the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide have hinge regions that do not contain an interchain disulfide bond, and the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide are not contacted in the absence of a reducing agent.

[0220] purification In one embodiment, i) a polypeptide chain comprising a CH3 domain in a first heterodimeric precursor polypeptide that comprises a knob mutation and a polypeptide chain comprising a CH3 domain in a second heterodimeric precursor polypeptide that comprises a hole mutation; or ii) a polypeptide chain comprising a CH3 domain in a first heterodimeric precursor polypeptide that comprises a hole mutation and a polypeptide chain comprising a CH3 domain in a second heterodimeric precursor polypeptide that comprises a knob mutation comprise a tagging moiety as defined above, and the method comprises recovering the third heterodimeric polypeptide by tag-specific affinity chromatography. In an alternative embodiment, the method comprises recovering the fourth heterodimeric polypeptide by tag-specific affinity chromatography.

[0221] In one embodiment, the tagging moiety is a poly(his) tag and the method comprises purifying the polypeptide by metal chelate affinity chromatography. In one embodiment, the tagging moiety is a poly(his) tag and the method comprises purifying the polypeptide by nickel chelate affinity chromatography.

[0222] In one embodiment, the tagging moiety is a C-tag and the method comprises purifying the polypeptide by C-tag affinity chromatography.

[0223] In one embodiment of the invention, no interchain disulfide bond is formed between the two polypeptide chains comprising the CH3 domains of the first and second heterodimeric polypeptides, and the contacting is performed in the absence of a reducing agent.

[0224] G) Heterodimeric Product Polypeptide One aspect of the invention is a heterodimeric product polypeptide obtained by the method of producing a heterodimeric product polypeptide of the invention.

[0225] One aspect of the invention is a heterodimeric polypeptide, and in one embodiment, the heterodimeric product polypeptide comprises at least two polypeptide chains comprising a CH3 domain, wherein the two polypeptide chains comprising a CH3 domain associate with each other via the CH3 domain to form a heterodimer, one of the CH3 domains comprising a knob mutation and the other CH3 domain comprising a hole mutation; the heterodimeric polypeptide comprises a first antigen-binding moiety, wherein at least a portion of the first antigen-binding moiety is located on one of the two polypeptide chains comprising the CH3 domain; the heterodimeric polypeptide comprises a second antigen-binding moiety, wherein at least a portion of the second antigen-binding moiety is located on the other of the two polypeptide chains comprising the CH3 domain; The CH3 domain with the hole mutation is - replacement of S354 with a hydrophobic amino acid; - replacement of D356 with a positively charged amino acid; - replacement of E357 with a positively charged or hydrophobic amino acid; - replacement of D356 with a positively charged amino acid and replacement of E357 with a positively charged or hydrophobic amino acid; - replacement of S364 with a hydrophobic amino acid; - replacement of A368 with a hydrophobic amino acid; - replacement of E392 with a negatively charged amino acid; - replacement of T394 with a hydrophobic amino acid; - replacement of D399 with a hydrophobic amino acid and replacement of S400 with a positively charged amino acid; - replacement of D399 with a hydrophobic amino acid and replacement of F405 with a positively charged amino acid; - replacement of V407 with a hydrophobic amino acid; and - replacement of K409 with a negatively charged amino acid; and - Replacement of K439 with a negatively charged amino acid at least one amino acid substitution selected from the group The CH3 domain with the knob mutation is - replacement of Q347 with a positively charged amino acid and replacement of K360 with a negatively charged amino acid; - replacement of Y349 with a negatively charged amino acid; - replacement of L351 with a hydrophobic amino acid and replacement of E357 with a hydrophobic amino acid; - replacement of S364 with a hydrophobic amino acid; - replacement of W366 with a hydrophobic amino acid and replacement of K409 with a negatively charged amino acid; - replacement of L368 with a hydrophobic amino acid; - replacement of K370 with a negatively charged amino acid; - replacement of K370 with a negatively charged amino acid and replacement of K439 with a negatively charged amino acid; - replacement of K392 with a negatively charged amino acid; - replacement of T394 with a hydrophobic amino acid; - replacement of V397 with a hydrophobic amino acid; - replacement of D399 with a positively charged amino acid and replacement of K409 with a negatively charged amino acid; - replacement of S400 with a positively charged amino acid; - F405W; - Y407W; and - Replacement of K439 with a negatively charged amino acid The amino acid sequence of the present invention comprises at least one amino acid substitution selected from the group consisting of:

[0226] A heterodimeric (product) polypeptide according to the invention comprises two polypeptide chains comprising a CH3 domain, and both CH3 domains comprise a destabilizing mutation as defined above. All of the embodiments listed above for destabilizing mutations in heterodimeric precursor polypeptides of the invention apply to the heterodimeric product polypeptide, with the difference that the heterodimeric product polypeptide comprises destabilizing mutations in both CH3 domains.

[0227] Another product of the method for producing a heterodimeric product polypeptide, and therefore another aspect of the present invention, is a heterodimeric product polypeptide comprising two polypeptide chains comprising CH3 domains, preferably obtained by the method of the present invention, wherein both CH3 domains do not comprise a destabilizing mutation.

[0228] In one embodiment of the invention, the heterodimeric product polypeptide comprises two polypeptide chains comprising a CH3 domain, wherein both CH3 domains comprise a cysteine ​​mutation as defined above. In one embodiment of the invention, the heterodimeric product polypeptide comprises two polypeptide chains comprising a CH3 domain, wherein both CH3 domains do not comprise a cysteine ​​mutation as defined above.

[0229] H) Methods for producing multispecific polypeptides In one aspect of the present invention, there is provided a method for identifying a multispecific heterodimeric polypeptide, the method comprising: a) A method for producing a heterodimeric product polypeptide according to the present invention, comprising: - a first heterodimeric precursor polypeptide from the plurality of first heterodimeric precursor polypeptides comprising an antigen-binding portion that specifically binds to a first antigen; and - a second heterodimeric precursor polypeptide from the plurality of second heterodimeric precursor polypeptides comprising an antigen-binding portion that specifically binds to a second antigen; and generating a plurality of multispecific heterodimeric polypeptides by providing each combination of b) individually identifying the desired properties of each multispecific heterodimeric polypeptide from the plurality of multispecific heterodimeric polypeptides generated in step a); and c) selecting the multispecific heterodimeric polypeptide Includes.

[0230] In one embodiment of the invention, the multispecific heterodimeric polypeptide specifically binds to a first antigen and specifically binds to a second antigen.

[0231] In one embodiment, the multispecific heterodimeric polypeptides are selected based on the desired properties detected in step b).

[0232] In one embodiment, the desired property detected in step b) is the binding affinity of the multispecific heterodimeric polypeptide to a first antigen. In one embodiment, the desired property detected in step b) is the binding affinity of the multispecific heterodimeric polypeptide to a second antigen. In one embodiment, the desired property detected in step b) is the thermal stability of the multispecific heterodimeric polypeptide.

[0233] Another aspect of the present invention is a multispecific heterodimeric polypeptide obtained by the method for identifying a multispecific heterodimeric polypeptide of the present invention.

[0234] I) Recombination method The precursor polypeptides according to the invention are prepared by recombinant methods. Thus, the present invention also relates to a method for the preparation of a heterodimeric precursor polypeptide according to the invention, which method comprises culturing a host cell comprising a nucleic acid for encoding the heterodimeric precursor polypeptide under conditions suitable for expression of the precursor polypeptide.

[0235] In one aspect, a method of making a heterodimeric precursor polypeptide of the invention is provided, the method comprising culturing a host cell comprising nucleic acid(s) encoding the heterodimeric precursor polypeptide as provided above under conditions suitable for expression of the heterodimeric precursor polypeptide, and optionally recovering the heterodimeric precursor polypeptide from the host cell (or host cell medium).

[0236] In one embodiment, the method comprises transforming a host cell with an expression vector comprising a nucleic acid encoding a heterodimeric precursor polypeptide, culturing the host cell under conditions that allow synthesis of the heterodimeric precursor polypeptide, and recovering the heterodimeric precursor polypeptide from the host cell culture.

[0237] For recombinant production of heterodimeric precursor polypeptides, e.g., as described above, nucleic acids encoding the heterodimeric precursor polypeptides are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the polypeptide chains of the heterodimeric precursor polypeptides), or can be produced by recombinant methods or obtained by chemical synthesis.

[0238] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, heterodimeric precursor polypeptides can be produced in bacteria. For bacterial expression of polypeptides, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, K.A., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the heterodimeric precursor polypeptides can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0239] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast, including fungal and yeast strains in which the glycosylation pathway has been "humanized," are suitable cloning or expression hosts for vectors encoding the heterodimeric precursor polypeptides of the invention, resulting in polypeptides with partially or fully human glycosylation patterns. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0240] Suitable host cells for the expression of the (glycosylated) heterodimeric precursor polypeptides are also obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0241] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 59,591,776, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (PLANTIBODIES FOR ANTIBODY PRODUCTION IN TRANSGENIC PLANTS). TM See (describe the technology).

[0242] Vertebrate cells may 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 lines (e.g., 293 cells or 293T cells as described in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine 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 (e.g., Mather, JP et al., Annals NY Acad. Sci. 383 (1982) 44-68; 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 specific mammalian host cells suitable for antibody production, see, for example, Yazaki, P. and Wu, A. M., Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0243] In one embodiment, the host cell is eukaryotic, for example, a Chinese hamster ovary (CHO) cell or a lymphocytic cell (e.g., a Y0, NS0, Sp20 cell).

[0244] In one aspect, the present invention provides an isolated nucleic acid encoding a heterodimeric precursor polypeptide of the present invention. In one aspect, the present invention provides an expression vector comprising a nucleic acid according to the present invention. In another aspect, the present invention provides a host cell comprising a nucleic acid of the present invention.

[0245] J) Therapeutic Applications The set of heterodimeric precursor polypeptides of the present invention can be used in therapy. Accordingly, one aspect of the present invention is a set of heterodimeric precursor polypeptides according to the present invention for use as a pharmaceutical. Another aspect of the present invention includes a pharmaceutical composition comprising the set of heterodimeric precursor polypeptides of the present invention and a pharmaceutically acceptable carrier. Another aspect of the present invention is a method of treating an individual having a disease, the method comprising administering to the individual an effective amount of the first and second heterodimeric precursor polypeptides of the present invention or a pharmaceutical composition of the present invention.

[0246] In one embodiment, the heterodimeric precursor polypeptides used in the method of treatment comprise a hinge region as defined above, and the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide do not comprise an interchain disulfide bond in the hinge region. In the absence of an interchain disulfide bond in the hinge region, exchange of polypeptide chains occurs in the absence of a reducing agent and thus can occur spontaneously; for example, when both heterodimeric precursor polypeptides bind to a target antigen or target cell.

[0247] In one embodiment, the heterodimeric precursor polypeptide used in the method of treatment comprises an activatable antigen binding site as defined above.

[0248] Thus, in one embodiment, the heterodimeric precursor polypeptide used in therapy comprises a hinge region that does not contain an interchain disulfide bond; and an activatable antigen-binding site as defined above.

[0249] 3. Specific Embodiments of the Invention Specific embodiments of the present invention are listed below.

[0250] 1. A set of heterodimeric precursor polypeptides comprising: a) a first heterodimeric precursor polypeptide comprising at least two polypeptide chains each comprising a CH3 domain, wherein the two polypeptide chains each comprising a CH3 domain associate with each other via the CH3 domain to form a heterodimer, wherein one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; wherein the first heterodimeric precursor polypeptide comprises a first antigen-binding portion, and at least a portion of the first antigen-binding portion is located on one of the two polypeptide chains comprising the CH3 domain; and b) a second heterodimeric precursor polypeptide comprising at least two polypeptide chains comprising CH3 domains, wherein the two polypeptide chains comprising CH3 domains associate with each other via the CH3 domains to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; a second heterodimeric precursor polypeptide, wherein the second heterodimeric precursor polypeptide comprises a second antigen-binding portion, and at least a portion of the second antigen-binding portion is located on one of the two polypeptide chains comprising the CH3 domain; where: A) i) in the first heterodimeric precursor polypeptide, a polypeptide chain comprising a CH3 domain that includes a knob mutation comprises at least a portion of a first antigen-binding portion, and in the second heterodimeric precursor polypeptide, a polypeptide chain comprising a CH3 domain that includes a hole mutation comprises at least a portion of a second antigen-binding portion; or ii) in the first heterodimeric precursor polypeptide, the polypeptide chain comprising the CH3 domain comprising the hole mutation comprises at least a portion of a first antigen-binding portion, and in the second heterodimeric precursor polypeptide, the polypeptide chain comprising the CH3 domain comprising the knob mutation comprises at least a portion of a second antigen-binding portion; and B) i) a CH3 domain of a first heterodimeric precursor polypeptide comprising a knob mutation and a CH3 domain of a second heterodimeric precursor polypeptide comprising a hole mutation; or ii) a CH3 domain of a first heterodimeric precursor polypeptide comprising a hole mutation, and a CH3 domain of a second heterodimeric precursor polypeptide comprising a knob mutation. contains the following amino acid substitutions, numbered according to the Kabat numbering system: - CH3 domain with hole mutations · Replacement of S354 with a hydrophobic amino acid; · Replacement of D356 with a positively charged amino acid; Replacement of E357 with a positively charged or hydrophobic amino acid; replacement of D356 with a positively charged amino acid and replacement of E357 with a positively charged or hydrophobic amino acid; · Replacement of S364 with a hydrophobic amino acid; ·Replacement of A368 with a hydrophobic amino acid; Replacement of E392 with a negatively charged amino acid; · Replacement of T394 with a hydrophobic amino acid; · Replacement of D399 with a hydrophobic amino acid and replacement of S400 with a positively charged amino acid; · Replacement of D399 with a hydrophobic amino acid and replacement of F405 with a positively charged amino acid; replacement of V407 with a hydrophobic amino acid; and Replacement of K409 with a negatively charged amino acid; and Replacement of K439 with a negatively charged amino acid at least one amino acid substitution selected from the group - CH3 domain with knob mutations · Replacement of Q347 with a positively charged amino acid and replacement of K360 with a negatively charged amino acid; · Replacement of Y349 with a negatively charged amino acid; replacement of L351 with a hydrophobic amino acid and replacement of E357 with a hydrophobic amino acid; · Replacement of S364 with a hydrophobic amino acid; · Replacement of W366 with a hydrophobic amino acid and replacement of K409 with a negatively charged amino acid; ·Replacement of L368 with a hydrophobic amino acid; · Replacement of K370 with a negatively charged amino acid; · Replacement of K370 with a negatively charged amino acid and replacement of K439 with a negatively charged amino acid; · Replacement of K392 with a negatively charged amino acid; · Replacement of T394 with a hydrophobic amino acid; · Replacement of V397 with a hydrophobic amino acid; Replacement of D399 with a positively charged amino acid and replacement of K409 with a negatively charged amino acid; · Replacement of S400 with a positively charged amino acid; ·F405W; Y407W; and Replacement of K439 with a negatively charged amino acid The amino acid sequence of the present invention comprises at least one amino acid substitution selected from the group consisting of:

[0251] 2. The set of heterodimeric polypeptides according to embodiment 1, wherein the CH3 domain as shown in B) comprises the following amino acid substitutions: - the CH3 domain with hole mutations comprises at least one amino acid substitution selected from the group consisting of S354V, S354I, S354L, D356K, D356R, E357K, E357R, E357F, S364L, S364I, A368F, K392D, K392E, T394L, T394I, V407Y, K409E, K409D, K439D, K439E, and double mutations D399A S400K, D399A S400R, D399A F405W; - CH3 domains with knob mutations Y349E, Y349D, S364V, S364I, S364L, L368F, K370E, K370D, K392E, K392D, T394L, T394I, V397Y, S400K, S400R, F405W, Y407W, K349E, K439D, as well as double mutations Q347K K360E, Q347R K360E, Q347K K360D, Q347R K360D, L351F E357F, W366I K409E, W366L K409E, W366K K409D, W366L K409D, D399K K409E, D399R It comprises at least one amino acid substitution selected from the group consisting of K409E, D399K K409D, and D399K K409E.

[0252] 3. A set of heterodimeric polypeptides according to embodiment 1 or 2, wherein the CH3 domain as shown in B) comprises the following amino acid substitutions: - the CH3 domain with a hole mutation comprises at least one amino acid substitution selected from the group consisting of S354V, D356K, E357K, E357F, S364L, A368F, K392E, T394I, V407Y, K409E, K439E, and the double mutation D399A S400K; - the CH3 domain with a knob mutation comprises at least one amino acid substitution selected from the group of Y349E, S364V, L368F, K370E, K392D, T394I, V397Y, S400K, F405W, Y407W, K349E, and the double mutations Q347K K360E, L351F E357F, W366I K409E, and D399K K409E.

[0253] 4. A set of heterodimeric polypeptides according to any one of embodiments 1 to 3, wherein the CH3 domain as shown in B) comprises the following amino acid substitutions: - the CH3 domain with a hole mutation comprises at least one amino acid substitution selected from the group consisting of D356K, D356R, E357K, E357R, E357F, S364L, S364I, V407Y, K409E, K409D, and the double mutations D399A S400K, D399A S400R; - the CH3 domain with a knob mutation comprises at least one amino acid substitution selected from the group of Y349E, Y349D, K370E, K370D, K392E, K392D, T394L, T394I, V397Y, F405W, Y407W, K349E, K439D, and the double mutations Q347K K360E, Q347R K360E, Q347K K360D, Q347R K360D, W366I K409E, W366L K409E, W366K K409D, W366L K409D, D399K K409E, D399R K409E, D399K K409D, and D399K K409E.

[0254] 5. A set of heterodimeric polypeptides according to any one of embodiments 1 to 4, wherein the CH3 domain as shown in B) comprises the following amino acid substitutions: - the CH3 domain with a hole mutation comprises at least one amino acid substitution selected from the group consisting of D356K, E357K, E357F, S364L, V407Y, K409E, and the double mutation D399A S400K; - the CH3 domain with a knob mutation comprises at least one amino acid substitution selected from the group of Y349E, K370E, K392D, T394I, V397Y, F405W, Y407W, K349E, and the double mutations Q347K K360E, W366I K409E, and D399K K409E.

[0255] 6.B) A set of heterodimeric polypeptides according to any one of the preceding embodiments, wherein the CH3 domain comprising the knob mutation and the CH3 domain comprising the hole mutation comprise one of the amino acid substitutions selected from the group shown in the table below. TIFF2026004426000010.tif254131TIFF2026004426000011.tif63131

[0256] 7.B) CH3 domains containing knob mutations and CH3 domains containing hole mutations are shown in the following table: 10. The set of heterodimeric polypeptides of any one of the preceding embodiments, comprising one of the amino acid substitutions selected from the group set forth in TIFF2026004426000012.tif81131.

[0257] 8. A set of heterodimeric polypeptides according to any one of the preceding embodiments, wherein the CH3 domain having a knob mutation as shown in B) contains the mutation E357K, and the CH3 domain having a hole mutation as shown in B) does not contain the mutation K370E.

[0258] 9. A set of heterodimeric polypeptides according to any one of the preceding embodiments, wherein the CH3 domain having a knob mutation as shown in B) contains the mutation D356K, and the CH3 domain having a hole mutation as shown in B) does not contain the mutation K439E.

[0259] 10. A set of heterodimeric polypeptides according to any one of the preceding embodiments, wherein i) the CH3 domain comprising the knob mutation of the first heterodimeric precursor polypeptide comprises a cysteine ​​mutation and the CH3 domain comprising the hole mutation of the second heterodimeric precursor polypeptide comprises a cysteine ​​mutation, or ii) the CH3 domain comprising the hole mutation of the first heterodimeric precursor polypeptide comprises a cysteine ​​mutation and the CH3 domain comprising the knob mutation of the second heterodimeric precursor polypeptide comprises a cysteine ​​mutation.

[0260] 11. A set of heterodimeric polypeptides according to embodiment 10, wherein i) the CH3 domain comprising the knob mutation of the first heterodimeric precursor polypeptide comprises the substitution S354C and the CH3 domain comprising the hole mutation of the second heterodimeric precursor polypeptide comprises the substitution Y349C, or ii) the CH3 domain comprising the hole mutation of the first heterodimeric precursor polypeptide comprises the substitution Y349C and the CH3 domain comprising the knob mutation of the second heterodimeric precursor polypeptide comprises the substitution S354C.

[0261] 12. A set of heterodimeric polypeptides according to embodiment 10 or 11, wherein in the first heterodimeric precursor polypeptide, the CH3 domain comprising the knob mutation comprises a substitution S354C and the CH3 domain comprising the hole mutation comprises a Y at position 349; and in the second heterodimeric precursor polypeptide, the CH3 domain comprising the hole mutation comprises a substitution Y349C and the CH3 domain comprising the knob mutation comprises an S at position 354.

[0262] 13. A set of heterodimeric polypeptides according to any one of the preceding embodiments, wherein the first antigen-binding moiety and / or the second antigen-binding moiety comprise a pair of VH and VL domains that form an antigen-binding site that specifically binds to a target antigen.

[0263] 14. The set of heterodimeric polypeptides according to any one of the preceding embodiments, wherein the first antigen-binding moiety and / or the second antigen-binding moiety is an antibody fragment.

[0264] 15. a) a first heterodimeric precursor polypeptide comprising: - a first heavy chain polypeptide comprising a CH3 domain and a first antibody variable domain; - a second heavy chain polypeptide comprising a CH3 domain, wherein the first heavy chain polypeptide and the second heavy chain polypeptide associate with each other via the CH3 domain to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and - a light chain polypeptide comprising a second antibody variable domain, wherein the first and second antibody variable domains together form a first antigen-binding site that specifically binds to a target antigen. Includes; b) the second heterodimeric precursor polypeptide is - a third heavy chain polypeptide comprising a CH3 domain and a third antibody variable domain; - a fourth heavy chain polypeptide comprising a CH3 domain, wherein the third heavy chain polypeptide and the fourth heavy chain polypeptide associate with each other via the CH3 domain to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and - a light chain polypeptide comprising a fourth antibody variable domain, wherein the third and fourth antibody variable domains together form a second antigen-binding site that specifically binds to a target antigen. where c) i) the first heavy chain polypeptide comprises a CH3 domain comprising a knob mutation, and the third heavy chain polypeptide comprises a CH3 domain comprising a hole mutation; or ii) the first heavy chain polypeptide comprises a CH3 domain comprising a hole mutation, and the third heavy chain polypeptide comprises a CH3 domain comprising a knob mutation. 10. A set of heterodimeric polypeptides according to any one of the preceding embodiments.

[0265] 16. The set of heterodimeric polypeptides of any one of the preceding embodiments, wherein the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide comprise at least two polypeptide chains comprising a CH2 domain and a CH3 domain.

[0266] 17. The set of heterodimeric polypeptides of any one of the preceding embodiments, wherein the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide comprise, in the N-terminal to C-terminal direction, at least two polypeptide chains comprising a CH2 domain and a CH3 domain.

[0267] 18. The set of heterodimeric polypeptides of any one of the preceding embodiments, wherein the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide comprise at least two polypeptide chains comprising, in the N-terminal to C-terminal direction, a hinge region, a CH2 domain, and a CH3 domain.

[0268] 19. a) a first heterodimeric precursor polypeptide comprising: - a first heavy chain polypeptide comprising, in N-terminal to C-terminal direction, a first VH domain and constant antibody domains CH1, CH2 and CH3; - a second heavy chain polypeptide comprising, from N-terminal to C-terminal, constant antibody domains CH2 and CH3 domains, wherein the first and second heavy chain polypeptides associate with each other via the CH3 domains to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and - a light chain polypeptide comprising, from N-terminal to C-terminal, a first VL domain and a CL domain, wherein the first VH domain and the first VL domain bind to each other to form an antigen-binding site that specifically binds to a target antigen. Includes; b) the second heterodimeric precursor polypeptide is - a third heavy chain polypeptide comprising, in N-terminal to C-terminal direction, a second VH domain and constant antibody domains CH1, CH2 and CH3; - a fourth heavy chain polypeptide comprising, from N-terminal to C-terminal, constant antibody domains CH2 and CH3 domains, wherein the third heavy chain polypeptide and the fourth heavy chain polypeptide associate with each other via the CH3 domains to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and - a light chain polypeptide comprising, from N-terminal to C-terminal, a second VL domain and a CL domain, wherein the second VH domain and the second VL domain associate with each other to form an antigen-binding site that specifically binds to a target antigen. where i) the first heavy chain polypeptide comprises a CH3 domain comprising a knob mutation, and the third heavy chain polypeptide comprises a CH3 domain comprising a hole mutation; or ii) the first heavy chain polypeptide comprises a CH3 domain comprising a hole mutation, and the third heavy chain polypeptide comprises a CH3 domain comprising a knob mutation. A set of heterodimeric polypeptides according to any one of embodiments 15 to 18.

[0269] 20. The set of heterodimeric polypeptides of embodiment 19, wherein the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide comprise an interchain disulfide bond in the hinge region.

[0270] 21. A set of heterodimeric polypeptides according to any one of the preceding embodiments, wherein the antigen-binding sites are antibody fragments.

[0271] twenty two. i) a polypeptide chain in a first heterodimeric precursor polypeptide comprising a CH3 domain that includes a knob mutation, and a polypeptide chain in a second heterodimeric precursor polypeptide comprising a CH3 domain that includes a hole mutation; or ii) a polypeptide chain in the first heterodimeric precursor polypeptide comprising a CH3 domain containing a hole mutation, and a polypeptide chain in the second heterodimeric precursor polypeptide comprising a CH3 domain containing a knob mutation. 12. The set of heterodimeric polypeptides according to any one of the preceding embodiments, wherein said set of heterodimeric polypeptides comprises a tagging moiety.

[0272] 23. The set of heterodimeric polypeptides according to embodiment 22, wherein the tagging moiety is fused to the C-terminus of the CH3 domain.

[0273] 24. The set of heterodimeric polypeptides according to embodiment 22 or 23, wherein the tagging moiety is a histidine tag or a C-tag.

[0274] 25. A set of heterodimeric polypeptides according to any one of the preceding embodiments, wherein the first heterodimeric precursor polypeptide comprises one polypeptide chain comprising a VL domain and a CH3 domain, and the second heterodimeric precursor polypeptide comprises one polypeptide chain comprising a VH domain and a CH3 domain, and wherein the VL domain and the VH domain specifically bind to an antigen when associated into a VH domain-VL domain pair.

[0275] 26. a) a first heterodimeric precursor polypeptide comprising: - a first heavy chain polypeptide comprising, from N-terminal to C-terminal, a first VH domain, a CH1 domain, a second antibody variable domain selected from a VH domain and a VL domain, and a CH3 domain; - a second heavy chain polypeptide comprising, from N-terminal to C-terminal, an antibody variable domain capable of associating with a second antibody variable domain of the first heavy chain polypeptide, and a CH3 domain, wherein the first heavy chain polypeptide and the second heavy chain polypeptide associate with each other via the CH3 domain to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and - a light chain polypeptide comprising, from N-terminal to C-terminal, a first VL domain and a CL domain, wherein the first VH domain and the first VL domain bind to each other to form an antigen-binding site that specifically binds to a target antigen. Includes; b) the second heterodimeric precursor polypeptide is - a third heavy chain polypeptide comprising, in N-terminal to C-terminal direction, a second VH domain, a CH1 domain, a third antibody variable domain selected from a VH domain and a VL domain, and a CH3 domain; - a fourth heavy chain polypeptide comprising, from N-terminal to C-terminal, an antibody variable domain capable of associating with a third antibody variable domain of a third heavy chain polypeptide, and a CH3 domain, wherein the third heavy chain polypeptide and the fourth heavy chain polypeptide associate with each other via the CH3 domain to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and - a light chain polypeptide comprising, from N-terminal to C-terminal, a second VL domain and a CL domain, wherein the second VH domain and the second VL domain associate with each other to form an antigen-binding site that specifically binds to a target antigen. where c) i) the first heavy chain polypeptide comprises a CH3 domain comprising a knob mutation and the third heavy chain polypeptide comprises a CH3 domain comprising a hole mutation; or ii) the first heavy chain polypeptide comprises a CH3 domain comprising a hole mutation and the third heavy chain polypeptide comprises a CH3 domain comprising a knob mutation; d) the variable domains of the first heavy chain polypeptide and the third heavy chain polypeptide are capable of forming an antigen-binding site that specifically binds to a target antigen; 10. A set of heterodimeric polypeptides according to any one of the preceding embodiments.

[0276] 27. A set of heterodimeric polypeptides according to any one of the preceding embodiments, wherein the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide comprise at least two polypeptide chains comprising, in an N-terminal to C-terminal direction, a CH2 domain and a CH3 domain; the first heterodimeric precursor polypeptide comprises one polypeptide chain comprising, in an N-terminal to C-terminal direction, a VL domain, a CH2 domain, and a CH3 domain; and the second heterodimeric precursor polypeptide comprises one polypeptide chain comprising, in an N-terminal to C-terminal direction, a VH domain, a CH2 domain, and a CH3 domain, wherein the VL domain and the VH domain are capable of forming an antigen-binding site that specifically binds to a target antigen.

[0277] 28. a) a first heterodimeric precursor polypeptide comprising: - a first heavy chain polypeptide comprising, from N-terminal to C-terminal, a first VH domain, a CH1 domain, a second antibody variable domain selected from a VH domain and a VL domain, a CH2 domain, and a CH3 domain; - a second heavy chain polypeptide comprising, from N-terminal to C-terminal, an antibody variable domain capable of associating with a second antibody variable domain of the first heavy chain polypeptide, a CH2 domain, and a CH3 domain, wherein the first heavy chain polypeptide and the second heavy chain polypeptide associate with each other via the CH3 domain to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and - a light chain polypeptide comprising, from N-terminal to C-terminal, a first VL domain and a CL domain, wherein the first VH domain and the first VL domain bind to each other to form an antigen-binding site that specifically binds to a target antigen. Includes; b) the second heterodimeric precursor polypeptide is - a third heavy chain polypeptide comprising, from N-terminal to C-terminal, a second VH domain, a CH1 domain, a third antibody variable domain selected from a VH domain and a VL domain, a CH2 domain, and a CH3 domain; - a fourth heavy chain polypeptide comprising, from N-terminal to C-terminal, an antibody variable domain capable of associating with a third antibody variable domain of a third heavy chain polypeptide, a CH2 domain, and a CH3 domain, wherein the third heavy chain polypeptide and the fourth heavy chain polypeptide associate with each other via the CH3 domain to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and - a light chain polypeptide comprising, from N-terminal to C-terminal, a second VL domain and a CL domain, wherein the second VH domain and the second VL domain associate with each other to form an antigen-binding site that specifically binds to a target antigen. where c) i) the first heavy chain polypeptide comprises a CH3 domain comprising a knob mutation and the third heavy chain polypeptide comprises a CH3 domain comprising a hole mutation; or ii) the first heavy chain polypeptide comprises a CH3 domain comprising a hole mutation and the third heavy chain polypeptide comprises a CH3 domain comprising a knob mutation; d) the variable domains of the first heavy chain polypeptide and the third heavy chain polypeptide are capable of forming an antigen-binding site that specifically binds to a target antigen; 10. A set of heterodimeric polypeptides according to any one of the preceding embodiments.

[0278] 29. The set of heterodimeric precursor polypeptides of any one of the preceding embodiments, wherein the antigen-binding portion of the first heterodimeric precursor polypeptide and the antigen-binding portion of the second heterodimeric precursor polypeptide bind to the same antigen.

[0279] 30. The set of heterodimeric precursor polypeptides of any one of the preceding embodiments, wherein the antigen-binding portion of the first heterodimeric precursor polypeptide and the antigen-binding portion of the second heterodimeric precursor polypeptide bind to different antigens.

[0280] 31. A heterodimeric polypeptide comprising at least two polypeptide chains comprising a CH3 domain, wherein the two polypeptide chains comprising the CH3 domain associate with each other via the CH3 domain to form a heterodimer, wherein one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; the heterodimeric polypeptide comprises a first antigen-binding moiety, wherein at least a portion of the first antigen-binding moiety is located on one of the two polypeptide chains comprising the CH3 domain; the heterodimeric polypeptide comprises a second antigen-binding moiety, wherein at least a portion of the second antigen-binding moiety is located on the other of the two polypeptide chains comprising the CH3 domain; The CH3 domain with the hole mutation is - replacement of S354 with a hydrophobic amino acid; - replacement of D356 with a positively charged amino acid; - replacement of E357 with a positively charged or hydrophobic amino acid; - replacement of D356 with a positively charged amino acid and replacement of E357 with a positively charged or hydrophobic amino acid; - replacement of S364 with a hydrophobic amino acid; -Replacement of A368 with a hydrophobic amino acid; - replacement of E392 with a negatively charged amino acid; - replacement of T394 with a hydrophobic amino acid; - replacement of D399 with a hydrophobic amino acid and replacement of S400 with a positively charged amino acid; - replacement of D399 with a hydrophobic amino acid and replacement of F405 with a positively charged amino acid; - replacement of V407 with a hydrophobic amino acid; and - replacement of K409 with a negatively charged amino acid; and - Replacement of K439 with a negatively charged amino acid at least one amino acid substitution selected from the group The CH3 domain with the knob mutation is - replacement of Q347 with a positively charged amino acid and replacement of K360 with a negatively charged amino acid; - replacement of Y349 with a negatively charged amino acid; - replacement of L351 with a hydrophobic amino acid and replacement of E357 with a hydrophobic amino acid; - replacement of S364 with a hydrophobic amino acid; - replacement of W366 with a hydrophobic amino acid and replacement of K409 with a negatively charged amino acid; - replacement of L368 with a hydrophobic amino acid; - replacement of K370 with a negatively charged amino acid; - replacement of K370 with a negatively charged amino acid and replacement of K439 with a negatively charged amino acid; - replacement of K392 with a negatively charged amino acid; - replacement of T394 with a hydrophobic amino acid; - replacement of V397 with a hydrophobic amino acid; - replacement of D399 with a positively charged amino acid and replacement of K409 with a negatively charged amino acid; - replacement of S400 with a positively charged amino acid; - F405W; - Y407W; and - Replacement of K439 with a negatively charged amino acid containing at least one amino acid substitution selected from the group The heterodimeric polypeptide.

[0281] 32. A method for producing a heterodimeric polypeptide, comprising: a) contacting a first heterodimeric precursor polypeptide with a second heterodimeric precursor polypeptide as defined in any one of embodiments 1 to 30 to form a third heterodimeric polypeptide, the third heterodimeric polypeptide comprising at least one polypeptide chain comprising a CH3 domain from the first heterodimeric precursor polypeptide and at least one polypeptide chain comprising a CH3 domain from the second heterodimeric polypeptide; and b) recovering the third heterodimeric polypeptide The method comprising:

[0282] 33. The method of embodiment 32, wherein the first heterodimeric precursor polypeptide comprises an antigen-binding portion that specifically binds to a first antigen, the second heterodimeric precursor polypeptide comprises an antigen-binding portion that specifically binds to a second antigen, and the third heterodimeric polypeptide comprises an antigen-binding portion that specifically binds to the first antigen and an antigen-binding portion that specifically binds to the second antigen.

[0283] 34. i) a polypeptide chain in a first heterodimeric precursor polypeptide comprising a CH3 domain that includes a knob mutation, and a polypeptide chain in a second heterodimeric precursor polypeptide comprising a CH3 domain that includes a hole mutation; or ii) a polypeptide chain in the first heterodimeric precursor polypeptide comprising a CH3 domain containing a hole mutation, and a polypeptide chain in the second heterodimeric precursor polypeptide comprising a CH3 domain containing a knob mutation. 34. The method of embodiment 32 or 33, wherein said third heterodimeric polypeptide comprises a tagging moiety, and said method comprises recovering the third heterodimeric polypeptide by tag-specific affinity chromatography.

[0284] 35. The method of any one of embodiments 32-34, wherein the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide comprise an interchain disulfide bond in the hinge region, and the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide are contacted in the presence of a reducing agent.

[0285] 36. A heterodimeric polypeptide obtainable by the method according to any one of embodiments 32 to 35.

[0286] 37. A method for identifying a multispecific heterodimeric polypeptide, comprising: a) The method according to any one of embodiments 32 to 35, - a first heterodimeric precursor polypeptide from the plurality of first heterodimeric precursor polypeptides comprising an antigen-binding portion that specifically binds to a first antigen; and - a second heterodimeric precursor polypeptide from the plurality of second heterodimeric precursor polypeptides comprising an antigen-binding portion that specifically binds to a second antigen; and generating a plurality of multispecific heterodimeric polypeptides by providing each combination of b) individually identifying the desired properties of each multispecific heterodimeric polypeptide from the plurality of multispecific heterodimeric polypeptides generated in step a); and c) selecting the multispecific heterodimeric polypeptide The method comprising:

[0287] 38. The method of embodiment 37, wherein the desired property is selected from affinity and thermal stability.

[0288] 39. A multispecific heterodimeric polypeptide obtainable by the method according to embodiment 37 or 38.

[0289] 40. A set of heterodimeric precursor polypeptides according to any one of embodiments 1 to 30 for use as a medicament.

[0290] 41. A set of heterodimeric precursor polypeptides according to any one of embodiments 26 to 30 for use as a medicament.

[0291] 42. A set of heterodimeric precursor polypeptides according to any one of embodiments 26 to 30, for use as a medicament, wherein the heterodimeric precursor polypeptides comprise a hinge region, and the hinge region does not comprise an interchain disulfide bond.

[0292] 43. A pharmaceutical composition comprising a set of heterodimeric precursor polypeptides according to any one of embodiments 1 to 30 and a pharmaceutically acceptable carrier.

[0293] 44. The first heterodimeric precursor polypeptide as defined in any one of embodiments 1 to 30.

[0294] 45. The second heterodimeric precursor polypeptide as defined in any one of embodiments 1 to 30.

[0295] 46. ​​Use of a heterodimeric precursor polypeptide according to embodiment 44 or 45 in a method according to any one of embodiments 32 to 36.

[0296] Description of amino acid sequence TIFF2026004426000013.tif160146TIFF2026004426000014.tif237146TIFF2026004426000015.tif237146TIFF2026004426000016.tif237146 TIFF2026004426000017.tif237146TIFF2026004426000018.tif230146TIFF2026004426000019.tif236146TIFF2026004426000020.tif243146 [Example]

[0297] The following examples are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit or scope of the invention.

[0298] Example 1: Generation of monospecific precursor polypeptides containing a complete Fc domain To assess the formation of bispecific anti-biocytinamide / anti-fluorescein antibodies from monospecific precursor polypeptides, monospecific precursor polypeptides were generated with the domain configurations shown for the first and second heterodimeric precursor polypeptides shown in Figure 1.

[0299] The first heterodimeric precursor polypeptide (also referred to as "anti-Bio precursor") contained a Fab fragment that specifically binds to biocytinamide ("Bio"), a biotin derivative having a VL domain of SEQ ID NO: 01 and a VH domain of SEQ ID NO: 02 (described in Dengl S, et al. Hapten-directed spontaneous disulfide shuffling: a universal technology for site-directed covalent coupling of payloads to antibodies. FASEB J 2015;29:1763-1779). The first precursor polypeptide contained a light chain polypeptide of SEQ ID NO: 03 (also referred to as "Bio LC"), a first heavy chain polypeptide of SEQ ID NO: 04 (also referred to as "Bio HC"), and a second heavy chain polypeptide based on SEQ ID NO: 05 (representing the basic amino acid sequence without destabilizing mutations), with the destabilizing mutations shown below and a histidine tag. The second heavy chain polypeptide (also referred to as the "dummy hole" polypeptide) contained, from N- to C-terminal, a hinge region, a CH2 domain, and a CH3 domain.

[0300] The second heterodimeric precursor polypeptide (also referred to as the "anti-fluo precursor") comprised a Fab fragment that specifically binds fluorescein ("fluo") having a VL domain of SEQ ID NO: 06 and a VH domain of SEQ ID NO: 07. The second precursor polypeptide comprised a light chain polypeptide of SEQ ID NO: 08 (also referred to as "fluo LC"), a first heavy chain polypeptide of SEQ ID NO: 09 (also referred to as "fluo HC"), and a second heavy chain polypeptide based on SEQ ID NO: 10 (representing the basic amino acid sequence without destabilizing mutations), with the destabilizing mutations shown below and a histidine tag. The second heavy chain polypeptide (also referred to as the "dummy knob" polypeptide) comprised, from the N-terminal to C-terminal direction, a hinge region, a CH2 domain, and a CH3 domain.

[0301] The CH3 domain of the polypeptide chain shown contains the following mutations:

[0302] Table 1. Amino acid substitutions in the CH3 domain of the precursor polypeptide TIFF2026004426000021.tif51150

[0303] Antibio precursors were generated containing a dummy hole polypeptide having the amino acid sequence of SEQ ID NO: 05, in which one of the following amino acid substitutions was made: E357K, D356K, C349Y, C349A, C349W, E357F, A368F, F405W, V407Y, D399A F405W, L441Y, K409E, T394I, D356K E357K, L351Y, Q347K, S354V, K370E, S364L, K392E, K439E, or D399A S400K.

[0304] An anti-fluo precursor was generated containing a dummy knob polypeptide having the amino acid sequence of SEQ ID NO: 10, in which one of the following amino acid substitutions was made: K370E, K439E, C354S, C354S N297Q, S354E, S364L, Y407W, F405W, W366I K409E, K370E K439E, D399K K409E, Y349E, S364V, L368F, K392D, T394I, Q347K K360E, E357F, S400K, or L351F E357F.

[0305] An expression plasmid for the precursor polypeptide was generated as follows: For the expression of the antibio and antifluo precursors reported herein, the following transcription unit was used, containing the following functional elements: - the immediate early enhancer and promoter from human cytomegalovirus (P-CMV) containing intron A, -human heavy chain immunoglobulin 5'-untranslated region (5'UTR), - mouse immunoglobulin heavy chain signal sequence, - nucleic acids encoding the respective precursor polypeptides, and - Bovine growth hormone polyadenylation sequence (BGH pA). - a basic / standard mammalian expression plasmid, besides the expression unit / cassette containing the desired gene to be expressed, - an origin of replication from the vector pUC18 that allows replication of this plasmid in E. coli, and -Beta-lactamase gene that confers ampicillin resistance to E. coli Includes.

[0306] Recombinant production of precursor polypeptides Transient expression of the anti-biotin and anti-fluotin precursors reported herein was performed using the transfection reagent mix ExpiFectamine. TM 293 Transfection Kit (A14524; Life Technologies TM ) using Expi293F TM Expression medium (A1435101; Life Technologies TM ) adapted to suspension Expi293F TM Cells (A14527; Life Technologies TM ) was carried out.

[0307] Cells were passaged by diluting at least four times (30 ml volume) after thawing in 125 ml shake flasks (incubated / shaken at 37°C, 7% CO2, 85% humidity, 135 rpm). Cells were diluted to 3x10 in a volume of 250 ml. 5 After 3 days, the cells were split and grown to 1.3*10 cells / ml in a volume of 250 ml in a 1 liter shake flask. 6 The cells were freshly seeded at a density of approximately 2.2-2.8 x 10 cells / ml. Transfections were performed at approximately 2.2-2.8 x 10 cells / ml. 6 The results were performed after 24 hours at a cell density of 100 cells / ml.

[0308] Prior to transfection, 30 μg of plasmid DNA was diluted to a final volume of 1.5 ml with preheated (water bath; 37°C) Opti-MEM (Gibco). The solution was gently mixed and incubated at room temperature for a maximum of 5 minutes. ExpiFectamine in Opti-MEM was then added. TM1.5 ml of the reagent pre-incubation solution was added to the DNA-OptiMEM solution. The resulting solution was mixed gently and incubated at room temperature for 20-30 minutes. The total volume of the mixture was then adjusted to 30 ml of Expi293F. TM Cultures were added to deep wells in 48-well deep well plates, 50 ml Falcon tubes or 100 ml shake flasks.

[0309] Transfected cells were incubated for 7 days at 37°C, 7% CO , and 85% humidity, shaking at 110 rpm for shake flasks and 205 rpm for Falcon tubes.

[0310] 16–24 hours after transfection, 20 μl of ExpiFectamine TM Enhancer 1 and 200 μl of ExpiFectamine TM Enhancer2 was added to 30 ml of cell culture.

[0311] The supernatant was collected by centrifugation at 4,000 rpm for 20 minutes at 4° C. The cell-free supernatant was then filtered through a 0.22 μm bottle-top filter and stored in a freezer (−20° C.).

[0312] Antibodies were purified from cell culture supernatants by affinity chromatography using MabSelectSure-Sepharose™ (GE Healthcare, Sweden).

[0313] Briefly, sterile-filtered cell culture supernatant was captured onto MabSelectSuRe resin equilibrated with PBS buffer (10 mM NaHPO, 1 mM KHPO, 137 mM NaCl, and 2.7 mM KCl, pH 7.4), washed with equilibration buffer, and eluted with 25 mM sodium citrate at pH 3.0. Eluted fractions for each precursor polypeptide were pooled and neutralized with 2 M Tris, pH 9.0.

[0314] Alternatively, the precursor polypeptide was purified from cell culture supernatant by affinity chromatography using anti-Ckappa resin (KappaSelect, GE Healthcare, Sweden).

[0315] Briefly, sterile-filtered cell culture supernatant was captured onto KappaSelect resin equilibrated with PBS buffer (10 mM NaHPO, 1 mM KHPO, 137 mM NaCl, and 2.7 mM KCl, pH 7.4), washed with equilibration buffer, and eluted with 25 mM sodium citrate at pH 3.0. Eluted precursor polypeptide fractions were pooled and neutralized with 2 M Tris, pH 9.0.

[0316] The identity of the precursor polypeptide was confirmed by mass spectrometry. For each individual sample, conserved Fc N-glycosylation was enzymatically removed (using N-glycosidase F), the protein was denatured (guanidine hydrochloride), and disulfide bonds were reduced (using DTT or TCEP). Samples were desalted by liquid chromatography (by size exclusion or reversed-phase chromatography) and analyzed by mass spectrometry (Bruker Maxis Q-ToF). The identity of each molecule was confirmed by accurate mass measurement and comparison with the theoretically predicted molecular mass.

[0317] Analytical size-exclusion chromatography was performed on a BioSuite High Resolution SEC column (250 Å, Waters, USA) using a running buffer of 200 mM KHPO / KHPO, 250 mM KCl, pH 7.0 at a flow rate of 1 mg / ml. The monomer content of all individual precursor polypeptides was assessed prior to reaction setup.

[0318] (Table 2) Monomer content of antibio precursor polypeptides containing dummy hole chains with the indicated destabilizing mutations within the CH3 domain (LMW··· large molecular weight by-products; monomer = desired heterodimeric precursor polypeptide; HMW··· high molecular weight by-products). TIFF2026004426000022.tif157128

[0319] Table 3. Monomer content of anti-fluo precursor polypeptides containing dummy knob chains with the indicated destabilizing mutations within the CH3 domain (LMW··· large molecular weight by-products; monomer = desired heterodimeric precursor polypeptide; HMW··· high molecular weight by-products). TIFF2026004426000023.tif144128

[0320] Example 2: Analysis of polypeptide chain exchange efficiency by direct detection of bispecific product polypeptide formation by ELISA. To assess the effect of different destabilizing mutations on polypeptide chain exchange, 460 exchange reactions were set up using the precursor polypeptides generated in Example 1. The structures of the predicted product polypeptides are shown in Figure 1. The presence of the bispecific anti-biocytinamide / anti-fluorescein product polypeptides was assessed by ELISA.

[0321] To initiate the exchange reaction, the anti-bio precursor polypeptide and the anti-fluo precursor polypeptide were mixed in equimolar amounts in a 384-well REMP® plate (Brooks, #1800030) at a protein concentration of 2 μM in a total volume of 48 μl of 1x PBS + 0.05% Tween 20 + 0.25 mM TCEP (normalized to % monomer SEC values ​​to ensure equal amounts of intact molecules in a single reaction). In particular, the addition of the reducing agent TCEP reduces hinge disulfides, thus supporting the dissociation of polypeptide chains. After centrifugation, the plate was sealed and incubated for 1 hour at 37°C. The resulting reaction mixture was analyzed by ELISA.

[0322] A biotin-fluorescein bridging ELISA was then used to quantify the bispecific antibodies: Therefore, white Nunc® MaxiSorp TMA 384-well plate was coated with 1 μg / ml albumin-fluorescent isothiocyanate conjugate (Sigma, #A9771) and incubated overnight at 4°C. After washing three times with 90 μl of PBST-buffer (PBST, double-distilled water, 10x PBS + 0.05% Tween 20) blocking buffer (1x PBS, 2% gelatin, 0.1% Tween-20), 90 μl / well was added and incubated for 1 hour at room temperature. After washing three times with 90 μl of PBST-buffer, 25 μl of each reaction mixture diluted 1:4 was added to each well. After incubation at room temperature for 1 hour, the plate was again washed three times with 90 μl of PBST-buffer. 25 μl / well of biotin-Cy5 conjugate in 0.5% BSA, 0.025% Tween-20, 1x PBS was added to a final concentration of 0.1 μg / ml, and the plate was incubated for 1 hour at room temperature. After six washes with 90 μl of PBST-buffer, 25 μl of 1x PBS was added to each well. Cy5 fluorescence was measured on a Tecan Safire 2 Reader at 670 nm (excitation at 649 nm).

[0323] A preformed anti-fluorescein / anti-biocytinamide bispecific reference antibody (bio light chain of SEQ ID NO: 03, bio heavy chain of SEQ ID NO: 04, fluo light chain of SEQ ID NO: 08 and fluo heavy chain of SEQ ID NO: 09) was used as a 100% control for the reaction results.

[0324] The preformed bispecific reference antibodies were analyzed by analytical size exclusion chromatography as described above:

[0325] Table 4. Monomer content of bispecific reference antibodies TIFF2026004426000024.tif27138

[0326] The absorbance signal from the reference antibody in the bridging ELISA setup was averaged based on 23 reactions. This average value was used as the 100% bridging signal for normalization of all polypeptide chain exchange reactions. The assay variability of the reference antibody in the bridging ELISA is 100 + / - 15.2%. Polypeptide chain exchange reactions greater than 100% may be within this variability. In addition, potential aggregation that may occur in the reaction mixture may lead to an increase in the bridging signal.

[0327] The results are shown in Table 5. Relative absorbance values ​​based on pairs of CH3 mutations considered to support polypeptide chain exchange are underlined.

[0328] Table 5. Formation of bispecific product polypeptides by polypeptide chain reaction from anti-bio and anti-fluo precursor polypeptides containing the indicated destabilizing mutation(s) in the CH3 domain of the dummy chain. Columns indicate destabilizing mutations in the dummy hole polypeptide of the anti-bio precursor; rows indicate destabilizing mutations in the dummy knob polypeptide of the anti-fluo precursor. Relative absorbance detected by bridging ELISA is shown. TIFF2026004426000025.tif81143TIFF2026004426000026.tif230143TIFF2026004426000027.tif111143

[0329] Example 3: Analysis of polypeptide chain exchange efficiency by biochemical quantification of bispecific product formation A subset of anti-bio and anti-fluo precursors were reacted to form 86 bispecific product polypeptides. Equimolar amounts of the precursor polypeptides described in Example 1 were combined for the reaction. Freshly prepared TCEP (60 equivalents of 0.5 mM TCEP in 1x PBS pH 7.4, 0.05% Tween 20) was added to the reaction mixture as a reducing agent, and the mixture was incubated at 37°C for 1 hour with gentle shaking at 300 rpm.

[0330] Bispecific product polypeptides were isolated as the flow-through from a cOmplete His-Tag column (Roche Diagnostics GmbH) equilibrated with 50 mM NaHPO, 300 mM NaCl, pH 8.0, and a flow of 1 ml / min. Unreacted residual precursor and product polypeptides consisting of dummy chain heterodimers were retained by their histidine-tags and were isolated from the column by 50 mM NaHPO. 4、 For analytical purposes, the product was eluted with 300 mM NaCl, 250 mM imidazole (pH 8.0). Samples were concentrated to protein concentrations of 0.2–1.5 mg / ml using Amicon Ultra centrifuge tubes (Millipore) and analyzed by size-exclusion chromatography (SE-HPLC) using BioSuite High Resolution SEC Columns (250 Å, 5 μm, Waters, USA) at a flow rate of 0.5 ml / min with a running buffer of 200 mM KHPO / KHPO, 250 mM KCl, pH 7.0. The purity of the bispecific product formation was determined. CE-SDS (LabChip GXII (Perkin Elmer)) was used to determine the quality of the dual-specific product polypeptides (correct amount and presence of protein chains) under non-reducing (reformation of disulfide bridges) and reducing conditions. For CE-SDS, samples were prepared as follows: 5 μl, c = 0.1–1 mg / ml, were combined with 35 μl of sample buffer or sample denaturing solution in a 96-well PCR plate and incubated at 70°C for 10 min with gentle shaking. For reducing conditions, the reducing agent (NuPage) was diluted 1:10 in HT Protein Express sample buffer (e.g., 100 μl reducing agent + 900 μl sample buffer). Then, 70 μl of purified water was added to the sample, and the sample plate was placed in the LAbChip System for analysis.

[0331] Results from 86 chain exchange reactions are shown in Table 6. Results from pairs of CH3 mutations with particularly high product yields are underlined and bolded. Shown is the total yield (mg) of bispecific product polypeptide after polypeptide chain exchange and purification steps. Product yield in % is calculated as the relative amount of recombinant product corrected to the maximum expected product mass. For this calculation, the amount of precursor polypeptide was corrected to its monomer content as analyzed by analytical size exclusion chromatography, since only monomers are expected to be available for recombination. In addition, it was taken into account that the maximum expected product mass is limited by the low abundance of the precursor polypeptide.

[0332] Table 6. Yields of bispecific product polypeptides after polypeptide chain exchange and purification from anti-bio and anti-fluo precursor polypeptides containing the indicated destabilizing mutation(s) within the CH3 domain of the dummy chain. TIFF2026004426000028.tif50141TIFF2026004426000029.tif236141TIFF2026004426000030.tif236141TIFF2026004426000031.tif79141

[0333] Example 4: Generation of monospecific precursor polypeptides for the generation of activatable binding sites during polypeptide chain exchange. To assess the formation of bispecific anti-LeY / anti-CD3 antibodies from monospecific precursor polypeptides, monospecific precursor polypeptides were generated with the domain configurations shown for the first and second heterodimeric precursor polypeptides shown in Figures 2 and 3.

[0334] Precursor polypeptide lacking the CH2 domain In a first set of experiments, a heterodimeric precursor polypeptide was provided having the domain organization shown in Figure 2. This precursor polypeptide lacks a CH2 domain and contains an antibody variable domain positioned N-terminal to the CH3 domain.

[0335] In a first alternative, the following precursor polypeptide was provided: The first heterodimeric precursor polypeptide (also referred to as "anti-LeY-CD3(VH)-knob precursor") comprised a Fab fragment that specifically bound to LeY. The anti-LeY-CD3(VH)-knob precursor comprised a light chain polypeptide of SEQ ID NO: 11 (also referred to as "LeY LC"), a first heavy chain polypeptide of SEQ ID NO: 12 (also referred to as "LeY-CD3(VH)-knob HC") comprising a VH domain (CD3(VH)") derived from an antibody that specifically bound to CD3, and a second heavy chain polypeptide based on SEQ ID NO: 13 (representing a basic amino acid sequence without destabilizing mutations), with the destabilizing mutations shown below and a histidine tag. The second heavy chain polypeptide (also referred to as "dummy-VL-hole" polypeptide) comprised, from the N- to C-terminus, a hinge region, a VL domain derived from an antibody that specifically bound to digoxigenin ("dig"), and a CH3 domain. The second heterodimeric precursor polypeptide (also referred to as "anti-LeY-CD3(VL)-hole precursor") contained a Fab fragment specifically binding to LeY. The anti-LeY-CD3(VL)-hole precursor contained a light chain polypeptide of SEQ ID NO: 11, i.e., LeY LC; a first heavy chain polypeptide of SEQ ID NO: 14 (also referred to as "LeY-CD3(VL)-holeHC") containing a VL domain ("CD3(VL)") derived from an antibody specifically binding to CD3; and a second heavy chain polypeptide based on SEQ ID NO: 15 (representing a basic amino acid sequence without destabilizing mutations), with destabilizing mutations and a histidine tag. The second heavy chain polypeptide (also referred to as "dummy-VH-knob" polypeptide) contained, from the N- to C-terminus, a hinge region, a VH domain derived from a non-binding antibody, and a CH3 domain.

[0336] In a second alternative, the following precursor polypeptide was provided: The first heterodimeric precursor polypeptide (also referred to as "anti-LeY-CD3(VL)-knob precursor") contained a Fab fragment specifically binding to LeY. The anti-LeY-CD3(VL)-knob precursor contained a light chain polypeptide of SEQ ID NO: 11, i.e., LeY LC, a first heavy chain polypeptide of SEQ ID NO: 16 containing a CD3(VL) domain (also referred to as "LeY-CD3(VL)-knob HC"), and a second heavy chain polypeptide based on SEQ ID NO: 17 (representing a basic amino acid sequence without destabilizing mutations), with destabilizing mutations and a histidine tag. The second heavy chain polypeptide (also referred to as "dummy-VH-hole" polypeptide) contained, from the N- to C-terminus, a hinge region, a VH domain derived from a non-binding antibody, and a CH3 domain. The second heterodimeric precursor polypeptide (also referred to as "anti-LeY-CD3(VH)-hole precursor") contained a Fab fragment specifically binding to LeY. The anti-LeY-CD3(VH)-hole precursor contained a light chain polypeptide of SEQ ID NO: 11, i.e., LeY LC; a first heavy chain polypeptide of SEQ ID NO: 18 (also referred to as "LeY-CD3(VH)-holeHC") containing the CD3(VH) domain; and a second heavy chain polypeptide based on SEQ ID NO: 19 (representing a basic amino acid sequence without destabilizing mutations), with destabilizing mutations and a histidine tag. The second heavy chain polypeptide (also referred to as "dummy-VL-knob" polypeptide) contained, from the N- to C-terminus, a hinge region, a VL domain derived from an anti-dig antibody, and a CH3 domain.

[0337] The polypeptide chain shown contains the following mutations:

[0338] Table 7. Amino acid substitutions in the CH3 domain of the precursor polypeptide TIFF2026004426000032.tif150134

[0339] Precursor polypeptide containing an Fc domain In a second set of experiments, a heterodimeric precursor polypeptide was provided having the domain organization shown in Figure 3. The precursor polypeptide contained a complete Fc domain and an antibody variable domain positioned N-terminal to the CH2 domain.

[0340] In a first alternative, the following precursor polypeptide was provided: The first heterodimeric precursor polypeptide (also referred to as "anti-LeY-CD3(VH)-Fc(knob) precursor") contained a Fab fragment that specifically bound to LeY. The anti-LeY-CD3(VH)-Fc(knob) precursor contained a light chain polypeptide of SEQ ID NO: 11, i.e., LeY LC, a first heavy chain polypeptide of SEQ ID NO: 20 (also referred to as "LeY-CD3(VH)-Fc(knob)HC") containing a CD3(VH) domain, and a second heavy chain polypeptide based on SEQ ID NO: 21 (representing a basic amino acid sequence without destabilizing mutations), with the destabilizing mutations shown below and a histidine tag. The second heavy chain polypeptide (also referred to as "dummy-VL-Fc(hole)" polypeptide) contained, from the N- to C-terminus, a hinge region, a VL domain derived from an antibody that specifically binds to digoxigenin ("dig"), a CH2 domain, and a CH3 domain. The second heterodimeric precursor polypeptide (also referred to as "anti-LeY-CD3(VL)-Fc(hole) precursor") contained a Fab fragment that specifically bound to LeY. The anti-LeY-CD3(VL)-Fc(hole) precursor comprised a first heavy chain polypeptide of SEQ ID NO: 22 (also referred to as "LeY-CD3(VL)-Fc(hole)HC") containing the LeY LC;CD3(VL) domain, and a second heavy chain polypeptide based on SEQ ID NO: 23 (representing a basic amino acid sequence without destabilizing mutations), with the destabilizing mutations shown below and a histidine tag. The second heavy chain polypeptide (also referred to as "dummy-VH-Fc(knob)" polypeptide) comprised, from the N-terminus to the C-terminus, a hinge region, a VH domain derived from an anti-dig antibody, a CH2 domain, and a CH3 domain.

[0341] In a second alternative, the following precursor polypeptide was provided: The first heterodimeric precursor polypeptide (also referred to as "anti-LeY-CD3(VL)-Fc(knob) precursor") contained a Fab fragment specifically binding to LeY. The anti-LeY-CD3(VL)-Fc(knob) precursor comprised a first heavy chain polypeptide of SEQ ID NO: 24 (also referred to as "LeY-CD3(VL)-Fc(knob)HC") comprising an LeY LC, a CD3(VL) domain, and a second heavy chain polypeptide based on SEQ ID NO: 25 (representing a basic amino acid sequence without destabilizing mutations), with the destabilizing mutations shown below and a histidine tag. The second heavy chain polypeptide (also referred to as "dummy-VH-Fc(hole)" polypeptide) comprised, from the N-terminus to the C-terminus, a hinge region, a VH domain derived from an anti-dig antibody, a CH2 domain, and a CH3 domain. The second heterodimeric precursor polypeptide (also referred to as "anti-LeY-CD3(VH)-Fc(hole) precursor") contained a Fab fragment specifically binding to LeY. The anti-LeY-CD3(VH)-Fc(hole) precursor comprised a first heavy chain polypeptide of SEQ ID NO: 26 (also referred to as "LeY-CD3(VH)-Fc(hole)HC") containing the LeY LC;CD3(VH) domain, and a second heavy chain polypeptide based on SEQ ID NO: 27 (representing a basic amino acid sequence without destabilizing mutations), with the destabilizing mutations shown below and a histidine tag. The second heavy chain polypeptide (also referred to as "dummy-VL-Fc(knob)" polypeptide) comprised, from the N-terminus to the C-terminus, a hinge region, a VL domain derived from an anti-dig antibody, a CH2 domain, and a CH3 domain.

[0342] The polypeptide chain shown contains the following mutations:

[0343] Table 8. Amino acid substitutions in the CH3 domain of the precursor polypeptide TIFF2026004426000033.tif142151

[0344] Heterodimeric precursor polypeptides were generated comprising the dummy VL-hole polypeptide of SEQ ID NO: 13 and the dummy VH-hole polypeptide of SEQ ID NO: 17 shown above, having the amino acid sequences of the respective dummy polypeptides shown above, and one of the following amino acid substitutions was made: E357K, A368F, D399A F405W, S364L, Y407W, or S354V.

[0345] Heterodimeric precursor polypeptides were generated comprising the dummy VH-knob polypeptide of SEQ ID NO: 15 shown above and the dummy VL-knob polypeptide of SEQ ID NO: 19 shown above, with the amino acid sequences of each dummy polypeptide shown above, and one of the following amino acid substitutions was made: K370E, no destabilizing mutation, W366I K409D, V397Y, or K392D.

[0346] Heterodimeric precursor polypeptides were generated comprising the dummy VL-Fc(hole) polypeptide of SEQ ID NO: 21 and the dummy-VH-Fc(hole) polypeptide of SEQ ID NO: 25 shown above, having the amino acid sequences of the respective dummy polypeptides shown above, and one of the following amino acid substitutions was made: E357K, A368F, D399A F405W, S364L, D356K, or S354V.

[0347] Heterodimeric precursor polypeptides were generated comprising the dummy-VH-Fc(knob) polypeptide of SEQ ID NO: 23 and the dummy-VL-Fc(knob) polypeptide of SEQ ID NO: 27 shown above, with the amino acid sequences of each dummy polypeptide shown above, and one of the following amino acid substitutions was made: K370E, no destabilizing mutation, W366I K409D, V397Y, K392D, or K370E K439E.

[0348] Recombinant production of precursor polypeptides Expression is achieved by infecting mammalian cells (e.g., HEK293 or Expi293F) with plasmids of the three polypeptide chains of each precursor polypeptide according to conventional techniques. TM ) by co-transfection.

[0349] For the expression of the precursor polypeptides shown above, a transcription unit was used containing the following functional elements: - the immediate early enhancer and promoter from human cytomegalovirus (P-CMV) containing intron A, -human heavy chain immunoglobulin 5'-untranslated region (5'UTR), - mouse immunoglobulin heavy chain signal sequence, - nucleic acids encoding the respective precursor polypeptides, and - 3'-untranslated region with polyadenylation signal sequence.

[0350] Besides the expression unit / cassette containing the desired gene to be expressed, a basic / standard mammalian expression plasmid contains: - an origin of replication that allows replication of this plasmid in E. coli, and - The beta-lactamase gene that confers ampicillin resistance to E. coli Includes.

[0351] Expression cassettes encoding precursor polypeptide chains were generated by PCR and / or gene synthesis and assembled by known recombinant methods and techniques, e.g., by joining matching nucleic acid segments using unique restriction sites in each plasmid. Subcloned nucleic acid sequences were confirmed by DNA sequencing. For transient transfections, large quantities of plasmids were prepared by plasmid preparation from transformed E. coli cultures (HiSpeed ​​Plasmid Maxi Kit, Qiagen).

[0352] Standard cell culture techniques were used 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.

[0353] Precursor polypeptide derivatives were synthesized using the HEK293-F system (Invitrogen) or Expi293F according to the manufacturer's instructions. TM The cells were generated by transient transfection with the respective plasmids using the serum-free FreeStyle system (Live Technologies). TM 293 expression medium (Invitrogen) or Expi293F TM HEK293-F cells (Invitrogen) or Expi293F grown in suspension in stirred fermentors or shake flasks in expression medium (Life Technologies) TM Cells (Live Technologies) were transfected with the respective expression plasmids and 293fectin TM , fectin (Invitrogen) or PEIpro (Polyplus) or the reagent mix ExpiFectamine TM HEK293-F cells or Expi293F cells were transfected using the 293 Transfection Kit (Life Technologies). For 1-2 L shake flasks (Corning), TM Cells were added at 1-1.3*10 in 250-600 mL. 6 Cells were seeded at a density of approximately 1.5*10 cells / mL and incubated at 120 rpm and 8% CO2. The following day, cells were transfected with the appropriate expression plasmid. HEK293-F cells were transfected at approximately 1.5*10 cells / mL using approximately 42 mL of a mix of A) 20 mL of Opti-MEM (Invitrogen) containing 300 μg total plasmid DNA (0.5 μg / mL) and B) 20 mL of Opti-MEM + 1.2 mL of 293fectin or fectin (2 μL / mL) or 750 μl of PEIpro (1.25 μL / mL). 6 Transfected at a cell density of 1000 cells / mL. TM The cells are approximately 2.2-2.8x10 6Cells were transfected at a cell density of 1000 cells / mL. Prior to transfection, 30 μg of plasmid DNA was diluted to a final volume of 1.5 ml with preheated (water bath; 37°C) Opti-MEM (Gibco). The solution was gently mixed and incubated at room temperature for a maximum of 5 minutes. ExpiFectamine in Opti-MEM was then added. TM 1.5 ml of the reagent pre-incubation solution was added to the DNA-OptiMEM solution. The resulting solution was mixed gently and incubated at room temperature for 20-30 minutes. The total volume of the mixture was then adjusted to 30 ml of Expi293F. TM The culture was incubated at 37°C, 7% CO2, 85% humidity, and 110 rpm for 7 days. TM For cultures, 20 μl ExpiFectamine TM Enhancer 1 and 200 μl of ExpiFectamine TM Enhancer2 was added to 30 ml cell cultures for 15–24 hours after transfection. Glucose solution was added over the course of fermentation according to glucose consumption. Correctly assembled split cytokine molecules were secreted into the culture supernatant like standard IgG. The supernatant containing the split cytokine molecules was harvested after 5–10 days, and the split cytokine molecules were either purified directly from the supernatant or frozen and stored at −20°C.

[0354] Precursor polypeptides with the complete Fc region (CH2-CH3) bind to Protein A. These precursors were purified by Protein A chromatography followed by SEC.

[0355] The precursor polypeptides lacked the CH2 domain and contained a kappa light chain. Therefore, these precursors were purified by standard kappa light chain affinity chromatography. The precursor polypeptides were purified from cell culture supernatants by affinity chromatography using KappaSelect (GE Healthcare, Sweden) and Superdex 200 size exclusion (GE Healthcare, Sweden) or ion exchange chromatography.

[0356] Briefly, sterile-filtered cell culture supernatant was captured onto KappaSelect resin equilibrated with PBS buffer (10 mM NaHPO, 1 mM KHPO, 137 mM NaCl, and 2.7 mM KCl, pH 7.4), washed with equilibration buffer, and eluted with 50 mM sodium citrate, 150 mM NaCl, pH 3.0. The eluted precursor polypeptide fractions were pooled and neutralized with 2 M Tris, pH 9.0. The precursor polypeptide pool was further purified by size-exclusion or ion-exchange chromatography. For size-exclusion chromatography, the resin was loaded onto a Superdex column equilibrated with 20 mM histidine, 140 mM NaCl, pH 6.0. TM 200pg HiLoad TM For ion exchange chromatography, the protein sample obtained from the KappaSelect purification was diluted 1:10 in 20 mM histidine, pH 6.0, and transferred to a HiTrap column equilibrated with buffer A (20 mM histidine, pH 6.0). TM The sample was loaded onto an SP HP ion exchange (GE Healthcare, Sweden) column, and a gradient of 0-100% buffer B (20 mM histidine, 1 M NaCl, pH 6.0) was applied to elute the different protein species.

[0357] Purity and integrity were analyzed after purification by SDS-PAGE. Protein solution (13 μl) was mixed with 5 μl 4x NuPAGE LDS sample buffer (Invitrogen) and 2 μl 10x NuPAGE sample reducing agent (Invitrogen) and heated to 95°C for 5 minutes. Samples were loaded onto NuPAGE 4-12% Bis-Tris gels (Invitrogen) and run using a Novex Mini-Cell (Invitrogen) and NuPAGE MES SDS running buffer (Life Technologies) according to the manufacturer's instructions. Gels were run using InstantBlue TM Coomassie protein stain was used for staining, and protein integrity and homogeneity were further analyzed using analytical size exclusion chromatography.

[0358] (CE-)SDS-PAGE revealed that all predicted polypeptide chains were present in the preparation; analytical size exclusion confirmed that the preparation was >90% pure. For a review of methods for assessing antibody purity, see, for example, Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0359] Example 5: Determination of polypeptide chain exchange by T cell activation assay To evaluate the effect of different destabilizing mutations on polypeptide chain exchange, exchange reactions were set up using the precursor polypeptides produced in Example 4. The structures of the predicted product polypeptides are shown in Figure 2 for the precursor polypeptide lacking the CH2 domain and in Figure 3 for the precursor polypeptide containing the complete Fc domain. The exchange of polypeptide chains results in the formation of an antigen-binding site that specifically binds to CD3. The presence of the bispecific anti-LeY / anti-CD3 product polypeptide was evaluated by a cell-based assay.

[0360] The effects of different CH3 interface mutations on the efficiency of this chain exchange reaction were evaluated in a cell-based reporter assay system consisting of LeY-expressing MCF7 cells and a Jurkat reporter cell line (Promega J1621) according to the following principle: binding of the first and second heterodimeric polypeptides to MCF7 cells and exchange of polypeptide chains results in the formation of an antigen-binding site that specifically binds to CD3. Jurkat cells expressing CD3 are bound by the antigen-binding site that specifically binds to CD3, resulting in the expression of luciferase from the Jurkat cells. Luminescence was detected after the addition of BioGlo substrate.

[0361] Briefly, cell-based assays were performed in 384-well plates as follows: RPMI1640 with 10% FCS was used as the assay medium. 4 Jurkat effector cells were cultured at 2 x 10 in a total volume of 10 μl. 4 The precursor polypeptides were applied alone or in combination at 200 nM and 2 nM in a final volume of 30 μl. The cells were incubated for 20 hours under cell culture conditions. 24 μl of Bioglo was added to each well and incubated for 5 minutes. Luminescence was measured using an Infinite® 200 PRO reader (TECAN).

[0362] Table 9. Formation of bispecific product polypeptides by polypeptide chain reaction from precursor polypeptides lacking the CH2 domain as defined above in Example 4, containing the indicated destabilizing mutation(s) in the CH3 domain of the dummy chain. Results are shown from exchange reactions at a precursor polypeptide concentration of 200 nM. Luminescence efficiency is assessed as follows: <10% "-", 10-29% "+", 30-50% "++", >50% "+++"). TIFF2026004426000034.tif148143

[0363] Table 10. Formation of bispecific product polypeptides by polypeptide chain reaction from precursor polypeptides lacking the CH2 domain as defined above in Example 4, containing the indicated destabilizing mutation(s) in the CH3 domain of the dummy chain. Results are shown from exchange reactions at a precursor polypeptide concentration of 2 nM. Luminescence efficiency is assessed as follows: <2% "-", 2-4% "+", 5-10% "++", >10% "+++" TIFF2026004426000035.tif148141

[0364] Table 11. Formation of bispecific product polypeptides by polypeptide chain reaction from precursor polypeptides comprising an Fc domain as defined above in Example 4, containing the indicated destabilizing mutation(s) in the CH3 domain of the dummy chain. Results are shown from exchange reactions at a precursor polypeptide concentration of 2 nM. Luminescence efficiency is assessed as follows: <10% "-", 10-19% "+", 20-50% "++", >50% "+++" TIFF2026004426000036.tif78141

[0365] Example 6: Generation of additional monospecific precursor polypeptides containing a complete Fc domain To assess the formation of bispecific anti-biocytinamide / anti-fluorescein antibodies from monospecific precursor polypeptides, we generated monospecific precursor polypeptides of the domain configurations shown for the first and second heterodimeric precursor polypeptides shown in Figure 1. Note that in this experiment, the knob and hole mutations were placed on opposite strands from each other.

[0366] The first heterodimeric precursor polypeptide (also referred to as the "anti-fluo precursor") comprised a Fab fragment that specifically bound to the biotin derivative fluorescein ("fluo"), having a VL domain of SEQ ID NO: 06 and a VH domain of SEQ ID NO: 07. The first precursor polypeptide comprised a light chain polypeptide of SEQ ID NO: 08 (also referred to as "fluo LC"), a first heavy chain polypeptide of SEQ ID NO: 29 (also referred to as "fluo HC"), and a second heavy chain polypeptide based on SEQ ID NO: 05 (representing the basic amino acid sequence without destabilizing mutations), with the destabilizing mutations shown below and a C-tag. The second heavy chain polypeptide (also referred to as the "dummy hole" polypeptide) comprised, from the N- to C-terminus, a hinge region, a CH2 domain, and a CH3 domain.

[0367] The second heterodimeric precursor polypeptide (also referred to as the "anti-Bio precursor") comprised a Fab fragment that specifically bound biocytinamide ("Bio") having a VL domain of SEQ ID NO: 01 and a VH domain of SEQ ID NO: 02. The second precursor polypeptide comprised a light chain polypeptide of SEQ ID NO: 03 (also referred to as "Bio LC"), a first heavy chain polypeptide of SEQ ID NO: 28 (also referred to as "Bio HC"), and a second heavy chain polypeptide based on SEQ ID NO: 10 (representing the basic amino acid sequence without destabilizing mutations), with the destabilizing mutations shown below and a C-tag. The second heavy chain polypeptide (also referred to as the "dummy knob" polypeptide) comprised, from the N- to C-terminus, a hinge region, a CH2 domain, and a CH3 domain.

[0368] The first and second heterodimeric precursor polypeptides were produced according to the methods disclosed in Example 1.

[0369] The CH3 domain of the polypeptide chain shown contains the following mutations:

[0370] Table 12. Purification yield and monomer content of anti-Fluo precursor polypeptides containing dummy hole chains with the indicated destabilizing mutations in the CH3 domain (purification yield [mg / ml] = amount of purified antibody / liter of expression volume, corrected for % monomer peak; monomer = desired heterodimeric precursor polypeptide). TIFF2026004426000037.tif105128

[0371] Table 13. Purification yield and monomer content of anti-bio precursor polypeptides containing dummy knob chains with the indicated destabilizing mutations in the CH3 domain (purification yield [mg / ml] = amount of purified antibody / liter of expression volume, corrected for % monomer peak; monomer = desired heterodimeric precursor polypeptide). TIFF2026004426000038.tif105128

[0372] Example 7: Analysis of polypeptide chain exchange efficiency of precursor polypeptides from Example 6 To assess the effect of different destabilizing mutations on polypeptide chain exchange, exchange reactions were performed between the precursor polypeptides produced in Example 6. The experiments were carried out according to the methods described in Example 2. The structures of the predicted product polypeptides are shown in Figure 1.

[0373] Table 14. Formation of bispecific product polypeptides by polypeptide chain exchange reaction from anti-bio and anti-fluo precursor polypeptides containing the indicated destabilizing mutation(s) in the CH3 domain of the dummy chain. Columns indicate destabilizing mutations in the dummy hole polypeptide of the anti-fluo precursor; rows indicate destabilizing mutations in the dummy knob polypeptide of the anti-bio precursor. Values ​​indicate exchange efficiency as product yield [%]. Experimental yields relate to the maximum possible yield of bispecific antibody. Because only monomers are expected to be effective for recombination, the maximum possible yield of bispecific antibody is corrected by the lowest % monomer peak SEC of the two respective input formats in each reaction. TIFF2026004426000039.tif152143TIFF2026004426000040.tif235143TIFF2026004426000041.tif170143

[0374] Example 8: Generation of additional monospecific precursor polypeptides containing a complete Fc domain, where the CH3 domain of the precursor polypeptide contains knob-into-hole mutations but no cysteine ​​mutations. To assess the formation of bispecific anti-biocytinamide / anti-fluorescein antibodies from monospecific precursor polypeptides, we generated monospecific precursor polypeptides of the domain configurations shown for the first and second heterodimeric precursor polypeptides shown in Figure 1. Note that in this experiment, the knob and hole mutations were placed on opposite strands from each other.

[0375] The first and second heterodimeric precursor polypeptides described in Example 6 were produced according to the structures and methods disclosed herein.

[0376] Further deviations from Example 1 include BioHC, which is based on SEQ ID NO: 28 but has a serine residue at position 354, and FluoHC, which is based on SEQ ID NO: 29 but has a tyrosine residue at position 349. Thus, the mutations in the CH3 domain are summarized as follows:

[0377] Table 15. Amino acid substitutions in the CH3 domain of the precursor polypeptide TIFF2026004426000042.tif59146

[0378] The CH3 domain of the polypeptide chain shown contains the following mutations:

[0379] Table 16. Purification yield and monomer content of anti-bio precursor polypeptides containing dummy knob chains with the indicated destabilizing mutations in the CH3 domain (purification yield [mg / ml] = amount of purified antibody / liter of expression volume, corrected for % monomer peak; monomer = desired heterodimeric precursor polypeptide). TIFF2026004426000043.tif105128

[0380] Table 17. Purification yield and monomer content of anti-Fluo precursor polypeptides containing dummy hole chains with the indicated destabilizing mutations in the CH3 domain (purification yield [mg / ml] = amount of purified antibody / liter of expression volume, corrected for % monomer peak; monomer = desired heterodimeric precursor polypeptide). TIFF2026004426000044.tif112128

[0381] Example 9: Analysis of polypeptide chain exchange efficiency of precursor polypeptides from Example 8 To assess the effect of different destabilizing mutations on polypeptide chain exchange, exchange reactions were performed between the precursor polypeptides produced in Example 8. The experiments were carried out according to the methods described in Example 2.

[0382] Table 18. Formation of bispecific product polypeptides by polypeptide chain exchange reaction from anti-bio and anti-fluo precursor polypeptides containing the indicated destabilizing mutation(s) in the CH3 domain of the dummy chain. Columns indicate destabilizing mutations in the dummy hole polypeptide of the anti-fluo precursor; rows indicate destabilizing mutations in the dummy knob polypeptide of the anti-bio precursor. Values ​​indicate exchange efficiency as product yield [%]. Experimental yields relate to the maximum possible yield of bispecific antibody. Because only monomers are expected to be effective for recombination, the maximum possible yield of bispecific antibody is corrected by the lowest % monomer peak SEC of the two respective input formats in each reaction. TIFF2026004426000045.tif111141TIFF2026004426000046.tif233141TIFF2026004426000047.tif123141

[0383] The results demonstrate that polypeptide chain exchange is detectable for the heterodimeric precursor polypeptide and that the knob-into-hole mutation is not stabilized by additional cysteine ​​mutations.

[0384] Example 10: Generation of further monospecific precursor polypeptides comprising a complete Fc domain with different mutations in the CH3 domain of the precursor polypeptide To assess the formation of bispecific anti-biocytinamide / anti-fluorescein antibodies from monospecific precursor polypeptides, we generated monospecific precursor polypeptides of the domain configurations shown for the first and second heterodimeric precursor polypeptides shown in Figure 1. Note that in this experiment, the knob and hole mutations were placed on opposite strands from each other.

[0385] The first and second heterodimeric precursor polypeptides described in Example 6 were produced according to the structures and methods disclosed herein, but with the following differences:

[0386] Deviating from Example 6, three precursor polypeptides were generated that specifically bind fluorescein, where the FluoHC is based on SEQ ID NO:29 and the DummyHole polypeptide is based on SEQ ID NO:05, with the following CH3 mutations: TIFF2026004426000048.tif127148

[0387] - Deviating from Example 1, three precursor polypeptides were generated that specifically bind to biocytinamide, where the BioHC is based on SEQ ID NO: 28 and the Dummy Knob polypeptide is based on SEQ ID NO: 10, with the following CH3 mutations: TIFF2026004426000049.tif176148

[0388] Table 19: Purification yield and monomer content of the indicated precursor polypeptides (purification yield [mg / ml] = amount of purified antibody / liter of expression volume, corrected for % monomer peak; monomer = desired heterodimeric precursor polypeptide). TIFF2026004426000050.tif53128

[0389] Example 11: Analysis of polypeptide chain exchange efficiency of precursor polypeptides from Example 10 To assess the effect of different destabilizing mutations on polypeptide chain exchange, exchange reactions were carried out between the precursor polypeptides produced in Example 10. The experiments were carried out according to the methods described in Example 2.

[0390] Table 20: Formation of bispecific product polypeptides by polypeptide chain exchange reactions from the indicated anti-bio and anti-fluo precursor polypeptides. Values ​​indicate exchange efficiency as product yield [%]. Experimental yields relate to the maximum possible yield of bispecific antibody. Because only monomers are expected to be available for recombination, the maximum possible yield of bispecific antibody is corrected by the lowest % monomer peak SEC of the two respective input formats in each reaction. TIFF2026004426000051.tif29128

[0391] The results show that polypeptide chains arise regardless of whether the cysteine ​​mutations are placed in the dummy polypeptide or in the polypeptide chain containing the antigen-binding moiety.

Claims

1. A set of heterodimeric precursor polypeptides comprising: a) a first heterodimeric precursor polypeptide comprising at least two polypeptide chains comprising a CH3 domain, wherein the two polypeptide chains comprising the CH3 domain associate with each other via the CH3 domain to form a heterodimer, one of the CH3 domains comprising a knob mutation and the other CH3 domain comprising a hole mutation; wherein the first heterodimeric precursor polypeptide comprises a first antigen-binding portion, and at least a portion of the first antigen-binding portion is located on one of the two polypeptide chains comprising a CH3 domain; and b) a second heterodimeric precursor polypeptide comprising at least two polypeptide chains comprising a CH3 domain, wherein the two polypeptide chains comprising the CH3 domain associate with each other via the CH3 domain to form a heterodimer, one of the CH3 domains comprising a knob mutation and the other CH3 domain comprising a hole mutation; a second heterodimeric precursor polypeptide, wherein the second heterodimeric precursor polypeptide comprises a second antigen-binding portion, and at least a portion of the second antigen-binding portion is located on one of the two polypeptide chains comprising the CH3 domain; where: A) i) in a first heterodimeric precursor polypeptide, a polypeptide chain comprising a CH3 domain that comprises a knob mutation comprises at least a portion of a first antigen-binding portion, and in a second heterodimeric precursor polypeptide, a polypeptide chain comprising a CH3 domain that comprises a hole mutation comprises at least a portion of a second antigen-binding portion; or ii) in the first heterodimeric precursor polypeptide, a polypeptide chain comprising a CH3 domain comprising a hole mutation comprises at least a portion of a first antigen-binding portion, and in the second heterodimeric precursor polypeptide, a polypeptide chain comprising a CH3 domain comprising a knob mutation comprises at least a portion of a second antigen-binding portion; B) i) a CH3 domain of a first heterodimeric precursor polypeptide comprising a knob mutation and a CH3 domain of a second heterodimeric precursor polypeptide comprising a hole mutation; or ii) a CH3 domain of a first heterodimeric precursor polypeptide comprising a hole mutation, and a CH3 domain of a second heterodimeric precursor polypeptide comprising a knob mutation. contains the following amino acid substitutions, numbered according to the Kabat numbering system: - CH3 domain with hole mutations Replacement of S354 with a hydrophobic amino acid; Replacement of D356 with a positively charged amino acid; replacement of E357 with a positively charged or hydrophobic amino acid; replacement of D356 with a positively charged amino acid and replacement of E357 with a positively charged or hydrophobic amino acid; Replacement of S364 with a hydrophobic amino acid; Replacement of A368 with a hydrophobic amino acid; Replacement of E392 with a negatively charged amino acid; Replacement of T394 with a hydrophobic amino acid; Replacement of D399 with a hydrophobic amino acid and replacement of S400 with a positively charged amino acid; Replacement of D399 with a hydrophobic amino acid and replacement of F405 with a positively charged amino acid; replacement of V407 with a hydrophobic amino acid; and replacement of K409 with a negatively charged amino acid; and Replacement of K439 with a negatively charged amino acid at least one amino acid substitution selected from the group - CH3 domain with knob mutations Replacement of Q347 with a positively charged amino acid and replacement of K360 with a negatively charged amino acid; Replacement of Y349 with a negatively charged amino acid; replacement of L351 with a hydrophobic amino acid and replacement of E357 with a hydrophobic amino acid; Replacement of S364 with a hydrophobic amino acid; Replacement of W366 with a hydrophobic amino acid and replacement of K409 with a negatively charged amino acid; Replacement of L368 with a hydrophobic amino acid; Replacement of K370 with a negatively charged amino acid; replacement of K370 with a negatively charged amino acid and replacement of K439 with a negatively charged amino acid; Replacement of K392 with a negatively charged amino acid; Replacement of T394 with a hydrophobic amino acid; Replacement of V397 with a hydrophobic amino acid; Replacement of D399 with a positively charged amino acid and replacement of K409 with a negatively charged amino acid; Replacement of S400 with a positively charged amino acid; · F405W; Y407W; and Replacement of K439 with a negatively charged amino acid The amino acid sequence of the present invention comprises at least one amino acid substitution selected from the group consisting of:

2. 3. The set of heterodimeric polypeptides according to claim 1 or 2, wherein the CH3 domain as shown in b) comprises the following amino acid substitutions: - the CH3 domain with a hole mutation comprises at least one amino acid substitution selected from the group of S354V, D356K, E357K, E357F, S364L, A368F, K392E, T394I, V407Y, K409E, K439E, and the double mutation D399A S400K; the CH3 domain with knob mutations comprises at least one amino acid substitution selected from the group of Y349E, S364V, L368F, K370E, K392D, T394I, V397Y, S400K, F405W, Y407W, K349E, and the double mutations Q347K K360E, L351F E357F, W366I K409E, and D399K K409E.

3. 5. The set of heterodimeric polypeptides according to any one of claims 1 to 4, wherein the CH3 domains shown in b) comprise the following amino acid substitutions: - the CH3 domain with a hole mutation comprises at least one amino acid substitution selected from the group of D356K, E357K, E357F, S364L, V407Y, K409E, and the double mutation D399A S400K; the CH3 domain with knob mutations comprises at least one amino acid substitution selected from the group of Y349E, K370E, K392D, T394I, V397Y, F405W, Y407W, K349E, and the double mutations Q347K K360E, W366I K409E, and D399K K409E.

4. A set of heterodimeric polypeptides according to any one of the preceding claims, wherein when the CH3 domain having the knob mutation shown in b) contains the mutation E357K, the CH3 domain having the hole mutation shown in b) does not contain the mutation K370E; and when the CH3 domain having the knob mutation shown in b) contains the mutation D356K, the CH3 domain having the hole mutation shown in b) does not contain the mutation K439E.

5. A set of heterodimeric polypeptides according to any one of the preceding claims, wherein i) the CH3 domain comprising the knob mutation of the first heterodimeric precursor polypeptide comprises a cysteine ​​mutation, and the CH3 domain comprising the hole mutation of the second heterodimeric precursor polypeptide comprises a cysteine ​​mutation, or ii) the CH3 domain comprising the hole mutation of the first heterodimeric precursor polypeptide comprises a cysteine ​​mutation, and the CH3 domain comprising the knob mutation of the second heterodimeric precursor polypeptide comprises a cysteine ​​mutation.

6. 6. The set of heterodimeric polypeptides of claim 5, wherein in the first heterodimeric precursor polypeptide, the CH3 domain comprising the knob mutation comprises a substitution S354C and the CH3 domain comprising the hole mutation comprises a Y at position 349; and in the second heterodimeric precursor polypeptide, the CH3 domain comprising the hole mutation comprises a substitution Y349C and the CH3 domain comprising the knob mutation comprises an S at position 354.

7. 10. A set of heterodimeric polypeptides according to any one of the preceding claims, wherein the first antigen-binding moiety and / or the second antigen-binding moiety is an antibody fragment.

8. a) a first heterodimeric precursor polypeptide comprising: a first heavy chain polypeptide comprising a CH3 domain and a first antibody variable domain, - a second heavy chain polypeptide comprising a CH3 domain, wherein the first heavy chain polypeptide and the second heavy chain polypeptide associate with each other via the CH3 domain to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and a light chain polypeptide comprising a second antibody variable domain, wherein the first and second antibody variable domains together form a first antigen-binding site that specifically binds to a target antigen; Including; b) the second heterodimeric precursor polypeptide comprises: a third heavy chain polypeptide comprising a CH3 domain and a third antibody variable domain, - a fourth heavy chain polypeptide comprising a CH3 domain, wherein the third heavy chain polypeptide and the fourth heavy chain polypeptide associate with each other via the CH3 domain to form a heterodimer, and one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation; and a light chain polypeptide comprising a fourth antibody variable domain, wherein the third and fourth antibody variable domains together form a second antigen-binding site that specifically binds to a target antigen; including c) i) the first heavy chain polypeptide comprises a CH3 domain comprising a knob mutation and the third heavy chain polypeptide comprises a CH3 domain comprising a hole mutation; or ii) the first heavy chain polypeptide comprises a CH3 domain comprising a hole mutation and the third heavy chain polypeptide comprises a CH3 domain comprising a knob mutation. A set of heterodimeric polypeptides according to any one of the preceding claims.

9. A set of heterodimeric polypeptides described in any one of the preceding claims, wherein the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide comprise at least two polypeptide chains comprising, in the N-terminal to C-terminal direction, a hinge region, a CH2 domain, and a CH3 domain.

10. 10. The set of heterodimeric polypeptides of claim 9, wherein the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide comprise an interchain disulfide bond in the hinge region.

11. A set of heterodimeric polypeptides according to any one of the preceding claims, wherein the first heterodimeric precursor polypeptide comprises one polypeptide chain comprising a VL domain and a CH3 domain, and the second heterodimeric precursor polypeptide comprises one polypeptide chain comprising a VH domain and a CH3 domain, and the VL domain and the VH domain specifically bind to an antigen when associated into a VH domain and VL domain pair.

12. A heterodimeric polypeptide comprising at least two polypeptide chains comprising a CH3 domain, wherein the two polypeptide chains comprising the CH3 domain associate with each other via the CH3 domain to form a heterodimer, one of the CH3 domains comprising a knob mutation and the other CH3 domain comprising a hole mutation; the heterodimeric polypeptide comprises a first antigen-binding portion, at least a portion of which is located on one of the two polypeptide chains comprising the CH3 domain; the heterodimeric polypeptide comprises a second antigen-binding portion, at least a portion of which is located on the other of the two polypeptide chains comprising the CH3 domain; The CH3 domain with the hole mutation is - replacement of S354 with a hydrophobic amino acid; - replacement of D356 with a positively charged amino acid; - replacement of E357 with a positively charged or hydrophobic amino acid; - replacement of D356 with a positively charged amino acid and replacement of E357 with a positively charged or hydrophobic amino acid; - replacement of S364 with a hydrophobic amino acid; - replacement of A368 with a hydrophobic amino acid; - replacement of E392 with a negatively charged amino acid; - replacement of T394 with a hydrophobic amino acid; - replacement of D399 with a hydrophobic amino acid and replacement of S400 with a positively charged amino acid; - replacement of D399 with a hydrophobic amino acid and replacement of F405 with a positively charged amino acid; - replacement of V407 with a hydrophobic amino acid; and - replacement of K409 with a negatively charged amino acid; and - Replacement of K439 with a negatively charged amino acid at least one amino acid substitution selected from the group The CH3 domain with the knob mutation is - replacement of Q347 with a positively charged amino acid and replacement of K360 with a negatively charged amino acid; - replacement of Y349 with a negatively charged amino acid; - replacement of L351 with a hydrophobic amino acid and replacement of E357 with a hydrophobic amino acid; - replacement of S364 with a hydrophobic amino acid; - replacement of W366 with a hydrophobic amino acid and replacement of K409 with a negatively charged amino acid; - replacement of L368 with a hydrophobic amino acid; - replacement of K370 with a negatively charged amino acid; - replacement of K370 with a negatively charged amino acid and replacement of K439 with a negatively charged amino acid; - replacement of K392 with a negatively charged amino acid; - replacement of T394 with a hydrophobic amino acid; - replacement of V397 with a hydrophobic amino acid; - replacement of D399 with a positively charged amino acid and replacement of K409 with a negatively charged amino acid; - replacement of S400 with a positively charged amino acid; - F405W; - Y407W; and - Replacement of K439 with a negatively charged amino acid containing at least one amino acid substitution selected from the group The heterodimeric polypeptide.

13. 1. A method for producing a heterodimeric polypeptide, comprising: a) contacting a first heterodimeric precursor polypeptide of any one of claims 1 to 12 with a second heterodimeric precursor polypeptide to form a third heterodimeric polypeptide comprising at least one polypeptide chain comprising a CH3 domain from the first heterodimeric precursor polypeptide and at least one polypeptide chain comprising a CH3 domain from the second heterodimeric polypeptide; and b) recovering the third heterodimeric polypeptide The method comprising:

14. i) a polypeptide chain in a first heterodimeric precursor polypeptide comprising a CH3 domain with a knob mutation and a polypeptide chain in a second heterodimeric precursor polypeptide comprising a CH3 domain with a hole mutation; or ii) a polypeptide chain comprising a CH3 domain with a hole mutation in a first heterodimeric precursor polypeptide, and a polypeptide chain comprising a CH3 domain with a knob mutation in a second heterodimeric precursor polypeptide.

14. The method of claim 13, wherein said third heterodimeric polypeptide comprises a tagging moiety, and said method comprises recovering the third heterodimeric polypeptide by tag-specific affinity chromatography.

15. A heterodimeric polypeptide obtainable by the method of claim 13 or 14.

16. 1. A method for identifying a multispecific heterodimeric polypeptide, comprising: a) the method according to any one of claims 13 to 15, a first heterodimeric precursor polypeptide from a plurality of first heterodimeric precursor polypeptides comprising an antigen-binding portion that specifically binds to a first antigen; and a second heterodimeric precursor polypeptide from a plurality of second heterodimeric precursor polypeptides comprising an antigen-binding portion that specifically binds to a second antigen; and generating a plurality of multispecific heterodimeric polypeptides by providing each combination of b) individually detecting the desired property of each multispecific heterodimeric polypeptide from the plurality of multispecific heterodimeric polypeptides generated in step a); and c) selecting a multispecific heterodimeric polypeptide The method comprising:

17. 38. The method of claim 37, wherein the desired property is selected from affinity and thermal stability.

18. A multispecific heterodimeric polypeptide obtainable by the method of claim 16 or 17.

19. The first heterodimeric precursor polypeptide according to any one of claims 1 to 12.

20. A second heterodimeric precursor polypeptide according to any one of claims 1 to 12.

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