Activatable therapeutic multispecific polypeptides with extended half-lives - Patents.com

Abstract

The present invention relates to a set of heterodimeric polypeptides and their use in therapy, for example to treat cancer.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a set of heterodimeric polypeptides and their use to generate multispecific antigen binding agents, for example by polypeptide chain exchange. [Background technology]

[0002] 2. Background of the Invention Cancer treatment with bispecific antibodies targeting antigens expressed on the surface of cancer and T cells, for example via CD3, and thereby mediating ADCC against cancer cells, presents a medication challenge due to off-target T cell activation, which is undesirable.

[0003] EP 3180361 discloses a precursor molecule in which a binding site that specifically binds to CD3 is activated on a target cell. Such a precursor molecule comprises a Fab fragment, in which a CH2 domain and, for example, a variable antibody domain that binds to CD3 are fused to the C-terminus of the Fab fragment. When two precursor molecules containing different variable domains bind to a target cell, a functional antigen binding site (e.g., binding to CD3) is formed by association of the variable domains.

[0004] Labrijn, AF, et al. disclose efficient generation of stable bispecific IgG1 by controlled Fab arm exchange (Proc. Natl. Acad. Sci. USA 110 (2013) 5145-5150 (Non-Patent Document 1)). Briefly, two monospecific precursor molecules of IgG-like domain sequences with point mutations in the CH3 domain are contacted and undergo polypeptide chain exchange to form bispecific product molecules of IgG-like domain sequences.

[0005] The unpublished prior art PCT / EP2018 / 078675 and PCT / EP2018 / 079523 disclose methods for generating multispecific antigen-binding agents from two different precursor molecules by polypeptide chain exchange. Both precursor molecules are heterodimeric polypeptides with asymmetric domain arrangements. Both precursor molecules contain CH3 domains modified according to the "knobs-into-holes" technique (WO 96 / 027011; Ridgway, JB, et al., Protein Eng. 9 (1996) 617-621; and Merchant, AM, et al., Nat. Biotechnol. 16 (1998) 677-681) and containing additional destabilizing mutations arranged in an asymmetric pattern. In each of the precursor molecules, only one of the CH3 domains contains such destabilizing mutations. During polypeptide chain exchange, two product molecules are formed, where each product molecule contains one polypeptide each from the precursor molecule. The precursor and product molecules are of different domain arrangements. PCT / EP2018 / 078675 (Patent Document 2) and PCT / EP2018 / 079523 (Patent Document 3) disclose the amino acid positions at the CH3 / CH3 interface of the precursor molecule to be replaced.

[0006] However, there remains a need for additional methods for generating multispecific antigen-binding agents by polypeptide chain exchange in therapy. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] European Patent No. 3180361 [Patent Document 2] PCT / EP2018 / 078675 [Patent Document 3] PCT / EP2018 / 079523 [Patent Document 4] International Publication No. 96 / 027011 [Non-patent literature]

[0008] [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; and Merchant, AM, et al., Nat. Biotechnol. 16 (1998) 677-681 Summary of the Invention

[0009] The present invention relates to a set of heterodimeric precursor polypeptides comprising: a) a first heterodimeric precursor polypeptide, - a first heavy chain polypeptide comprising, from N-terminus to C-terminus, an antibody variable domain selected from a VH domain and a VL domain, and a CH3 domain, the first heavy chain polypeptide comprising at least a portion of a first antigen-binding portion; and - a second heavy chain polypeptide comprising, from the N-terminus to the C-terminus, a CH2 domain and a CH3 domain; Including, wherein the first heavy chain polypeptide and the second 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; b) a second heterodimeric precursor polypeptide, - a third heavy chain polypeptide comprising, from N-terminal to C-terminal, an antibody variable domain selected from a VH domain and a VL domain and a CH3 domain, wherein the antibody variable domain is capable of forming an antigen-binding site that specifically binds to a target antigen together with an antibody variable domain comprised in a first heavy chain polypeptide of the first heterodimeric precursor polypeptide, and wherein the third heavy chain polypeptide comprises at least a portion of a second antigen-binding portion; and - a fourth heavy chain polypeptide comprising, from the N-terminus to the C-terminus, a CH2 domain and a CH3 domain; Including, a second heterodimeric precursor polypeptide, wherein the third heavy chain polypeptide and the fourth heavy chain polypeptide 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; Where: A) i) the first heavy chain polypeptide comprises a CH3 domain having a knob mutation and the third heavy chain polypeptide comprises a CH3 domain having a hole mutation; or ii) the first heavy chain polypeptide comprises a CH3 domain with a hole mutation and the third heavy chain polypeptide comprises a CH3 domain with a knob mutation; 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 said first heterodimeric precursor polypeptide comprising a hole mutation, and a CH3 domain of said second heterodimeric precursor polypeptide comprising said knob mutation. any of the above 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.

[0010] One embodiment of the present invention relates to a set of heterodimeric polypeptides of the invention, wherein the amino acid substitutions that destabilize the CH3 / CH3 interface are selected from one or more of the following amino acid substitutions, the numbering being according to the Kabat numbering system: - the CH3 domain having a hole mutation Substitution of S354 with a hydrophobic amino acid, Substitution of D356 with a positively charged amino acid, Substitution of E357 with a positively charged or hydrophobic amino acid, Substitution of D356 with a positively charged amino acid and substitution of E357 with a positively charged or hydrophobic amino acid, Substitution of S364 with a hydrophobic amino acid, Substitution of A368 with a hydrophobic amino acid, Substitution of K392 with a negatively charged amino acid, Substitution of T394 with a hydrophobic amino acid, Substitution of D399 with a hydrophobic amino acid and substitution of S400 with a positively charged amino acid, Substitution of D399 with a hydrophobic amino acid and substitution of F405 with a positively charged amino acid, Substitution of V407 with a hydrophobic amino acid, Substitution of K409 with a negatively charged amino acid, and Substitution of K439 with a negatively charged amino acid At least one amino acid substitution selected from the group - said CH3 domain having a knob mutation Substitution of Q347 with a positively charged amino acid and K360 with a negatively charged amino acid, Substitution of Y349 with a negatively charged amino acid, Substitution of L351 with a hydrophobic amino acid and substitution of E357 with a hydrophobic amino acid, Substitution of S364 with a hydrophobic amino acid, Substitution of W366 with a hydrophobic amino acid and K409 with a negatively charged amino acid, Substitution of L368 with a hydrophobic amino acid, Substitution of K370 with a negatively charged amino acid, Substitution of K370 with a negatively charged amino acid and substitution of K439 with a negatively charged amino acid, Substitution of K392 with a negatively charged amino acid, Substitution of T394 with a hydrophobic amino acid, Substitution of V397 with a hydrophobic amino acid, Substitution of D399 with a positively charged amino acid and K409 with a negatively charged amino acid, Substitution of S400 with a positively charged amino acid, 〇 F405W, 〇 Y407W, and Substitution of K439 with a negatively charged amino acid The present invention comprises at least one amino acid substitution selected from the group consisting of:

[0011] One embodiment of the present invention relates to a set of heterodimeric polypeptides of the invention, wherein the amino acid substitutions that destabilize the CH3 / CH3 interface are selected from one or more of the following amino acid substitutions, the numbering being according to the Kabat numbering system: - the CH3 domain having a hole mutation Substitution of E357 with a positively charged amino acid, Substitution of S364 with a hydrophobic amino acid, Substitution of A368 with a hydrophobic amino acid, and Substitution of V407 with a hydrophobic amino acid and comprising at least one amino acid substitution selected from the group consisting of - said CH3 domain having a knob mutation Substitution of K370 with a negatively charged amino acid, Substitution of K370 with a negatively charged amino acid and substitution of K439 with a negatively charged amino acid, Substitution of K392 with a negatively charged amino acid, and Substitution of V397 with a hydrophobic amino acid The present invention comprises at least one amino acid substitution selected from the group consisting of:

[0012] One embodiment of the present invention relates to a set of heterodimeric polypeptides of the invention, wherein the first antigen-binding portion and / or the second antigen-binding portion is an antibody fragment.

[0013] One embodiment of the present invention relates to a set of heterodimeric polypeptides of the invention, wherein in a first heterodimeric polypeptide, no interchain disulfide bond is formed between the two polypeptide chains comprising a CH3 domain, and in a second heterodimeric polypeptide, no interchain disulfide bond is formed between the two polypeptide chains comprising a CH3 domain.

[0014] One embodiment of the present invention relates to a set of heterodimeric polypeptides of the invention, wherein the antibody variable domains comprised in the first heavy chain polypeptide and the third heavy chain polypeptide are capable of forming an antigen binding site that specifically binds to CD3.

[0015] Another aspect of the invention is a method for producing a heterodimeric polypeptide comprising contacting a first heterodimeric precursor polypeptide according to the invention with a second heterodimeric precursor polypeptide to form a third heterodimeric polypeptide comprising a first heavy chain polypeptide and a third heavy chain polypeptide.

[0016] One embodiment of the present invention relates to a method of producing a heterodimeric polypeptide of the invention comprising contacting a first heterodimeric precursor polypeptide with a second heterodimeric precursor polypeptide to form a fourth heterodimeric polypeptide comprising a second heavy chain polypeptide and a fourth heavy chain polypeptide.

[0017] One embodiment of the present invention relates to a method, wherein in the first heterodimeric polypeptide, no interchain disulfide bond is formed between the two polypeptide chains comprising a CH3 domain, and in the second heterodimeric polypeptide, no interchain disulfide bond is formed between the two polypeptide chains comprising a CH3 domain, and wherein said contacting is performed in the absence of a reducing agent.

[0018] Another aspect of the present invention is a heterodimeric polypeptide obtainable using the method according to the present invention.

[0019] Another aspect of the present invention is a set of heterodimeric precursor polypeptides according to the invention for use as pharmaceuticals.

[0020] Another aspect of the present invention is a set of heterodimeric precursor polypeptides according to the present invention, wherein the antibody variable domains contained in the first heavy chain polypeptide and the third heavy chain polypeptide in the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide are capable of forming an antigen-binding site that specifically binds to CD3, for use in the treatment of cancer.

[0021] In the invention disclosed herein, precursor polypeptides are provided that can undergo polypeptide chain exchange to form product polypeptides. Multispecific antigen-binding polypeptides can thereby be generated. The generation of multispecific antigen-binding polypeptides includes activating antigen-binding sites due to polypeptide chain exchange that results in the association of antibody variable domains that specifically bind to antigens. Also, multispecific antigen-binding polypeptides are formed upon combination and polypeptide chain exchange between two precursor polypeptides that contain antigen-binding moieties that specifically bind to different antigens. Furthermore, both heterodimeric precursor polypeptides contain CH2 domains, allowing them to bind to FcRn and undergo FcRn recycling, and the half-life of the precursor polypeptides in circulation is extended (compared to precursor polypeptides that do not contain CH2 domains). In particular, upon polypeptide chain exchange between two precursor polypeptides, one product polypeptide is formed that lacks a CH2 domain. This product polypeptide contains an activated antigen-binding site and can be rapidly removed upon dissociation from the target or in the event of off-target activation, thereby reducing undesirable off-target effects.

[0022] The methods and sets of polypeptides of the present invention can be advantageously used to provide antigen-binding polypeptides for therapeutic use, for example, the treatment of cancer.

[0023] Therapeutic application of the set of precursor polypeptides of the invention allows for the production of desired product polypeptides at the target site, thus reducing undesirable off-target effects of the product polypeptides. [The present invention 1001] A set of heterodimeric precursor polypeptides comprising: a) a first heterodimeric precursor polypeptide, - a first heavy chain polypeptide comprising, from N-terminal to C-terminal, an antibody variable domain selected from a VH domain and a VL domain and a CH3 domain, the first heavy chain polypeptide comprising at least a portion of a first antigen-binding portion; and - a second heavy chain polypeptide comprising, from N-terminus to C-terminus, a CH2 domain and a CH3 domain; Including, wherein the first heavy chain polypeptide and the second 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; b) a second heterodimeric precursor polypeptide, - a third heavy chain polypeptide comprising, from N-terminal to C-terminal, an antibody variable domain selected from a VH domain and a VL domain and a CH3 domain, wherein the antibody variable domain is capable of forming an antigen-binding site that specifically binds to a target antigen together with an antibody variable domain comprised in a first heavy chain polypeptide of the first heterodimeric precursor polypeptide, and wherein the third heavy chain polypeptide comprises at least a portion of a second antigen-binding portion; and a fourth heavy chain polypeptide comprising, from N-terminus to C-terminus, a CH2 domain and a CH3 domain; Including, a second heterodimeric precursor polypeptide, wherein the third heavy chain polypeptide and the fourth heavy chain polypeptide 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; Where: A) i) the first heavy chain polypeptide comprises a CH3 domain having the knob mutation and the third heavy chain polypeptide comprises a CH3 domain having the hole mutation; or ii) the first heavy chain polypeptide comprises a CH3 domain having the hole mutation and the third heavy chain polypeptide comprises a CH3 domain having the knob mutation; and B) i) the CH3 domain of the first heterodimeric precursor polypeptide comprising the knob mutation and the CH3 domain of the second heterodimeric precursor polypeptide comprising the hole mutation; or ii) the CH3 domain of the first heterodimeric precursor polypeptide comprising the hole mutation and the CH3 domain of the second heterodimeric precursor polypeptide comprising the knob mutation. any of the above contains one or more amino acid substitutions that destabilize the CH3 / CH3 interface, and the amino acid substitutions are positioned such that the substituted amino acids interact at the CH3 / CH3 interface within the pair of CH3 domains. [The present invention 1002] B) the CH3 domain comprising the knob mutation and the CH3 domain comprising the hole mutation comprise one or more of the following amino acid substitutions, wherein the numbering is according to the Kabat numbering system: - said CH3 domain having said hole mutation, Substitution of S354 with a hydrophobic amino acid, Substitution of D356 with a positively charged amino acid, Substitution of E357 with a positively charged or hydrophobic amino acid, Substitution of D356 with a positively charged amino acid and substitution of E357 with a positively charged or hydrophobic amino acid, Substitution of S364 with a hydrophobic amino acid, Substitution of A368 with a hydrophobic amino acid, Substitution of K392 with a negatively charged amino acid, Substitution of T394 with a hydrophobic amino acid, Substitution of D399 with a hydrophobic amino acid and substitution of S400 with a positively charged amino acid, Substitution of D399 with a hydrophobic amino acid and substitution of F405 with a positively charged amino acid, Substitution of V407 with a hydrophobic amino acid, and Substitution of K409 with a negatively charged amino acid, and Substitution of K439 with a negatively charged amino acid At least one amino acid substitution selected from the group - said CH3 domain having said knob mutation, Substitution of Q347 with a positively charged amino acid and K360 with a negatively charged amino acid, Substitution of Y349 with a negatively charged amino acid, Substitution of L351 with a hydrophobic amino acid and substitution of E357 with a hydrophobic amino acid, Substitution of S364 with a hydrophobic amino acid, o substitution of W366 with a hydrophobic amino acid and K409 with a negatively charged amino acid; o Substitution of L368 with a hydrophobic amino acid; o Substitution of K370 with a negatively charged amino acid; o substitution of K370 with a negatively charged amino acid and substitution of K439 with a negatively charged amino acid; Substitution of K392 with a negatively charged amino acid, Substitution of T394 with a hydrophobic amino acid, Substitution of V397 with a hydrophobic amino acid, o Substitution of D399 with a positively charged amino acid and substitution of K409 with a negatively charged amino acid; Substitution of S400 with a positively charged amino acid, 〇 F405W 〇 Y407W, and Substitution of K439 with a negatively charged amino acid The present invention comprises at least one amino acid substitution selected from the group consisting of: [The present invention 1003] B) any of the heterodimeric polypeptides of the present invention, wherein the CH3 domain comprising the knob mutation and the CH3 domain comprising the hole mutation comprise one or more of the following amino acid substitutions, wherein the numbering is according to the Kabat numbering system: - said CH3 domain having said hole mutation, Substitution of E357 with a positively charged amino acid, Substitution of S364 with a hydrophobic amino acid, Substitution of A368 with a hydrophobic amino acid, and Substitution of V407 with a hydrophobic amino acid and comprising at least one amino acid substitution selected from the group consisting of - said CH3 domain having said knob mutation, Substitution of K370 with a negatively charged amino acid, Substitution of K370 with a negatively charged amino acid and substitution of K439 with a negatively charged amino acid, Substitution of K392 with a negatively charged amino acid, and Substitution of V397 with a hydrophobic amino acid The present invention comprises at least one amino acid substitution selected from the group consisting of: [The present invention 1004] Any of the above sets of heterodimeric polypeptides of the present invention, wherein the first antigen-binding portion and / or the second antigen-binding portion is an antibody fragment. [The present invention 1005] a) the first heterodimeric precursor polypeptide is - a further antibody variable domain (a first antibody variable domain) within said first heavy chain polypeptide comprising a CH3 domain, and - a further polypeptide chain which is a light chain polypeptide comprising a second antibody variable domain. wherein the first antibody variable domain and the second antibody variable domain together form a first antigen-binding site that specifically binds to a target antigen; and b) the second heterodimeric precursor polypeptide comprises: - a further antibody variable domain (a third antibody variable domain) within said third heavy chain polypeptide comprising a CH3 domain, and - a further polypeptide chain which is a light chain polypeptide comprising a fourth antibody variable domain wherein the third antibody variable domain and the fourth antibody variable domain together form a second antigen-binding site that specifically binds to a target antigen. A set of any of the heterodimeric polypeptides of the present invention. [The present invention 1006] A set of any of the heterodimeric polypeptides of the present invention, wherein in the first heterodimeric polypeptide, no interchain disulfide bond is formed between the two polypeptide chains containing the CH3 domain, and in the second heterodimeric polypeptide, no interchain disulfide bond is formed between the two polypeptide chains containing the CH3 domain. [The present invention 1007] A set of any of the heterodimeric precursor polypeptides of the present invention, 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. [The present invention 1008] A set of any of the heterodimeric precursor polypeptides of the present invention, wherein the antibody variable domains contained in the first heavy chain polypeptide and the third heavy chain polypeptide are capable of forming an antigen-binding site that specifically binds to CD3. [The present invention 1009] 1009. A method for producing a heterodimeric polypeptide comprising contacting a first heterodimeric precursor polypeptide of any of claims 1001 to 1008 with a second heterodimeric precursor polypeptide to form a third heterodimeric polypeptide comprising the first heavy chain polypeptide and the third heavy chain polypeptide. [The present invention 1010] 1009. The method of claim 1009, comprising contacting said first heterodimeric precursor polypeptide with said second heterodimeric precursor polypeptide to form a fourth heterodimeric polypeptide comprising said second heavy chain polypeptide and said fourth heavy chain polypeptide. [The present invention 1011] The method of any one of claims 1009 to 1010, wherein in said first heterodimeric polypeptide, no interchain disulfide bond is formed between said two polypeptide chains comprising said CH3 domains, and in said second heterodimeric polypeptide, no interchain disulfide bond is formed between said two polypeptide chains comprising said CH3 domains, and said contacting is performed in the absence of a reducing agent. [The present invention 1012] The first heterodimeric precursor polypeptide of any of 1001 to 1008. [The present invention 1013] The second heterodimeric precursor polypeptide of any of 1001 to 1008. [The present invention 1014] A set of heterodimeric precursor polypeptides according to any one of 1001 to 1008 for use as a medicament. [The present invention 1015] A pharmaceutical composition comprising a set of heterodimeric precursor polypeptides according to any one of claims 1001 to 1008 and a pharma- ceutically acceptable carrier. [The present invention 1016] The set of heterodimeric precursor polypeptides of any of claims 1001 to 1008, wherein the antibody variable domains contained in the first heavy chain polypeptide and the third heavy chain polypeptide in the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide are capable of forming an antigen binding site that specifically binds to CD3 for use in the treatment of cancer. [Brief description of the drawings]

[0024] [Figure 1]Exemplary structures of heterodimeric precursor polypeptides according to the present invention and corresponding product polypeptides formed upon polypeptide chain exchange, where the polypeptide chain exchange results 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-terminal to C-terminal, antibody domains VH, CH1, a peptide connector, a VH domain from a first antibody, and CH3. The CH3 domain comprises a knob mutation and does not comprise a destabilizing mutation. 2. A second heavy chain polypeptide comprising, from N-terminal to C-terminal, the following antibody domains: CH2 and CH3. The CH3 domain comprises a hole mutation and a destabilizing mutation. 3. A light chain polypeptide comprising, from N-terminal 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 are associated with each other via their CH3 domains. No interchain disulfide bonds are formed between the first and second heavy chain polypeptides. The second heterodimeric precursor polypeptide comprises three polypeptide chains: 1. a first heavy chain polypeptide comprising, from N-terminal to C-terminal, antibody domains VH, CH1, a peptide connector, a VL domain from a first antibody, and CH3. The CH3 domain comprises a hole mutation and does not comprise a destabilizing mutation. 2. a second heavy chain polypeptide comprising, from N-terminal to C-terminal, the following antibody domains: CH2 and CH3. The CH3 domain comprises a knob mutation and a destabilizing mutation. 3. a light chain polypeptide comprising, from N-terminal 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 bonds are formed between the first and second heavy chain polypeptides. Upon polypeptide chain exchange, a heterodimeric product polypeptide is formed.The first product polypeptide comprises two antigen-binding sites from precursor polypeptides, namely, an antigen-binding site from the first heterodimeric precursor polypeptide and an antigen-binding site from the second heterodimeric precursor polypeptide. The first product polypeptide comprises a first heavy chain polypeptide from the first and second heterodimeric precursor polypeptides, which are associated through their CH3 domains. Both heavy chain polypeptides contained in the first product polypeptide comprise a CH3 domain that does not contain a destabilizing mutation. The association of the first heavy chain polypeptide from the first and second heterodimeric precursor polypeptides forms a pair of a VH domain from the first antibody and a VL domain 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 any of the precursor polypeptides and is only formed (activated) upon polypeptide chain exchange. 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 through their CH3 domains. Both CH3 domains contain destabilizing mutations that interact and favor the formation of a heterodimeric product polypeptide. [Diagram 2]Exemplary structures of heterodimeric precursor polypeptides used in proof-of-concept experiments according to Example 4 and corresponding product polypeptides formed upon polypeptide chain exchange, where the polypeptide chain exchange results 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-terminal to C-terminal direction, antibody domains VH, CH1, peptide connector, VH domain from a first antibody and CH3. The CH3 domain comprises a knob mutation and does not comprise a destabilizing mutation. 2. A second heavy chain polypeptide comprising, from N-terminal to C-terminal direction, the next antibody domain, VL domain 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-terminal to C-terminal direction, 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 are associated with each other via their CH3 domains. No interchain disulfide bond is formed between the first and second heavy chain polypeptides. A pair of VH and VL domains is formed between the VH domain 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 the N-terminus to the C-terminus, the antibody domains VH, CH1, a peptide connector, the VL domain from the first antibody and CH3. The CH3 domain comprises a hole mutation and does not comprise a destabilizing mutation. 2. A second heavy chain polypeptide comprising, from the N-terminus to the C-terminus, the following antibody domains, a VH domain from a third antibody and CH3. The CH3 domain comprises a knob mutation and a destabilizing mutation. 3. A light chain polypeptide comprising, from the N-terminus to the C-terminus, the 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 the target antigen.The heavy chain polypeptides of the second heterodimeric precursor polypeptide are associated with each other through their CH3 domains. No interchain disulfide bond is formed between the first and second heavy chain polypeptides. A pair of VH and VL domains is formed between the VL domain from the first antibody and the VH domain from the second heavy chain polypeptide. Both variable domains are associated with each other but do not form an antigen binding site that specifically binds to the antigen. Upon polypeptide chain exchange, a heterodimeric product polypeptide is formed. The first product polypeptide comprises two antigen binding sites from the precursor polypeptides, namely, an antigen binding site from the first heterodimeric precursor polypeptide and an antigen binding site from the second heterodimeric precursor polypeptide. The first product polypeptide comprises a first heavy chain polypeptide from the first and second heterodimeric precursor polypeptides, which are associated with each other through their CH3 domains. Both heavy chain polypeptides in the first product polypeptide comprise a CH3 domain that does not contain a destabilizing mutation. The association of the first heavy chain polypeptide from the first and second heterodimeric precursor polypeptides forms a pair of the VH domain from the first antibody and the VL domain 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 any of the precursor polypeptides and is only formed (activated) upon polypeptide chain exchange. 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 and support the formation of the heterodimeric product polypeptide. The association of the second heavy chain polypeptide from the first and second heterodimeric precursor polypeptides forms a new pair of the VH and VL domains. Both variable domains are associated in the second product polypeptide. [Diagram 3]Exemplary structures of heterodimeric precursor polypeptides used in proof-of-concept experiments according to Example 4 and corresponding product polypeptides formed upon polypeptide chain exchange, where the polypeptide chain exchange results 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-terminal to C-terminal direction, antibody domains VH, CH1, a peptide connector, a VH domain from a first antibody, CH2 and CH3. The CH3 domain comprises a knob mutation and does not comprise a destabilizing mutation. 2. A second heavy chain polypeptide comprising, from N-terminal to C-terminal direction, the following antibody domains: a VL domain 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-terminal to C-terminal direction, 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 are associated with each other via their CH3 domains. No interchain disulfide bond is formed between the first and second heavy chain polypeptides. A pair of VH and VL domains is formed between the VH domain 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 an antigen. The second heterodimeric precursor polypeptide comprises three polypeptide chains: 1. A first heavy chain polypeptide comprising, from N-terminal to C-terminal, antibody domains VH, CH1, a peptide connector, a VL domain from said first antibody, CH2 and CH3. The CH3 domain comprises a hole mutation and does not comprise a destabilizing mutation. 2. A second heavy chain polypeptide comprising, from N-terminal to C-terminal, the following antibody domains: a VH domain 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-terminal 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 a target antigen.The heavy chain polypeptides of the second heterodimeric precursor polypeptide are associated with each other through their CH3 domains. No interchain disulfide bond is formed between the first and second heavy chain polypeptides. A pair of VH and VL domains is formed between the VL domain from the first antibody and the VH domain from the second heavy chain polypeptide. Both variable domains are associated with each other but do not form an antigen binding site that specifically binds to the antigen. Upon polypeptide chain exchange, a heterodimeric product polypeptide is formed. The first product polypeptide comprises two antigen binding sites from the precursor polypeptides, namely, an antigen binding site from the first heterodimeric precursor polypeptide and an antigen binding site from the second heterodimeric precursor polypeptide. The first product polypeptide comprises a first heavy chain polypeptide from the first and second heterodimeric precursor polypeptides, which are associated with each other through their CH3 domains. Both heavy chain polypeptides in the first product polypeptide comprise a CH3 domain that does not contain a destabilizing mutation. The association of the first heavy chain polypeptide from the first and second heterodimeric precursor polypeptides forms a pair of the VH domain from the first antibody and the VL domain 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 any of the precursor polypeptides and is only formed (activated) upon polypeptide chain exchange. 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 and support the formation of the heterodimeric product polypeptide. The association of the second heavy chain polypeptide from the first and second heterodimeric precursor polypeptides forms a new pair of the VH domain and the VL domain. Both variable domains are associated in the second product polypeptide. [Figure 4]Exemplary structures of heterodimeric precursor polypeptides used in proof-of-concept experiments according to Example 1 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-terminal to C-terminal direction, antibody domains VH, CH1, hinge, CH2 and CH3. The CH3 domain comprises a knob mutation and a cysteine ​​mutation and does not comprise a destabilizing mutation. 2. A second heavy chain polypeptide comprising, from N-terminal to C-terminal direction, 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-terminal to C-terminal direction, 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 are associated 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-terminal to C-terminal, antibody domains VH, CH1, hinge, CH2 and CH3. The CH3 domain comprises a hole mutation and a cysteine ​​mutation, and does not comprise a destabilizing mutation. 2. a second heavy chain polypeptide comprising, from N-terminal 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-terminal 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 are associated with each other through 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 favoring polypeptide chain exchange, resulting in the formation of a heterodimeric product polypeptide.The first product polypeptide comprises two antigen binding sites, an antigen binding site from the first heterodimeric precursor polypeptide and an antigen binding site from the second heterodimeric precursor polypeptide. The first product polypeptide comprises a first heavy chain polypeptide from the first and second heterodimeric precursor polypeptides, which are associated through 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 and support the formation of heterodimeric product polypeptides. The second product polypeptide, which does not comprise an antigen binding site in this embodiment, 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 through their CH3 domains. Both CH3 domains contain destabilizing mutations that interact and support the formation of heterodimeric product polypeptides. The product polypeptide comprises a tagging moiety that allows purification by tag-specific chromatography. [Diagram 5] SDS-PAGE of the anti-LeY-CD3(VH)-knob precursor and the anti-LeY-CD3(VL)-hole precursor of the invention as described in Example 6. [Figure 6] Size-exclusion chromatography of the anti-LeY-CD3(VH)-knob precursor of the invention as described in Example 6. [Figure 7] Size-exclusion chromatography of the anti-LeY-CD3(VL)-hole precursor of the invention described in Example 6. [Figure 8] FcRn affinity chromatography of the anti-LeY-CD3(VH)-knob precursor and the anti-LeY-CD3(VL)-hole precursor of the invention, and of the bispecific anti-LeY / anti-CD3 product polypeptide resulting from polypeptide chain exchange between the two precursor polypeptides described in Example 6. [Figure 9]T cell activation upon activation of bispecific anti-LeY / anti-CD3 bispecific antibodies resulting from polypeptide chain exchange between monospecific anti-LeY-CD3(VH)-knob precursors and anti-LeY-CD3(VL)-hole precursors as described in Example 6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Detailed Description of the Invention 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 skilled in the art.Furthermore, unless otherwise required by context, singular terms shall include plurals and plural terms shall include singulars.The methods and techniques of the present disclosure are generally carried out according to conventional methods well known in the art.Generally, the nomenclatures and techniques used in connection with biochemistry, enzymology, molecular and cell biology, microbiology, genetics, and chemistry of proteins and nucleic acids, and hybridization described herein are well known and commonly used in the art.

[0026] The terms "a," "an," and "the" generally include plural referents unless the context clearly dictates otherwise.

[0027] Unless otherwise defined herein, the term "comprising" is intended to include the term "consisting of."

[0028] Unless the context clearly dictates otherwise, the provision of two alternatives using the term "or" indicates mutually exclusive alternatives.

[0029] 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 molecules (e.g., receptors, ligands) or synthetic molecules (e.g., DARPins) that can specifically bind to a target antigen.

[0030] 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.

[0031] The term "binding site" or "antigen-binding site" as used herein refers to one or more regions of an antigen-binding portion to which an antigen actually binds. If 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 may be derived from a) a known antibody that specifically binds to the antigen, or b) a new antibody or antibody fragment obtained by de novo immunization methods using, inter alia, an antigen protein or nucleic acid or a fragment thereof, or by phage display methods.

[0032] When derived from an antibody, the antigen-binding site of the antibody according to the invention may comprise six complementarity determining regions (CDRs) that contribute to various 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 the regions are defined according to the variability between sequences. Functional antigen-binding sites composed of fewer CDRs (i.e., binding specificity is determined by three, four or five CDRs) are also included within the scope of the present invention. For example, less than a complete set of six CDRs may be sufficient for binding.

[0033] The term "valency" as used herein refers to the presence of a certain number of antigen binding sites in 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 in an antibody molecule.

[0034] An "antibody fragment" is a molecule other than an intact antibody that contains a portion of an 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.

[0035] "Specificity" refers to the selective recognition of a particular epitope of an antigen by an antigen-binding moiety, e.g., an antibody. For example, a natural antibody is monospecific. As used herein, the term "monospecific antibody" refers to an antibody having one or more binding sites that each bind 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 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 has more than one specificity, the recognized epitope can be associated with a single antigen or with two or more antigens.

[0036] An epitope is a region of an antigen that is bound by an antigen-binding moiety, such as an antibody. The term "epitope" includes any polypeptide determinant capable of specific binding to an antibody or antigen-binding moiety. In certain embodiments, epitopic determinants include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl, and in certain embodiments may have specific three-dimensional structural characteristics, and / or specific charge characteristics.

[0037] As used herein, the terms "binding" and "specific binding" refer to the binding of an antibody or antigen-binding portion 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 certain embodiments, an antibody or antigen-binding portion is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0038] The affinity of binding of an antibody to an antigen is expressed by the term k a (rate constant for association of the antibody from the antibody / antigen complex), k D (dissociation constant), and K D (k D In one embodiment, binding or specifically binding is defined as 10- 8 Binding affinity (K D ), in one embodiment, 10 -8 M~10 -13 Thus, an antigen-binding moiety, particularly an antibody-binding site, will bind 10 -8 Binding affinity (K D ), e.g., 10 -8 ~10 -13 Binding affinity in mol / l (K D ), in one embodiment, 10 -9 ~10 -13 Binding affinity in mol / l (K D ) and binds specifically to it.

[0039] The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of native antibodies generally have a similar structure, 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., WH 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 particular antigen may be isolated by screening a library of complementary VL or VH domains, respectively, using the VH or VL domain of 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).

[0040] 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.

[0041] Depending on the amino acid sequence of the constant region of the heavy chain, antibodies are classified into "classes" such as IgA, IgD, IgE, IgG and IgM, some of which may be further divided into subclasses, e.g., IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called α, δ, ε, γ and μ, respectively. The light chain constant regions (CL) found in all five antibody classes are called κ (kappa) and λ (lambda).

[0042] As used herein, a "constant domain" is preferably derived from human origin, 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, in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0043] In wild-type antibodies, the "hinge region" is a flexible amino acid stretch in the central part of the heavy chains of IgG and IgA immunoglobulin classes, which connects the two heavy chains by disulfide bonds, i.e., "interchain disulfide bonds" formed between the two heavy chains. The hinge region of human IgG1 is generally defined as the stretch from about Glu at position 216 or Cys at position 226 to about Pro at position 230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). Disulfide bond formation in the hinge region is avoided by deleting the cysteine ​​residues in the hinge region or by substituting them with other amino acids, such as serine.

[0044] 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 sequences of their constant domains. Wild-type light chains usually contain two immunoglobulin domains, usually one variable domain (VL) that is important for binding to an antigen, and a constant domain (CL).

[0045] There are several different types of "heavy chains" that define the class or isotype of an antibody. Wild-type heavy chains contain a series of immunoglobulin domains, usually one variable domain (VH) that is important for antigen binding, and several constant domains (CH1, CH2, CH3, etc.).

[0046] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The 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, the 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.

[0047] The "CH2 domain" of a human IgG Fc region usually extends from about amino acid residue 231 to about amino acid residue 340. Multispecific antibodies lack a CH2 domain. By "lacking a CH2 domain" is meant that the antibody according to the invention does not comprise a CH2 domain.

[0048] A "CH3 domain" comprises an extension 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 different amino acid substitution (i.e., modification of the amino acid sequence of the CH3 domain) to promote heterodimerization of two CH3 domains opposite each other in a multispecific antibody.

[0049] Typically, in heterodimerization approaches known in the art, the CH3 domain of one heavy chain and the CH3 domain of the other heavy chain are both engineered in a complementary manner such that the heavy chain containing one engineered CH3 domain can no longer homodimerize with other heavy chains of the same structure, thereby forcing the heavy chain containing one engineered CH3 domain to heterodimerize with the other heavy chain containing a CH3 domain engineered in a complementary manner.

[0050] One of the heterodimerization approaches known in the art is the so-called "knobs-into-holes" technique, which is described in detail with some 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, within the interface formed between the two CH3 domains in the tertiary structure of the antibody, specific amino acids on each CH3 domain are engineered to create a protrusion ("knob") on one of the CH3 domains and a cavity ("hole") on the other CH3 domain. In the tertiary structure of a multispecific antibody, the introduced protuberance of one CH3 domain can be positioned within the introduced cavity of the other CH3 domain.

[0051] In combination with the knob-into-hole technology substitution, additional interchain disulfide bonds can be introduced into the CH3 domain 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 domain: D399C in one CH3 domain and K392C in the other CH3 domain; Y349C in one CH3 domain and S354C in the other CH3 domain; Y349C in one CH3 domain and E356C in the other CH3 domain; Y349C in one CH3 domain and E357C in the other CH3 domain; L351C in one CH3 domain and S354C in the other CH3 domain; T394C in one CH3 domain and V397C in the other CH3 domain. As used herein, a "cysteine ​​mutation" refers to the substitution of one amino acid in the CH3 domain with a cysteine ​​that is capable of forming an interchain disulfide bond with the other, matching amino acid substitution of the amino acid in the second CH3 domain with a cysteine.

[0052] Apart from the aforementioned "knobs-into-holes" technique, further techniques are known in the art for modifying CH3 domains to perform heterodimerization. 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 contemplated herein as alternatives to the "knobs-into-holes technology" for the polypeptides provided by the invention. All of these techniques involve engineering the CH3 domain in a complementary manner by the introduction of amino acids of opposite charge or different side chain volume, thereby supporting heterodimerization.

[0053] The precursor polypeptide of the present invention comprises, in only one of its CH3 domains, an amino acid substitution that "destabilizes the CH3 / CH3 interface", also referred to herein as a "destabilizing mutation". By these terms, it is meant an amino acid substitution that is located in only one of the CH3 domains associated in the heterodimeric precursor polypeptide. In said CH3 domain, one or more amino acid positions known to interact within the CH3 / CH3 interface are replaced by amino acids with other site chain properties, for example 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 in the associated CH3 domains are typically replaced (i.e., one or more amino acid residues in one CH3 domain involved in the heterodimer and one or more amino acid residues in the other CH3 domain involved in the heterodimer), the destabilizing mutation is located in only one of the CH3 domains involved in the heterodimeric precursor polypeptide according to the present invention. Exemplary amino acid substitutions that destabilize the CH3 / CH3 interface are listed below in the "destabilizing mutation" section. All of the exemplary amino acid substitutions specifically disclosed herein are positioned such that the substituted amino acids interact at the CH3 / CH3 interface within the pair of CH3 domains.

[0054] The term "polypeptide chain" as used herein refers to a linear organic polymer comprising a number of amino acids linked together via peptide bonds. One or more polypeptide chains form a "polypeptide" or a "protein", both terms being used interchangeably herein. The heterodimeric precursor polypeptides provided in the set according to the present invention comprise at least two polypeptide chains comprising a CH3 domain. Thus, a first polypeptide chain comprising a first CH3 domain "associates" with a second polypeptide chain comprising a second CH3 domain to form a dimeric polypeptide. Because the first CH3 domain and the second CH3 domain comprise amino acid substitutions by knob-into-hole technology, the two polypeptide chains form a "heterodimer", i.e., a dimer formed by two non-identical polypeptides.

[0055] The polypeptide chains contained in the heterodimeric polypeptides, i.e., the heterodimeric precursor polypeptides and the heterodimeric product polypeptides, contain one or two polypeptide domains. The order of the polypeptide domains, when presented herein, is presented in the N-terminal to C-terminal direction.

[0056] Each heterodimeric precursor polypeptide comprises at least two polypeptide chains that comprise a CH3 domain.

[0057] When the antigen-binding moieties present in the two heterodimeric precursor polypeptides are antibody-derived antigen-binding sites, such as antibody fragments, the polypeptide chain containing the CH3 domain is also referred to herein as a "heavy chain polypeptide". In this case, the heterodimeric precursor polypeptide may also contain a "light chain polypeptide", which typically contains an antibody variable domain and an antibody constant domain, such as VL and CL.

[0058] The present invention provides a set comprising at least two polypeptides. The set comprises at least two heterodimeric "precursor" polypeptides. When the precursor polypeptides are reacted to undergo polypeptide chain exchange with each other, a "product" polypeptide is formed. The present invention also provides a method for producing a heterodimeric polypeptide, i.e., a heterodimeric product polypeptide, by contacting at least two heterodimeric precursor polypeptides. The contacting step may be carried out in any suitable condition 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 CH3 domain from another precursor polypeptide. The mechanism of polypeptide chain exchange is also illustrated in FIG. 1.

[0059] An "isolated" heterodimeric polypeptide is, for example, one that is separated from components 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, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0060] As used herein, the amino acid positions of all constant regions and domains of the heavy and light chains are numbered according to the Kabat numbering system as described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). In particular, for the variable domains and the light chain constant domains CL of the kappa and lambda isotypes, the 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:

[0061] Amino acid "substitutions" or "replacements" or "mutations" (all terms used interchangeably herein) in 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, e.g., as described above. For example, modifications do not alter the above-mentioned antibody properties, such as IgG isotype or antigen binding, but may further improve the yield of recombinant production, protein stability, or facilitate purification. In certain 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 the respective polypeptide chain.

[0062] 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" or "negatively charged" amino acids refer to the amino acid side chain charge at pH 7.4. Amino acids have the following common side chain characteristics: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, Trp, Tyr, Phe; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic or negatively charged: Asp, Glu; (4) Basic or positively charged: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro The items may be grouped according to:

[0063] (Table) Amino acids with specific properties TIFF0007638900000001.tif116155

[0064] As used herein, a "tagging moiety" is a peptide sequence genetically grafted onto a polypeptide chain for various purposes, for example to support purification. In one embodiment, the tagging moiety is an affinity tag. Thus, a polypeptide comprising said affinity tag can be purified via an appropriate affinity technique, for example by 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 composed of a pattern of glycine and serine residues.

[0065] The term "purified" as used herein refers to polypeptides that have been removed or otherwise isolated or separated from their natural environment or recombinant production source and are at least 60%, e.g., at least 80%, free from other components with which they are naturally associated, e.g., membranes and microsomes. Purification of antibodies (recovery of antibodies from host cell culture) is performed to remove cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others known in the art. See Ausubel, F., et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987). For protein purification, various methods are well established and widely used, such as affinity chromatography with microbial proteins (e.g., using affinity media for purification of kappa or lambda isotype constant light chain domains, e.g., KappaSelect or LambdaSelect), ion exchange chromatography (e.g., cation exchange (carboxymethyl resin), anion exchange (aminoethyl resin) and mixed mode exchange), thiophene adsorption (e.g., using β-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g., using phenyl-Sepharose, aza-aromatic resins, 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).

[0066] 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, in particular nickel chelate affinity chromatography.

[0067] The term "peptide linker" as used herein means a peptide of amino acid sequence, preferably of synthetic origin. Within the heterodimeric polypeptides used in the present invention, peptide connectors can be used to fuse additional polypeptide domains, such as antibody fragments, to the C-terminus or N-terminus of the individual polypeptide chains. In one embodiment, the peptide connector is a peptide having an amino acid sequence having a length of at least 5 amino acids, in another embodiment a length of 5 to 100 amino acids, and in yet another embodiment a length of 10 to 50 amino acids. 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 and where 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, and m=0, 1, 2 or 3.

[0068] In one embodiment, x=4 and n=2 or 3, and in another embodiment, x=4, n=2. In one embodiment, the peptide connector is (G4S)2.

[0069] The term "valency" as used herein refers to the presence of a certain number of antigen-binding sites in an antigen-binding molecule. A natural antibody, for example, has two binding sites and is bivalent. Thus, the term "trivalent" means that there are three binding sites in an antigen-binding molecule.

[0070] The polypeptides according to the invention are produced by recombinant means. Methods for the recombinant production of polypeptides, such as antibodies, are widely known in the state of 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 purity. For expression of the polypeptides as described above in host cells, the 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 antibody 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 state of the art and are described, for example, in the reviews by 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.

[0071] The polypeptide produced by the host cell may be subjected to post-translational truncation of one or more, particularly one or two amino acids, from the C-terminus of the polypeptide chain that contains the CH3 domain at the C-terminus. Thus, by expression of a particular nucleic acid molecule that encodes such a polypeptide, the polypeptide produced by the host cell may contain a full-length polypeptide chain that contains a full-length CH3, or may contain a truncated variant of the full-length heavy chain (also referred to herein as a truncated variant polypeptide). This may be the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447).

[0072] "Polynucleotide" or "nucleic acid", as used interchangeably herein, refers to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides may 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 may contain modified nucleotides, such as methylated nucleotides and their analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may contain modifications made after synthesis, such as conjugation to a label. Other types of modifications include, for example, "caps", substitution of one or more analogs of naturally occurring nucleotides, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), intercalators (e.g., acridine, psoralen, 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 polynucleotides. Additionally, any hydroxyl groups normally present in the sugar may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or conjugated to solid or semi-solid supports. The 5' and 3' terminal oH may be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. The other hydroxyls may also 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, acrylic acid analogs, and abasic 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 a substituted or unsubstituted alkyl (1-20C), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl, optionally including an ether (-o-) linkage. 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.

[0073] An "isolated" nucleic acid refers to a nucleic acid molecule that is 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.

[0074] 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, and includes such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations within a host cell.

[0075] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating other nucleic acids to which it is linked. The term also includes vectors as self-replicating nucleic acid structures, as well as vectors integrated into the genome of a host cell into which the vector is introduced. The term includes vectors that function primarily to insert DNA or RNA into a cell (e.g., chromosomal integration), replicating vectors that function primarily for DNA or RNA replication, and expression vectors that function for DNA or RNA transcription and / or translation. Vectors that provide more than one of the listed functions are also included.

[0076] An "expression vector" is a vector capable of directing the expression of an operably linked nucleic acid. When an expression vector is introduced into a suitable host cell, it can be transcribed and translated into a polypeptide. When transforming a host cell in the method according to the invention, an "expression vector" is used, whereby the term "vector" in relation to the transformation of a host cell as described herein means an "expression vector". An "expression system" usually refers to a suitable host cell comprised of an expression vector that can function to produce a desired expression product.

[0077] As used herein, "expression" refers to the process by which a nucleic acid is transcribed into mRNA and / or the transcribed mRNA (also called a transcript) is subsequently translated into a peptide or polypeptide. The transcript and the encoded polypeptide are individually or collectively called gene product. If the nucleic acid is derived from genomic DNA, expression in eukaryotic cells may include splicing of the corresponding mRNA.

[0078] The term "transformation" as used herein refers to the process of transferring a vector or nucleic acid into a host cell. When cells that do not have a formidable cell wall barrier are used as host cells, transfection is performed by the calcium phosphate precipitation method described, for example, in Graham and Van der Eh, Virology 52 (1978) 546ff. However, other methods for introducing DNA into cells, such as by nuclear injection or protoplast fusion, can also be used. When prokaryotic cells or cells that contain substantial cell wall structures are used, for example, one method of transfection is the calcium treatment using calcium chloride, as described in Cohen, FN, et al., PNAS 69 (1972) 7110 et seq.

[0079] The term "host cell" as used in this application means any type of cell system that can be engineered to produce the polypeptides provided by the present invention.

[0080] As used herein, the terms "cell", "cell line" and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the terms "transformant" and "transformed cell" include the primary subject cell and the culture derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be exactly identical in DNA content due to deliberate or unintended mutations. Mutant progeny that have the same function or biological activity as screened for in the original transformed cell are included. Where a different designation is intended, it will be clear from the context.

[0081] Transient expression is described, for example, in Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. Cloning of variable domains is described 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 (HEK 293) is described in Schlaeger, E.-J., and Christensen, K., Cytotechnology 30 (1999) 71-83 and Schlaeger, E.-J., J. Immunol. Methods 194 (1996) 191-199.

[0082] The term "pharmaceutical composition" refers to a formulation in a form that allows the biological activity of the active ingredient contained therein to be effective and does not contain additional components that are unacceptably toxic to the subject to which the composition is administered. The pharmaceutical composition of the present invention can be administered by various methods known in the art. As will be understood by those skilled in the art, the route and / or method of administration can vary depending on the desired results. To administer the antibody of the present invention by a certain route of administration, it may be necessary to coat the antibody with a material to prevent its inactivation or to co-administer the antibody with said material. For example, the heterodimeric polypeptide can be administered to the subject in a suitable carrier, such as a liposome or diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.

[0083] The pharmaceutical composition comprises an effective amount of the heterodimeric polypeptide provided by the present invention. An "effective amount" of an agent, such as a heterodimeric polypeptide, refers to an amount effective at dosages and for periods of time necessary to achieve a desired therapeutic or prophylactic result. In particular, an "effective amount" refers to an amount of the heterodimeric polypeptide of the present invention that, when administered to a subject, (i) treats or prevents a particular disease, condition or disorder, (ii) attenuates, improves 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 described herein. A therapeutically effective amount will vary depending on the heterodimeric polypeptide molecule used, the disease state being treated, the severity of the disease being treated, the age and relative health of the subject, the route and form of administration, the judgment of the attending physician or veterinary professional, and other factors.

[0084] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation other than the active ingredient that is non-toxic to a subject. Pharmaceutically acceptable carriers include any physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. In a preferred embodiment, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).

[0085] The pharmaceutical composition 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 by both the above-mentioned sterilization procedures and the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may be desirable to include isotonic agents, such as sugars and sodium chloride, in the composition. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0086] The phrases "parenteral administration" and "administered parenterally" as used herein 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.

[0087] Regardless of the route of administration selected, 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 pharma- ceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0088] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The selected dosage level depends on various pharmacokinetic factors, including the activity of the particular composition of the present invention used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0089] The composition must be sterile and fluid to the extent that the composition is deliverable by syringe, hi one embodiment, the carrier is an isotonic buffered saline solution in addition to water.

[0090] 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.

[0091] As used herein, "treatment" (and grammatical variants 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 either for prophylaxis or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, remission or alleviation of disease symptoms, and improving recovery or prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of disease or to delay the progression of disease.

[0092] 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 certain embodiments, an individual or subject is a human.

[0093] 2. Detailed Description of the Invention The invention provides precursor polypeptides that are applicable to the in vivo generation of resulting polypeptides, for example, by polypeptide chain exchange. One application is the on-cell generation of antigen binding sites by association of newly formed VH and VL domain pairs.

[0094] Each precursor polypeptide comprises a pair of CH3 domains arranged on two individual polypeptide chains that associate with each other via said CH3 domains. The CH3 domains comprise several amino acid substitutions. This allows the two polypeptide chains comprising the CH3 domains in the precursor polypeptide to form a heterodimer. The CH3 domains of the precursor polypeptides provided by the present invention comprise at least two patterns of mutations with different functionalities. The first pattern of mutations are mutations that support heterodimerization of the two polypeptide chains comprising the CH3 domains, i.e. knob-into-hole mutations. Thus, one CH3 domain of the precursor polypeptide comprises a knob mutation and the other CH3 domain of the precursor polypeptide comprises a hole mutation. The second pattern of mutations are one or more mutations provided in only one of the CH3 domains associated with the heterodimer of the precursor polypeptide, said mutations destabilizing the interaction of the two polypeptides comprising the CH3 domains. Thus, each precursor polypeptide comprises one CH3 domain with one or more destabilizing mutations, selected and positioned to support correct assembly of the product polypeptide upon polypeptide chain exchange between the precursor polypeptides.

[0095] Each precursor polypeptide comprises one heavy chain polypeptide that comprises one antibody variable domain associated in each precursor polypeptide with a CH2 domain located in the corresponding heavy chain polypeptide. The antibody variable domain is derived from an antibody that specifically binds to a target antigen, e.g., CD3, and is arranged in the precursor polypeptide such that upon polypeptide chain exchange and assembly of the first and third heavy chain polypeptides from two different heterodimeric precursor polypeptides, a new antigen binding site is formed in the resulting product polypeptide that specifically binds to the target antigen, e.g., CD3.

[0096] Precursor Polypeptides In one aspect, the present invention provides a set of heterodimeric precursor polypeptides comprising: a) a first heterodimeric precursor polypeptide, - a first heavy chain polypeptide comprising, from N-terminal to C-terminal, an antibody variable domain selected from a VH domain and a VL domain and a CH3 domain, the first heavy chain polypeptide comprising at least a portion of a first antigen-binding portion; and - a second heavy chain polypeptide comprising, from N-terminus to C-terminus, a CH2 domain and a CH3 domain; Including, wherein the first heavy chain polypeptide and the second 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; b) a second heterodimeric precursor polypeptide, - a third heavy chain polypeptide comprising, from N-terminal to C-terminal, an antibody variable domain selected from a VH domain and a VL domain and a CH3 domain, wherein the antibody variable domain is capable of forming an antigen-binding site that specifically binds to a target antigen together with an antibody variable domain comprised in a first heavy chain polypeptide of the first heterodimeric precursor polypeptide, and wherein the third heavy chain polypeptide comprises at least a portion of a second antigen-binding portion; and a fourth heavy chain polypeptide comprising, from N-terminus to C-terminus, a CH2 domain and a CH3 domain; Including, a second heterodimeric precursor polypeptide, wherein the third heavy chain polypeptide and the fourth heavy chain polypeptide 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; Where: A) i) the first heavy chain polypeptide comprises a CH3 domain having a knob mutation and the third heavy chain polypeptide comprises a CH3 domain having a hole mutation; or ii) the first heavy chain polypeptide comprises a CH3 domain with a hole mutation and the third heavy chain polypeptide comprises a CH3 domain with a knob mutation; 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 said first heterodimeric precursor polypeptide comprising said hole mutation, and a CH3 domain of said second heterodimeric precursor polypeptide comprising said knob mutation. contains one or more amino acid substitutions that destabilize the CH3 / CH3 interface.

[0097] In another aspect, the present invention provides a method for producing a method for manufacturing a pharmaceutical composition comprising: (i) a first heavy chain polypeptide comprising, from N-terminal to C-terminal, an antibody variable domain selected from a VH domain and a VL domain and a CH3 domain, the first heavy chain polypeptide comprising at least a portion of a first antigen-binding portion; and (ii) a second heavy chain polypeptide comprising, from N-terminus to C-terminus, 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 domains to form a heterodimer, one of the CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation, and one of the CH3 domains (but not the other CH3 domain) comprises one or more amino acid substitutions that destabilize the CH3 / CH3 interface.

[0098] In another aspect, the present invention provides a method for producing a method for manufacturing a pharmaceutical composition comprising: (i) a third heavy chain polypeptide comprising, from N-terminal to C-terminal, an antibody variable domain selected from a VH domain and a VL domain and a CH3 domain, the third heavy chain polypeptide comprising at least a portion of a second antigen-binding portion; and (ii) a fourth heavy chain polypeptide comprising, from N-terminus to C-terminus, a CH2 domain and a CH3 domain; wherein said third heavy chain polypeptide and said fourth heavy chain polypeptide associate with each other via said CH3 domains to form a heterodimer, one of said CH3 domains comprises a knob mutation and the other CH3 domain comprises a hole mutation, and one of the CH3 domains (but not the other CH3 domain) comprises one or more amino acid substitutions that destabilize the CH3 / CH3 interface.

[0099] 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 to form a heterodimeric product polypeptide. In one embodiment, the first heterodimeric precursor polypeptide according to the present invention is used in combination with a second heterodimeric polypeptide according to the present invention for the formation of a heterodimeric product polypeptide by polypeptide chain exchange.

[0100] 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. In 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 for the formation of a heterodimeric product polypeptide by polypeptide chain exchange.

[0101] In another aspect of the invention, there is provided the use of a first heterodimeric precursor polypeptide according to the invention in a set of heterodimeric precursor polypeptides according to the invention.In another aspect of the invention, there is provided the use of a second heterodimeric precursor polypeptide according to the invention in a set of heterodimeric precursor polypeptides according to the invention.

[0102] Another aspect of the invention is the use of a first heterodimeric precursor polypeptide according to the invention in a method for producing a heterodimeric polypeptide according to the invention.Another aspect of the invention is the use of a second heterodimeric precursor polypeptide according to the invention in a method for producing a heterodimeric polypeptide according to the invention.

[0103] 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.

[0104] 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 a 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, 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, and 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 a CH3 domain does not comprise an antigen-binding portion that specifically binds to an antigen. In other words, according to this embodiment of the invention, one or more functional antigen-binding portions are located only in one of the two polypeptide chains comprising a CH3 domain, and no functional antigen-binding portion 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 the other polypeptide chain that contains a CH3 domain, i.e., the polypeptide chain in the heterodimer, and not with any other (eg, third) polypeptide chain.In one embodiment, the dummy polypeptide is a polypeptide chain comprising a CH2 domain and a CH3 domain.One advantage of such an arrangement, for example, the combination of a dummy polypeptide comprising a CH3 domain and a polypeptide chain comprising a CH3 domain involved in the formation of one or more functional antigen binding sites, is that the product polypeptide formed during polypeptide chain exchange is a different size from the heterodimeric precursor molecule, thereby allowing the improvement of the product polypeptide from the unreacted precursor polypeptide.This arrangement also allows the unreacted precursor polypeptide to bind to FcRn, thereby extending the half-life of these polypeptides, while the product polypeptide comprising the activated antigen binding site does not comprise a CH2 domain and is rapidly removed from circulation when not associated with the target antigen on cells.

[0105] As shown, in each one 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 polypeptide chain exchange, the polypeptide chain comprising a CH3 domain with a knob mutation from the first precursor polypeptide forms a heterodimer (i.e., a first heterodimeric product polypeptide) with a polypeptide chain comprising a CH3 domain with a hole mutation from the second precursor polypeptide, and the polypeptide chain comprising a CH3 domain with a hole mutation from the first precursor polypeptide forms a heterodimer (i.e., a second heterodimeric product polypeptide) with a polypeptide chain comprising a CH3 domain with a knob mutation from the second precursor polypeptide.

[0106] As shown, one CH3 domain of the first heterodimeric precursor polypeptide contains one or more destabilizing mutations as described above, the other CH3 domain of the first heterodimeric precursor polypeptide does not contain destabilizing mutations, and one CH3 domain of the second heterodimeric precursor polypeptide contains one or more destabilizing mutations as described above, and the other CH3 domain of the second heterodimeric precursor polypeptide does not contain destabilizing mutations. The destabilizing mutations present in the precursor polypeptides are arranged to be 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 the knob mutation, and in the other precursor polypeptide, one or more destabilizing mutations are arranged in the CH3 domain containing the hole mutation. In one embodiment, the destabilizing mutations of the first heterodimeric precursor polypeptide are located in the CH3 domain of the dummy polypeptide of the first heterodimeric precursor polypeptide, and the destabilizing mutations of the second heterodimeric precursor polypeptide are located in the CH3 domain of the dummy polypeptide of the second heterodimeric precursor polypeptide.

[0107] In one embodiment, in a first heterodimeric precursor polypeptide, a polypeptide chain comprising a CH3 domain comprising 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 having a hole mutation comprises at least a portion of a second antigen binding portion.

[0108] In one embodiment, in a 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 a second heterodimeric precursor polypeptide, a polypeptide chain comprising a CH3 domain having a knob mutation comprises at least a portion of a second antigen binding portion.

[0109] In one embodiment, the heterodimeric precursor polypeptide comprises exactly two polypeptide chains that contain a CH3 domain.

[0110] In one embodiment, the CH3 domain comprising the destabilizing mutation comprises one, two or three destabilizing mutations. In one embodiment, the CH3 domain comprising the destabilizing mutation comprises one or two destabilizing mutations.

[0111] In one embodiment of the present invention, no interchain disulfide bond is formed between the two polypeptide chains comprising the 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 the 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 the CH3 domain of the first heterodimeric polypeptide and the second heterodimeric polypeptide. Heterodimeric precursor polypeptides lacking interchain disulfide bond between the two polypeptide chains comprising the CH3 domain can undergo polypeptide chain exchange in the absence of reducing agent. Thus, in the absence of interchain disulfide bond between the polypeptide chains comprising the CH3 domain, the heterodimeric precursor polypeptide is suitable for applications where the presence of reducing agent is not possible or desirable, for example, therapeutic applications.

[0112] 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 can 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, rather than a tryptophan (W) at position 366 of the CH3 domain. However, heterodimeric precursor polypeptides having such a substitution are considered to be encompassed by the present invention.

[0113] The present invention also includes heterodimeric precursor polypeptides in which one or more mutations in the CH3 domain with hole mutations are replaced by destabilizing mutations. For example, the destabilizing mutation can be located at position 368 of the CH3 domain with hole mutations, e.g., T366S L368A Y407V. Thus, the heterodimeric precursor polypeptide contains a destabilizing mutation, i.e., other hydrophobic amino acid, rather than an alanine (A) at position 368 in the CH3 domain. In another example, the destabilizing mutation can be located at position 407 of the CH3 domain with hole mutations, e.g., T366S L368A Y407V. Thus, the heterodimeric precursor polypeptide contains a destabilizing mutation, i.e., other hydrophobic amino acid, rather than a valine (V) at position 407 in the CH3 domain. However, such heterodimeric precursor polypeptides with such substitutions or substitutions are considered to be encompassed by the present invention.

[0114] A) Amino acid substitutions in the CH3 domain The precursor polypeptide provided by the invention contains an amino acid substitution in its CH3 domain.

[0115] 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.

[0116] In one embodiment, the knob mutation is T366W. In one embodiment, the hole mutation is T366S L368A Y407V.

[0117] Destabilizing mutations As noted above, only one CH3 domain of each precursor polypeptide contains one or more destabilizing mutations.

[0118] According to the present invention, 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. any of the heterodimeric precursor polypeptides comprises one or more destabilizing mutations. 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 polypeptide chain exchange between the precursor polypeptides.

[0119] If a CH3 domain of a heterodimeric precursor polypeptide that includes a knob mutation includes a destabilizing mutation, then a CH3 domain of said heterodimeric precursor polypeptide that includes a hole mutation does not include a destabilizing mutation. If a CH3 domain "does not include a destabilizing mutation," then it includes a wild-type amino acid residue at a position that interacts with an amino acid residue at the position of a destabilizing mutation included in a corresponding CH3 domain in a wild-type immunoglobulin CH3 domain of the same class.

[0120] In one embodiment of the present invention, the CH3 domain having a hole mutation comprises at least one amino acid mutation, i.e., a destabilizing mutation, selected from the group consisting of: substitution of S354 with a hydrophobic amino acid, substitution of D356 with a positively charged amino acid, substitution of E357 with a positively charged amino acid or a hydrophobic amino acid, substitution of D356 with a positively charged amino acid, and substitution of E357 with a positively charged amino acid or a hydrophobic amino acid, substitution of S364 with a hydrophobic amino acid, substitution of A368 with a hydrophobic amino acid, substitution of K392 with a negatively charged amino acid, substitution of T394 with a hydrophobic amino acid, substitution of D399 with a hydrophobic amino acid, substitution of S400 with a positively charged amino acid, substitution of D399 with a hydrophobic amino acid, substitution of F405 with a positively charged amino acid, substitution of V407 with a hydrophobic amino acid, substitution of K409 with a negatively charged amino acid, and substitution of K439 with a negatively charged amino acid. and substitution of K360 with a negatively charged amino acid, substitution of Y349 with a negatively charged amino acid, substitution of L351 with a hydrophobic amino acid and substitution of E357 with a hydrophobic amino acid, substitution of S364 with a hydrophobic amino acid, substitution of W366 with a hydrophobic amino acid, substitution of K409 with a negatively charged amino acid, substitution of L368 with a hydrophobic amino acid, substitution of K370 with a negatively charged amino acid, substitution of K370 with a negatively charged amino acid, substitution of K439 with a negatively charged amino acid, substitution of K392 with a negatively charged amino acid, substitution of T394 with a hydrophobic amino acid, substitution of V397 with a hydrophobic amino acid, substitution of D399 with a positively charged amino acid and substitution of K409 with a negatively charged amino acid, substitution of S400 with a positively charged amino acid, F405W, Y407W, and substitution of K439 with a negatively charged amino acid.

[0121] In one embodiment of the invention, the CH3 domain having a hole mutation comprises at least one amino acid substitution, i.e., a destabilizing mutation, selected from the group consisting of substitution of E357 with a positively charged amino acid, substitution of S364 with a hydrophobic amino acid, substitution of A368 with a hydrophobic amino acid, and substitution of V407 with a hydrophobic amino acid, and the CH3 domain having a knob mutation either does not comprise a destabilizing mutation or comprises at least one amino acid substitution, i.e., a destabilizing mutation, selected from the group consisting of substitution of K370 with a negatively charged amino acid, substitution of K370 with a negatively charged amino acid, and substitution of K439 with a negatively charged amino acid, substitution of K392 with a negatively charged amino acid, and substitution of V397 with a hydrophobic amino acid.

[0122] 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.

[0123] 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.

[0124] In one embodiment, the negatively charged amino acid is Asp or Glu. In one embodiment, the negatively charged amino acid residue is Asp. In one embodiment, the negatively charged amino acid residue is Glu.

[0125] Amino acid substitutions at the indicated amino acid positions in the CH3 domain with amino acids having the respective side chain properties were found to support polypeptide chain exchange and product polypeptide formation from two precursor polypeptides.

[0126] In one embodiment of the present invention, the CH3 domain with hole mutations are S354V, S354I, S354L, D356K, D356R, E357K, E357R, E357F, S364L, S364I, A368F, K392D, K392E, T394L, T394I, V407Y, K409E, K409D, K439D, K439E and double mutation D399A S400K, D399A S400R, D399A and F405W, Y349E, Y349D, S364V, S364I, S364L, L368F, K370E, K370D, K392E, K392D, T394L, T394I, V397Y, S400K, S400R, F405W, Y407W, K349E, K439D and 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.

[0127] In one embodiment of the invention, the CH3 domain with hole mutations 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, and 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 mutation Q347K K360E, L351F E357F, W366I K409E, and D399K K409E.

[0128] In one embodiment of the present invention, the CH3 domain with hole mutations comprises at least one amino acid substitution selected from the group of D356K, D356R, E357K, E357R, E357F, S364L, S364I, V407Y, K409E, K409D and double mutation D399A S400K, D399A S400R, and the CH3 domain with knob mutations 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 double mutation 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.

[0129] In one embodiment of the invention, 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, and 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 mutation Q347K K360E, W366I K409E, and D399K K409E.

[0130] In one embodiment of the invention, the CH3 domain with hole mutations and the CH3 domain with knob mutations, including the destabilizing mutations, comprise one of the amino acid substitutions selected from the group shown in the table below. TIFF0007638900000002.tif118143TIFF0007638900000003.tif211143

[0131] For clarity, this table should be understood in 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.

[0132] In one embodiment of the invention, the CH3 domain with hole mutations and the CH3 domain with knob mutations, including the destabilizing mutations, comprise one of the amino acid substitutions selected from the group shown in the table below. TIFF0007638900000004.tif87141

[0133] In one embodiment of the invention, the CH3 domain with a hole mutation comprises at least one amino acid substitution selected from the group of E357K, E357R, S364L, S364I, V407Y, V407F and A368F, and the CH3 domain with a knob mutation does not comprise a destabilizing mutation or comprises at least one amino acid substitution selected from the group of K370E, K370D, K392E, K392D, V397Y, and the double mutations K370E K439E, K370D K439E, K370E K439D, and K370D K439D.

[0134] In one embodiment of the invention, the CH3 domain with a hole mutation comprises at least one amino acid substitution selected from the group of E357K, S364L, V407Y and A368F, and the CH3 domain with a knob mutation comprises at least one amino acid substitution selected from the group of K370E, K392D, V397Y and the double mutation K370E K439E.

[0135] In one embodiment of the invention, the CH3 domain with a hole mutation comprises at least one amino acid substitution selected from the group of E357K, E357R, S364L, S364I, V407Y and V407F, and the CH3 domain with a knob mutation comprises at least one amino acid substitution selected from the group of K370E, K370D, K392E, K392D, V397Y, and the double mutations K370E K439E, K370D K439E, K370E K439D, and K370D K439D.

[0136] In one embodiment of the invention, the CH3 domain having a hole mutation comprises at least one amino acid substitution selected from the group of E357K, S364L and V407Y, and the CH3 domain having a knob mutation comprises at least one amino acid substitution selected from the group of K370E, K392D, V397Y and the double mutation K370E K439E.

[0137] In one embodiment of the invention, the CH3 domain with hole mutations and the CH3 domain with knob mutations, including the destabilizing mutations, comprise one of the amino acid substitutions selected from the group shown in the table below. TIFF0007638900000005.tif99143

[0138] For clarity, this table should be understood in that a CH3 domain containing a hole mutation contains a destabilizing mutation shown in the first column of the table above, and a CH3 domain containing a knob mutation contains a destabilizing mutation listed in the right column of the table above shown in the same row.

[0139] In one embodiment of the invention, the CH3 domain with hole mutations and the CH3 domain with knob mutations, including the destabilizing mutations, comprise one of the amino acid substitutions selected from the group shown in the table below. TIFF0007638900000006.tif62141

[0140] In one embodiment of the invention, the CH3 domain with hole mutations and the CH3 domain with knob mutations, including the destabilizing mutations, comprise one of the amino acid substitutions selected from the group shown in the table below. TIFF0007638900000007.tif124141

[0141] For clarity, this table should be understood in that the CH3 domain containing the hole mutation contains the destabilizing mutation shown in the first column of the table above, and the CH3 domain containing the knob mutation contains the destabilizing mutation listed in the right column of the table above shown in the same row. Precursor molecules with this combination of destabilizing mutations exhibit certain beneficial polypeptide chain exchanges.

[0142] In one embodiment of the invention, the CH3 domain with hole mutations and the CH3 domain with knob mutations, including the destabilizing mutations, comprise one of the amino acid substitutions selected from the group shown in the table below. TIFF0007638900000008.tif161141

[0143] For clarity, this table should be understood in that the CH3 domain containing the hole mutation contains the destabilizing mutation shown in the first column of the table above, and the CH3 domain containing the knob mutation contains the destabilizing mutation listed in the right column of the table above shown in the same row. Precursor molecules with this combination of destabilizing mutations can be produced in high yields while exhibiting certain beneficial polypeptide chain exchange.

[0144] Cysteine ​​mutations In one embodiment of the invention, the CH3 domain of the heterodimeric precursor polypeptide comprises a third pattern of mutation, i.e., substitution of different amino acids with cysteines at the CH3 / CH3 interface, to allow the formation of interchain disulfide bonds between the two CH3 domains with cysteine ​​substitutions at interacting positions.

[0145] Thus, in one embodiment of the present invention, either 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, either 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.

[0146] In one embodiment, either 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: TIFF0007638900000009.tif51141

[0147] In one embodiment, the first cysteine ​​mutation is Y349C and the second cysteine ​​mutation is S354C.

[0148] In one embodiment of the invention, i) the CH3 domain comprising the knob mutation of the first heterodimeric precursor polypeptide comprises a substitution S354C and the CH3 domain comprising the hole mutation of the second heterodimeric precursor polypeptide comprises a substitution Y349C, or ii) the CH3 domain comprising the hole mutation of the first heterodimeric precursor polypeptide comprises a substitution Y349C and the CH3 domain comprising the knob mutation of the second heterodimeric precursor polypeptide comprises a substitution S354C.

[0149] In one embodiment of the invention, in a 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 said 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.

[0150] 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.

[0151] In one embodiment of the invention, in a first heterodimeric precursor polypeptide, the CH3 domain comprising the knob mutation comprises the substitution T366W S354C, the CH3 domain comprising the hole mutation comprises a Y at position 349, and the substitution T366S L368A Y407V, and in a 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.

[0152] In one embodiment of the invention, the CH3 domain of the heterodimeric precursor polypeptide does not comprise an interchain disulfide bond.

[0153] B) Antigen binding portion In one embodiment of the invention, the antigen-binding moiety is a polypeptide that specifically binds to an antigen. In one embodiment, the antigen-binding moiety is selected from the group of antibodies, receptors, ligands and DARPins, which are capable of specifically binding to an antigen.

[0154] In one embodiment of the invention, the antigen-binding moiety comprised in the (precursor) polypeptide according to the invention is an antibody fragment.

[0155] In one embodiment of the invention, the antigen-binding portion comprises a pair of a VH domain and a VL domain, which form an antigen-binding site that specifically binds to a target antigen.

[0156] In one embodiment of the invention, the antibody fragment comprised in the (precursor) polypeptide according to the invention is an antibody fragment selected from the group of Fv, Fab, Fab', Fab'-SH, F(ab')2, diabody, scFv and scFab. In one embodiment, the antibody fragment comprised in the (precursor) polypeptide according to the invention is an Fv or Fab.

[0157] In one embodiment of the invention, the target antigen binding portion is a Fab fragment.

[0158] In one embodiment of the invention, the first antigen-binding portion is a first Fab fragment and the second antigen-binding portion is a second Fab fragment. In one embodiment of the invention, the first Fab fragment, the second Fab fragment, or both, the first Fab fragment and the second Fab fragment are linked by domain crossing: a) only the CH1 and CL domains are replaced with each other; b) only the VH and VL domains are replaced with each other, or c) The CH1 and CL domains are altered such that they are substituted for each other, and the VH and VL domains are substituted for each other.

[0159] 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.

[0160] 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.

[0161] 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 different antigens, in which case, upon polypeptide chain exchange between the two heterodimeric precursor polypeptides, a multispecific product polypeptide is formed that comprises an antigen-binding portion derived from the first heterodimeric precursor polypeptide and an antigen-binding portion derived from the second heterodimeric precursor polypeptide.

[0162] Additional antigen-binding moieties may be present in 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.

[0163] Such further antigen-binding moieties are fused to the polypeptide chain via a suitable peptide connector, in one embodiment the peptide connector is a glycine-serine linker.

[0164] In one embodiment of the present invention, in the 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 the heterodimeric precursor polypeptide, one of the polypeptide chains comprising a CH3 domain of the antigen-binding site that specifically binds to the target antigen. In one embodiment of the present invention, in the heterodimeric precursor polypeptide, one of the polypeptide chains comprising a CH3 domain comprises, from the N-terminus to the C-terminus, a hinge region, an antibody variable domain, and a CH3 domain, and the polypeptide chain is not part of the antigen-binding site that specifically binds to the target antigen. In one embodiment of the present invention, in the heterodimeric precursor polypeptide, one of the polypeptide chains comprising a CH3 domain comprises, from the N-terminus to the C-terminus, a hinge region, an antibody variable domain, a CH2 domain, and a CH3 domain, and the polypeptide chain is not part of the antigen-binding site that specifically binds to the target antigen.

[0165] C) Domain arrangement of the precursor polypeptide The precursor polypeptides according to the invention are suitable for the generation of product polypeptides in a variety of formats and in a variety of domain arrangements. 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.

[0166] In one embodiment, the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide comprise exactly two polypeptide chains that contain a CH3 domain. Thus, an additional polypeptide chain that lacks a CH3 domain may be included in the first and second heterodimeric precursor polypeptides.

[0167] Precursor Polypeptides Containing Antibody Fragments In one embodiment of the invention, the antigen-binding portion comprises a pair of a VH domain and a VL domain, which form an antigen-binding site that specifically binds to a target antigen, and a) a first heterodimeric precursor polypeptide comprising: - a further antibody variable domain (first antibody variable domain) in a first heavy chain polypeptide comprising a CH3 domain, and - a further polypeptide chain which is a light chain polypeptide comprising a second antibody variable domain. wherein the first antibody variable domain and the second antibody variable domain together form a first antigen-binding site that specifically binds to a target antigen; and b) the second heterodimeric precursor polypeptide comprises - a further antibody variable domain in a third heavy chain polypeptide comprising a CH3 domain (a third antibody variable domain), and - a further polypeptide chain which is a light chain polypeptide comprising a fourth antibody variable domain wherein said third antibody variable domain and said fourth antibody variable domain together form a second antigen-binding site that specifically binds to a target antigen.

[0168] Precursor Polypeptides Containing an Activatable Antigen Binding Site According to the invention, each precursor polypeptide comprises a portion of an antigen-binding moiety, said antigen-binding moiety being non-functional in the precursor polypeptide, and being functional in a product polypeptide formed by polypeptide chain exchange between the precursor polypeptides, which specifically binds to a target antigen. Exemplary structures of such precursor polypeptides are shown in FIG.

[0169] In one embodiment of the invention, said antigen binding portion is an antigen binding site comprising a pair of antibody variable domains.

[0170] In one embodiment of the 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 said VL domain and said VH domain specifically bind to an antigen when associated with the pair of VH and VL domains. In one embodiment, the antigen specifically bound by the pair of VH and VL domains is CD3.

[0171] In one embodiment of the 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 with the pair of VH domain and VL domain.

[0172] In one embodiment of the present invention, a) a first heterodimeric precursor polypeptide comprising: - a first heavy chain polypeptide comprising, from N-terminal to C-terminal direction, 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, 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, one of the CH3 domains comprising a knob mutation and the other CH3 domain comprising 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 associate with each other to form an antigen-binding site that specifically binds to a target antigen. and b) the second heterodimeric precursor polypeptide comprises - a third heavy chain polypeptide comprising, from 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, 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, one of the CH3 domains comprising a knob mutation and the other CH3 domain comprising 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. and c) the variable domains of said first heavy chain polypeptide and said third heavy chain polypeptide are capable of forming an antigen-binding site that specifically binds to a target antigen.

[0173] In one embodiment, the first heavy chain polypeptide comprises, from N-terminal to C-terminal, a first VH domain, a second antibody variable domain selected from a CH1 domain, 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, 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, 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 third antibody variable domain selected from a second VH domain, a CH1 domain, 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, a CH2 domain and a CH3 domain; the third heavy chain polypeptide and the fourth heavy chain polypeptide comprise, In one embodiment, the peptide connectors included in the first and third heavy chain polypeptides are identical.

[0174] Precursor Polypeptide Containing the Hinge Region In one embodiment of the invention, the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide comprise, from N-terminal to C-terminal direction, at least two heavy chain polypeptides comprising a hinge region and a CH3 domain.

[0175] 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 can be advantageously used in therapy.

[0176] In one embodiment of the invention, the first and second heterodimeric precursor polypeptides 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.

[0177] The heterodimeric precursor polypeptide may comprise a peptide connector linking (a part of) the antigen binding moiety with the antibody domain (i.e., VL, VH or CH2) in place of a hinge region without an interchain disulfide bond. In one embodiment of the invention, no interchain disulfide bond is formed between the first and second peptide connectors. In one embodiment of the invention, the first and second peptide connectors are identical to each other.

[0178] In one embodiment of the invention, the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide comprise, from N-terminal to C-terminal direction, at least two polypeptide chains comprising a peptide connector and a CH3 domain.

[0179] 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. Depending on, for example, the type of antigen bound by the activatable binding site, shorter (or even longer) peptide connectors may also be applicable to the heterodimeric precursor polypeptides according to the invention.

[0180] In yet another embodiment of the invention, the first and second peptide connectors are approximately the same length as the native hinge region (about 15 amino acids for native antibody molecules of IgG1 isotype and about 62 amino acids for IgG3 isotype). Thus, in one embodiment where the first and second heterodimeric precursor polypeptides are of IgG1 isotype, the peptide connector is a peptide of 10-20 amino acids, in a preferred embodiment 12-17 amino acids. In another embodiment where the first and second heterodimeric precursor polypeptides are of IgG3 isotype, the peptide connector is a peptide of 55-70 amino acids, in a preferred embodiment 60-65 amino acids.

[0181] 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 glycine and serine residues. 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.

[0182] 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 one preferred embodiment, x=4, n=2 or 3, and m=0. In one yet another preferred embodiment, x=4, and n=2. In one embodiment, the peptide connector is (G4S)4 or (G4S)6.

[0183] D) Antibody isotype and binding valency In one embodiment of the invention, the precursor polypeptide comprises an immunoglobulin constant region of one or more immunoglobulin classes, including IgG, IgM, IgA, IgD, and IgE isotypes, including, in the case of IgG and IgA, their subtypes. In one embodiment of the invention, the precursor polypeptide has the constant domain structure of an IgG type antibody.

[0184] In one embodiment of the invention, the CH3 domain contained in the precursor polypeptide is of the mammalian IgG class. In one embodiment of the invention, the CH3 domain contained in the precursor polypeptide is of the mammalian IgG1 subclass. In one embodiment of the invention, the CH3 domain contained in the precursor polypeptide is of the mammalian IgG4 subclass.

[0185] 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.

[0186] In one embodiment, the constant domain of the precursor polypeptide according to the invention is of the human IgG class. 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.

[0187] In one embodiment, the precursor polypeptide lacks a CH4 domain.

[0188] In one embodiment of the invention, the constant domains of the precursor polypeptides 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 polypeptides according to the invention are of the same immunoglobulin subclass.

[0189] 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.

[0190] In one embodiment, a heterodimeric precursor polypeptide or a 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.

[0191] 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 bispecific or trispecific.

[0192] 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.

[0193] In one embodiment, the first heterodimeric precursor polypeptide is bispecific. In one embodiment, the second heterodimeric precursor polypeptide is monospecific.

[0194] In one embodiment, the first heterodimeric precursor polypeptide is bispecific. In one embodiment, the second heterodimeric precursor polypeptide is bispecific.

[0195] In one embodiment, the first heterodimeric precursor polypeptide is monovalent. In one embodiment, the second heterodimeric precursor polypeptide is monovalent.

[0196] In one embodiment, the first heterodimeric precursor polypeptide is bivalent. In one embodiment, the second heterodimeric precursor polypeptide is bivalent.

[0197] In one embodiment, the first heterodimeric precursor polypeptide is trivalent. In one embodiment, the second heterodimeric precursor polypeptide is trivalent.

[0198] In one embodiment, the heterodimeric product polypeptide is trivalent. In one embodiment, the heterodimeric product polypeptide is tetravalent.

[0199] E) Methods for Producing Product Polypeptides In one embodiment, the present invention provides a method of producing a heterodimeric product polypeptide, comprising contacting a first heterodimeric precursor polypeptide of the present invention with a second heterodimeric precursor polypeptide to form a third heterodimeric polypeptide comprising a first heavy chain polypeptide and a third heavy chain polypeptide. In one embodiment of the present invention, the method comprises recovering the third heterodimeric polypeptide. In one embodiment of the present invention, the third heterodimeric polypeptide comprises at least three antigen binding sites.

[0200] In one embodiment of the invention, the method comprises contacting a first heterodimeric precursor polypeptide with a second heterodimeric precursor polypeptide to form a fourth heterodimeric polypeptide comprising a second heavy chain polypeptide and a fourth heavy chain polypeptide. In one embodiment of the invention, the method comprises recovering the fourth heterodimeric polypeptide. In one embodiment of the invention, the fourth heterodimeric polypeptide does not comprise an antigen binding site that specifically binds to an antigen.

[0201] In one embodiment of the invention, a first heterodimeric precursor polypeptide comprises an antigen binding portion that specifically binds to a first antigen and comprises a portion of a second antigen binding site, a second heterodimeric precursor polypeptide comprises an antigen binding portion that specifically binds to a third antigen and comprises another portion of the second antigen binding site, and a third heterodimeric polypeptide comprises an antigen binding portion that specifically binds to the first antigen, an antigen binding portion that specifically binds to the second antigen, and an antigen binding portion that specifically binds to said third antigen.

[0202] In one embodiment of the present invention, the first heterodimer precursor polypeptide and the second heterodimer precursor polypeptide comprise a hinge region that does not contain interchain disulfide bonds.In this case, polypeptide chain exchange can occur in the absence of reducing agent.Thus, in one embodiment, the first heterodimer precursor polypeptide and the second heterodimer precursor polypeptide comprise a hinge region that does not contain interchain disulfide bonds, and the first heterodimer precursor polypeptide and the second heterodimer precursor polypeptide are contacted in the absence of reducing agent.

[0203] In one embodiment of the invention, in the first heterodimeric polypeptide, no interchain disulfide bonds are formed between the two polypeptide chains comprising a CH3 domain, and in the second heterodimeric polypeptide, no interchain disulfide bonds are formed between the two polypeptide chains comprising a CH3 domain, and said contacting is performed in the absence of a reducing agent.

[0204] F) 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.

[0205] One aspect of the present invention is a heterodimeric polypeptide, which in one embodiment is a heterodimeric product polypeptide comprising a first heavy chain polypeptide and a third heavy chain polypeptide as defined above.

[0206] Another aspect of the invention is a heterodimeric polypeptide, which in one embodiment is a heterodimeric product polypeptide comprising a second heavy chain polypeptide and a fourth heavy chain polypeptide as defined above.

[0207] The heterodimeric (product) polypeptide according to the present invention comprises (i) two heavy chain polypeptides that contain destabilizing mutations, or (ii) two heavy chain polypeptides that do not contain destabilizing mutations. In option (i), in contrast to the precursor polypeptide, the heterodimeric (product) polypeptide comprises destabilizing mutations in both CH3 domains. In this configuration, the destabilizing mutations no longer destabilize the CH3 / CH3 interface, but support the formation of heterodimers between the heavy chain polypeptides. All the embodiments listed above for destabilizing mutations in the heterodimeric precursor polypeptide of the present invention apply to the heterodimeric product polypeptide.

[0208] Another product of the method for producing a heterodimeric product polypeptide, and therefore another aspect of the present invention, is a heterodimeric product polypeptide, preferably obtainable by the method of the present invention, which comprises two polypeptide chains comprising a CH3 domain, and both of the CH3 domains do not comprise a destabilizing mutation.

[0209] G) Recombination Methods The precursor polypeptides according to the invention are prepared by recombinant methods. Thus, the present invention also relates to a method for preparing a heterodimeric precursor polypeptide according to the invention, comprising culturing a host cell comprising a nucleic acid encoding the heterodimeric precursor polypeptide under conditions suitable for expression of the precursor polypeptide.

[0210] In one aspect, a method for producing a heterodimeric precursor polypeptide of the invention is provided, the method comprising culturing a host cell comprising a nucleic acid 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).

[0211] 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 allowing synthesis of the heterodimeric precursor polypeptide, and recovering the heterodimeric precursor polypeptide from the host cell culture.

[0212] For recombinant production of heterodimeric precursor polypeptides, nucleic acids encoding the heterodimeric precursor polypeptides, e.g., as described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids may 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 may be produced by recombinant methods, or may be obtained by chemical synthesis.

[0213] 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 expression of polypeptides in bacteria, see, for example, U.S. Patent No. 5,648,237, U.S. Patent No. 5,789,199 and U.S. Patent No. 5,840,523 (also see Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, heterodimeric precursor polypeptides can be isolated from bacterial cell paste in soluble fractions and can be further purified.

[0214] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding the heterodimeric precursor polypeptides of the invention, including fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of polypeptides having partial or complete human glycosylation patterns. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0215] Suitable host cells for the expression of the (glycosylated) heterodimeric precursor polypeptides are also derived 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 the transfection of insect cells, particularly Spodoptera frugiperda cells.

[0216] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PLANTIBoDIES™ technology for producing antibodies in transgenic plants).

[0217] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are 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), MRC5 cells and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHo) cells, including DHFR-CHo cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220), and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cells suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0218] In one aspect, the host cell is eukaryotic, such as a Chinese hamster ovary (CHo) cell or a lymphocytic cell (eg, Y0, NS0, Sp20 cell).

[0219] In one aspect, the invention provides an isolated nucleic acid encoding a heterodimeric precursor polypeptide of the invention. In one aspect, the invention provides an expression vector comprising a nucleic acid according to the invention. In another aspect, the invention provides a host cell comprising a nucleic acid of the invention.

[0220] I) Therapeutic use The set of heterodimeric precursor polypeptides of the invention may be used therapeutically. The heterodimeric precursor polypeptides used therapeutically comprise an activatable antigen-binding site as defined above.

[0221] Thus, one aspect of the present invention is a set of heterodimeric precursor polypeptides according to the present invention for use as a medicament. Another aspect of the present invention is a pharmaceutical composition comprising a set of heterodimeric precursor polypeptides of the present invention and a pharma- ceutically acceptable carrier. Another aspect of the present invention is a method of treating an individual with a disease, comprising administering to said individual an effective amount of the first and second heterodimeric precursor polypeptides of the present invention or the pharmaceutical composition of the present invention.

[0222] One aspect of the invention is a set of heterodimeric precursor polypeptides according to the invention, wherein the variable domains of the first and third heavy chain polypeptides are capable of forming an antigen binding site that specifically binds to CD3, for use in the treatment of cancer. Another aspect of the invention is a method of treating an individual with cancer, comprising administering to said individual an effective amount of the first and second heterodimeric precursor polypeptides of the invention, wherein the variable domains of the first and third heavy chain polypeptides are capable of forming an antigen binding site that specifically binds to CD3.

[0223] In one embodiment, in the heterodimeric precursor polypeptide used for treatment, no interchain disulfide bond is formed in the first heterodimeric polypeptide between the two polypeptide chains comprising the CH3 domain, and an interchain disulfide bond is formed in the second heterodimeric polypeptide between the two polypeptide chains comprising the CH3 domain.If there is no interchain disulfide bond between the heavy chain polypeptides, polypeptide chain exchange occurs in the absence of a reducing agent, and therefore can occur spontaneously, for example, when both heterodimeric precursor polypeptides are bound to a target antigen or target cell.

[0224] 3. Specific Embodiments of the Invention 1. A set of heterodimeric precursor polypeptides comprising: a) a first heterodimeric precursor polypeptide, - a first heavy chain polypeptide comprising, from N-terminal to C-terminal, an antibody variable domain selected from a VH domain and a VL domain and a CH3 domain, the first heavy chain polypeptide comprising at least a portion of a first antigen-binding portion; and - a second heavy chain polypeptide comprising, from N-terminus to C-terminus, a CH2 domain and a CH3 domain; Including, wherein the first heavy chain polypeptide and the second 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; b) a second heterodimeric precursor polypeptide, - a third heavy chain polypeptide comprising, from N-terminal to C-terminal, an antibody variable domain selected from a VH domain and a VL domain and a CH3 domain, wherein the antibody variable domain is capable of forming an antigen-binding site that specifically binds to a target antigen together with an antibody variable domain comprised in a first heavy chain polypeptide of the first heterodimeric precursor polypeptide, and wherein the third heavy chain polypeptide comprises at least a portion of a second antigen-binding portion; and a fourth heavy chain polypeptide comprising, from N-terminus to C-terminus, a CH2 domain and a CH3 domain; Including, a second heterodimeric precursor polypeptide, wherein the third heavy chain polypeptide and the fourth heavy chain polypeptide 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; Where: A) i) the first heavy chain polypeptide comprises a CH3 domain having a knob mutation and the third heavy chain polypeptide comprises a CH3 domain having a hole mutation; or ii) the first heavy chain polypeptide comprises a CH3 domain with a hole mutation and the third heavy chain polypeptide comprises a CH3 domain with a knob mutation; 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. Any of the above contains one or more amino acid substitutions that destabilize the CH3 / CH3 interface, and the amino acid substitutions are positioned such that the substituted amino acids interact at the CH3 / CH3 interface within the pair of CH3 domains.

[0225] 2. A set of heterodimeric polypeptides according to embodiment 1, wherein the CH3 domain comprising the knob mutation and the CH3 domain comprising the hole mutation as shown in B) comprise one or more of the following amino acid substitutions, wherein the numbering is according to the Kabat numbering system: - the CH3 domain having a hole mutation Substitution of S354 with a hydrophobic amino acid, Substitution of D356 with a positively charged amino acid, Substitution of E357 with a positively charged or hydrophobic amino acid, Substitution of D356 with a positively charged amino acid and substitution of E357 with a positively charged or hydrophobic amino acid, Substitution of S364 with a hydrophobic amino acid, Substitution of A368 with a hydrophobic amino acid, Substitution of K392 with a negatively charged amino acid, Substitution of T394 with a hydrophobic amino acid, Substitution of D399 with a hydrophobic amino acid and substitution of S400 with a positively charged amino acid, Substitution of D399 with a hydrophobic amino acid and substitution of F405 with a positively charged amino acid, at least one amino acid substitution selected from the substitution of V407 with a hydrophobic amino acid, Substitution of K409 with a negatively charged amino acid, and Substitution 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 Substitution of Q347 with a positively charged amino acid and K360 with a negatively charged amino acid, Substitution of Y349 with a negatively charged amino acid, Substitution of L351 with a hydrophobic amino acid and substitution of E357 with a hydrophobic amino acid, Substitution of S364 with a hydrophobic amino acid, Substitution of W366 with a hydrophobic amino acid and K409 with a negatively charged amino acid, Substitution of L368 with a hydrophobic amino acid, Substitution of K370 with a negatively charged amino acid, Substitution of K370 with a negatively charged amino acid and substitution of K439 with a negatively charged amino acid, Substitution of K392 with a negatively charged amino acid, Substitution of T394 with a hydrophobic amino acid, Substitution of V397 with a hydrophobic amino acid, Substitution of D399 with a positively charged amino acid and K409 with a negatively charged amino acid, Substitution of S400 with a positively charged amino acid, 〇 F405W 〇 Y407W, and Substitution of K439 with a negatively charged amino acid The present invention comprises at least one amino acid substitution selected from the group consisting of:

[0226] 3. A set of heterodimeric polypeptides according to embodiment 1 or 2, in which the CH3 domain comprising the knob mutation and the CH3 domain comprising the hole mutation as shown in B) comprise one or more of the following amino acid substitutions, wherein the numbering is according to the Kabat numbering system: a) the CH3 domain with hole mutations comprises at least one amino acid substitution selected from the group of S354V, S354I, S354L, D356K, D356R, E357K, E357R, E357F, S364L, S364I, A368F, K392D, K392E, T394L, T394I, V407Y, K409E, K409D, K439D, K439E and the double mutations D399A S400K, D399A S400R, D399A F405W; b) CH3 domains with knob mutations are Y349E, Y349D, S364V, S364I, S364L, L368F, K370E, K370D, K392E, K392D, T394L, T394I, V397Y, S400K, S400R, F405W, Y407W, K349E, K439D and double mutations Q347K K360E, Q347R K360E, Q347K K360D, Q347R K360D, L351F E357F, W366I K409E, W366L K409E, W366K K409D, W366L K409D, D399K K409E, D399R and at least one amino acid selected from K409E, D399K K409D and D399K K409E.

[0227] 4. A set of heterodimeric polypeptides according to one of the preceding embodiments, wherein the CH3 domain comprising the knob mutation and the CH3 domain comprising the hole mutation as shown in B) comprise one or more of the following amino acid substitutions, wherein the numbering is according to the Kabat numbering system: a) the CH3 domain with hole mutations 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; b) 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.

[0228] 5. A set of heterodimeric polypeptides according to one of the preceding embodiments, wherein the CH3 domain comprising the knob mutation and the CH3 domain comprising the hole mutation as shown in B) comprise one or more of the following amino acid substitutions, wherein the numbering is according to the Kabat numbering system: a) 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 the double mutation D399A S400K, D399A S400R; b) The CH3 domain with knob mutations 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.

[0229] 6. A set of heterodimeric polypeptides according to one of the preceding embodiments, wherein the CH3 domain comprising the knob mutation and the CH3 domain comprising the hole mutation as shown in B) comprise one or more of the following amino acid substitutions, wherein the numbering is according to the Kabat numbering system: a) 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; and b) The CH3 domain with the 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.

[0230] 7.B) The CH3 domain containing the knob mutation and the CH3 domain containing the hole mutation are shown in the following table: A set of heterodimeric polypeptides according to one of the preceding embodiments, comprising one or more amino acid substitutions selected from the group set forth in TIFF0007638900000010.tif112136TIFF0007638900000011.tif217136, wherein the numbering is according to the Kabat numbering system.

[0231] 8.B) The CH3 domain containing the knob mutation and the CH3 domain containing the hole mutation are shown in the following table: A set of heterodimeric polypeptides described in one of the preceding embodiments, comprising one or more amino acid substitutions selected from the group set forth in TIFF0007638900000012.tif87133, wherein the numbering is according to the Kabat numbering system.

[0232] 9. The set of heterodimeric polypeptides according to one of the preceding embodiments, wherein the CH3 domain comprising the knob mutation and the CH3 domain comprising the hole mutation as shown in B) comprise one or more of the following amino acid substitutions, wherein the numbering is according to the Kabat numbering system: - the CH3 domain having a hole mutation Substitution of E357 with a positively charged amino acid, Substitution of S364 with a hydrophobic amino acid, Substitution of A368 with a hydrophobic amino acid, and Substitution of V407 with a hydrophobic amino acid At least one amino acid substitution selected from the group - The CH3 domain with the knob mutation Substitution of K370 with a negatively charged amino acid, Substitution of K370 with a negatively charged amino acid and substitution of K439 with a negatively charged amino acid, Substitution of K392 with a negatively charged amino acid, and Substitution of V397 with a hydrophobic amino acid The present invention comprises at least one amino acid substitution selected from the group consisting of:

[0233] 10.B) The CH3 domain containing the knob mutation and the CH3 domain containing the hole mutation are shown in the following table: A set of heterodimeric polypeptides according to one of the preceding embodiments, comprising one of the amino acid substitutions selected from the group set forth in TIFF0007638900000013.tif74131.

[0234] 11.B) The CH3 domain containing the knob mutation and the CH3 domain containing the hole mutation are shown in the following table: A set of heterodimeric polypeptides according to any one of the preceding embodiments, comprising one of the amino acid substitutions selected from the group set forth in TIFF0007638900000014.tif44131.

[0235] 12. The set of heterodimeric polypeptides according to one of the preceding embodiments, wherein the first antigen-binding moiety and / or the second antigen-binding moiety comprises a pair of VH and VL domains that form an antigen-binding site that specifically binds to a target antigen.

[0236] 13. The set of heterodimeric polypeptides according to one of the preceding embodiments, wherein the first antigen-binding portion and / or the second antigen-binding portion is an antibody fragment.

[0237] 14.a) the first heterodimeric precursor polypeptide comprises - a further antibody variable domain (first antibody variable domain) within said first heavy chain polypeptide comprising a CH3 domain, - a further polypeptide chain which is a light chain polypeptide comprising a second antibody variable domain. wherein the first antibody variable domain and the second antibody variable domain together form a first antigen-binding site that specifically binds to a target antigen; b) the second heterodimeric precursor polypeptide comprises - a further antibody variable domain (a third antibody variable domain) within said third heavy chain polypeptide comprising a CH3 domain, - a further polypeptide chain which is a light chain polypeptide comprising a fourth antibody variable domain wherein the third antibody variable domain and the fourth antibody variable domain together form a second antigen-binding site that specifically binds to a target antigen. 2. A set of heterodimeric polypeptides according to one of the preceding embodiments.

[0238] 15. The set of heterodimeric polypeptides according to one of the preceding embodiments, wherein the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide comprise a hinge region.

[0239] 16. The set of heterodimeric polypeptides according to embodiment 15, wherein the first heterodimeric precursor polypeptide and the second heterodimeric precursor polypeptide do not contain an interchain disulfide bond in the hinge region.

[0240] 17. A set of heterodimeric polypeptides according to one of the preceding embodiments, wherein in the first heterodimeric polypeptide, no interchain disulfide bond is formed between the two polypeptide chains comprising a CH3 domain, and in the second heterodimeric polypeptide, no interchain disulfide bond is formed between the two polypeptide chains comprising a CH3 domain.

[0241] 18.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; and - a second heavy chain polypeptide comprising, from N-terminal to C-terminal, 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, one of the CH3 domains comprising a knob mutation and the other CH3 domain comprising 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 associate with each other to form an antigen-binding site that specifically binds to a target antigen. Including, 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, and a CH3 domain; and - a fourth heavy chain polypeptide comprising, in an N-terminal to C-terminal direction, 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, one of the CH3 domains comprising a knob mutation and the other CH3 domain comprising 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. Including, c) 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; 2. A set of heterodimeric polypeptides according to one of the preceding embodiments.

[0242] 19. The set of heterodimeric precursor polypeptides according to 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.

[0243] 20. The set of heterodimeric precursor polypeptides according to 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.

[0244] 21. The set of heterodimeric precursor polypeptides according to one of the preceding embodiments, wherein the antibody variable domains comprised in the first heavy chain polypeptide and the third heavy chain polypeptide are capable of forming an antigen binding site that specifically binds to CD3.

[0245] 22. A method for producing a heterodimeric polypeptide, comprising contacting a first heterodimeric precursor polypeptide and a second heterodimeric precursor polypeptide as defined in one of embodiments 1 to 21 to form a third heterodimeric polypeptide comprising the first heavy chain polypeptide and the third heavy chain polypeptide.

[0246] 23. The method of embodiment 23, comprising recovering the third heterodimeric polypeptide.

[0247] 24. The method of embodiment 22 or 23, comprising contacting the first heterodimeric precursor polypeptide with the second heterodimeric precursor polypeptide to form a fourth heterodimeric polypeptide comprising the second heavy chain polypeptide and the fourth heavy chain polypeptide.

[0248] 25. The method of embodiment 24, comprising recovering the fourth heterodimeric polypeptide.

[0249] 26. The method according to one of embodiments 22 to 25, wherein the fourth heterodimeric polypeptide does not comprise an antigen-binding site.

[0250] 27. The method of one of embodiments 22 to 26, wherein the third heterodimeric polypeptide comprises at least three antigen-binding sites.

[0251] 28. The method of one of embodiments 22 to 27, wherein the first heterodimeric precursor polypeptide comprises an antigen-binding portion that specifically binds to the first antigen and comprises a portion of the second antigen-binding site, the second heterodimeric precursor polypeptide comprises an antigen-binding portion that specifically binds to a third antigen and comprises another portion of the second antigen-binding site, and the third heterodimeric polypeptide comprises an antigen-binding portion that specifically binds to the first antigen, an antigen-binding portion that specifically binds to the second antigen, and an antigen-binding portion that specifically binds to the third antigen.

[0252] 29. The method according to one of embodiments 22 to 28, wherein in the first heterodimeric polypeptide, no interchain disulfide bond is formed between the two polypeptide chains comprising a CH3 domain, and in the second heterodimeric polypeptide, no interchain disulfide bond is formed between the two polypeptide chains comprising a CH3 domain, and the contacting is performed in the absence of a reducing agent.

[0253] 30. The method according to one of embodiments 22 to 28, wherein in the first heterodimeric polypeptide, at least one interchain disulfide bond is formed between the two polypeptide chains comprising a CH3 domain, and in the second heterodimeric polypeptide, at least one interchain disulfide bond is formed between the two polypeptide chains comprising a CH3 domain, and the contacting is performed in the presence of a reducing agent.

[0254] 31. A third heterodimeric polypeptide obtainable by the method according to any one of embodiments 22 to 30.

[0255] 32. A fourth heterodimeric polypeptide obtainable by a method according to any one of embodiments 22 to 30.

[0256] 33. A first heterodimeric precursor polypeptide as defined in any one of embodiments 1 to 21.

[0257] 34. A second heterodimeric precursor polypeptide as defined in any one of embodiments 1 to 21.

[0258] 35. A set of heterodimeric precursor polypeptides according to any one of embodiments 1 to 21 for use as a medicament.

[0259] 36. A pharmaceutical composition comprising a set of heterodimeric precursor polypeptides according to any one of embodiments 1 to 21 and a pharma- ceutically acceptable carrier.

[0260] 37. A method for treating an individual having a disease, comprising administering to the individual an effective amount of a first and a second heterodimeric precursor polypeptide according to any one of embodiments 1 to 21 or a pharmaceutical composition according to embodiment 36.

[0261] 38. A set of heterodimeric precursor polypeptides according to any one of embodiments 1 to 21, wherein in the first and second heterodimeric precursor polypeptides, the antibody variable domains comprised in the first heavy chain polypeptide and the third heavy chain polypeptide are capable of forming an antigen binding site that specifically binds to CD3 for use in the treatment of cancer.

[0262] 39. A method of treating an individual having cancer, comprising administering to the individual an effective amount of a first and a second heterodimeric precursor polypeptide according to any one of embodiments 1 to 21, wherein in the first and second heterodimeric precursor polypeptides, the antibody variable domains contained in the first heavy chain polypeptide and the third heavy chain polypeptide are capable of forming an antigen-binding site that specifically binds to CD3.

[0263] Explanation of amino acid sequence TIFF0007638900000015.tif218146TIFF0007638900000016.tif218146TIFF00076389000 00017.tif173146TIFF0007638900000018.tif198146TIFF0007638900000019.tif173146 TIFF0007638900000020.tif173146TIFF0007638900000021.tif173146TIFF00076389000 00022.tif166146TIFF0007638900000023.tif224146TIFF0007638900000024.tif205146 EXAMPLES

[0264] The following examples are provided to aid in the understanding of the present invention, the actual scope of which is set forth in the appended claims. It is understood that variations can be made in the procedures set forth without departing from the spirit of the invention.

[0265] Example 1: Generation of monospecific precursor polypeptides containing an entire Fc domain for determination of destabilizing mutations that favor polypeptide chain exchange. This example is a proof-of-concept example for identifying destabilizing mutations suitable for supporting polypeptide chain exchange and therefore applicable to the present invention.

[0266] To assess the efficacy of polypeptide chain exchange to yield bispecific anti-biocytinamide / anti-fluorescein antibodies from monospecific precursor polypeptides via polypeptide chain exchange, the following monospecific precursor polypeptides were generated:

[0267] The first heterodimeric precursor polypeptide (also referred to as "anti-bio precursor") contained a Fab fragment that specifically binds to the biotin derivative biocytinamide ("bio") and had 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 a basic amino acid sequence without destabilizing mutations), with the destabilizing mutations and histidine tag shown below. The second heavy chain polypeptide (also referred to as the "dummy hole" polypeptide) contained, from N- to C-terminus, a hinge region, a CH2 domain and a CH3 domain.

[0268] The second heterodimeric precursor polypeptide (also referred to as "anti-fluoro 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 with destabilizing mutations as shown below (representing a basic amino acid sequence without destabilizing mutations) and a histidine tag. The second heavy chain polypeptide (also referred to as "dummy knob" polypeptide) comprised, from the N-terminus to the C-terminus, a hinge region, a CH2 domain and a CH3 domain.

[0269] The CH3 domain of the polypeptide chain shown contains the following mutations:

[0270] Table 1 Amino acid substitutions in the CH3 domain of the precursor polypeptide TIFF0007638900000025.tif30165

[0271] An anti-bio precursor was generated that contained a dummy hole polypeptide having the amino acid sequence of SEQ ID NO:05 and 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.

[0272] An anti-fluo precursor was generated that contains a dummy knob polypeptide having the amino acid sequence of SEQ ID NO:10 and any of the following amino acid substitutions were 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.

[0273] An expression plasmid for the precursor polypeptide was generated as follows. For the expression of the anti-bio and anti-fluo precursors reported herein, a transcription unit was used that contains the following functional elements: - the immediate early enhancer and promoter from human cytomegalovirus (P-CMV) including intron A, - a human heavy chain immunoglobulin 5' untranslated region (5'UTR), - mouse immunoglobulin heavy chain signal sequence, - a nucleic acid encoding each of the precursor polypeptides, and - bovine growth hormone polyadenylation sequence (BGH pA). - A basic / standard mammalian expression plasmid, in addition to the expression unit / cassette containing the desired gene to be expressed - the origin of replication from the vector pUC18 allowing replication of this plasmid in E. coli, and - The beta-lactamase gene that confers ampicillin resistance to E. coli Includes.

[0274] Recombinant production of precursor polypeptides Transient expression of the anti-bio and anti-fluo precursors described herein was performed in suspension adapted Expi293F™ cells (A14527, Life Technologies™) in Expi293F™ Expression Medium (A1435101, Life Technologies™) using the transfection reagent mixture ExpiFectamine™ 293 Transfection Kit (A14524; Life Technologies™).

[0275] After thawing in a 125 ml shake flask (incubated / shaken at 37°C, 7% CO2, 85% humidity, 135 rpm), cells were passaged by dilution at least four times (volume 30 ml). Cells were diluted to 3 × 10 in a volume of 250 ml. 5 After 3 days, the cells were split and grown to 1.3 × 10 cells / ml in a 250 ml volume in a 1 liter shake flask. 6 Freshly seeded at a density of 1.2–2.8 × 10 cells / ml, 24 h after transfection, 6 Cell densities were measured in cells / ml.

[0276] Prior to transfection, 30 μg of plasmid DNA was diluted in pre-warmed (water bath, 37 °C) opti-MEM (Gibco) to a final volume of 1.5 ml. The solution was gently mixed and incubated at room temperature for no more than 5 min. Then, 1.5 ml of a pre-incubated solution of ExpiFectamine™ reagent in opti-MEM was added to the DNA-optiMEM solution. The resulting solution was gently mixed and incubated at room temperature for 20-30 min. The entire volume of the mixture was added to a 100 ml shake flask, a 50 ml Falcon tube or a deep well in a 48-well deep well plate containing 30 ml Expi293F™ culture.

[0277] Transfected cells were incubated at 37° C., 7% CO 2 , 85% humidity for 7 days, shaking at 110 rpm for shake flasks and 205 rpm for Falcon tubes.

[0278] 16-24 hours after transfection, 20 μl of ExpiFectamine™ Enhancer 1 and 200 μl of ExpiFectamine™ Enhancer 2 were added to 30 ml of cell culture.

[0279] The supernatant was collected by centrifugation at 4,000 rpm for 20 min 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.).

[0280] Antibodies were purified from cell culture supernatants by affinity chromatography using MabSelectSure-Sepharose™ (GE Healthcare, Sweden) chromatography.

[0281] Briefly, sterile filtered cell culture supernatants were captured onto MabSelectSuRe resin equilibrated with PBS buffer (10 mM Na2HPo4, 1 mM KH2Po4, 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. Elution fractions for each precursor polypeptide were pooled and neutralized with 2 M Tris, pH 9.0.

[0282] Alternatively, precursor polypeptides were purified from cell culture supernatants by affinity chromatography using ani-Ckappa resin (KappaSelect, GE Healthcare, Sweden).

[0283] Briefly, sterile filtered cell culture supernatant was captured onto KappaSelect resin equilibrated with PBS buffer (10 mM Na2HPo4, 1 mM KH2Po4, 137 mM NaCl and 2.7 mM KCl, pH 7.4), washed with equilibration buffer and eluted with 25 mM sodium citrate, pH 3.0. Eluted antibody fractions were pooled and neutralized with 2 M Tris, pH 9.0.

[0284] The identity of the precursor polypeptides was confirmed by mass spectrometry. For each individual sample, the 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 (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 theoretically predicted molecular masses.

[0285] Analytical size exclusion chromatography was performed on a BioSuite High Resolution SEC Column (250 Å, Waters, USA) with a 200 mM K2HPo4 / KH2Po4, 250 mM KCl, pH 7.0 running buffer at a flow rate of 1 mg / ml. Prior to reaction set-up, the monomer content of all individual precursor polypeptides was assessed.

[0286] Example 2: Analysis of polypeptide chain exchange efficiency by direct detection of bispecific product polypeptide formation by ELISA. To evaluate the effect of different destabilizing mutations on polypeptide chain exchange, an exchange reaction was set up using the precursor polypeptide generated in Example 1. The polypeptide chain exchange in this experiment does not result in the activation of additional antigen binding sites.

[0287] The presence of the bispecific anti-biocytinamide / anti-fluorescein product polypeptide was assessed by ELISA.

[0288] To initiate the exchange reaction, anti-bio and anti-fluo precursor polypeptides were mixed in equimolar amounts at a protein concentration of 2 μM in a total volume of 48 μl of 1×PBS+0.05% Tween 20+0.25 mM TCEP on a 384-well REMP® plate (Brooks, #1800030) (normalized to monomeric SEC values ​​to ensure the same amount of intact molecules in a single reaction). Of note, the addition of the reducing agent TCEP reduces the hinge disulfides, thus favoring the dissociation of the polypeptide chains. After centrifugation, the plate was sealed and incubated at 37° C. for 1 h. The resulting reaction mixture was analyzed by ELISA.

[0289] Bispecific antibodies were then quantified using a biotin-fluorescein bridging ELISA.

[0290] Therefore, white Nunc® MaxiSorp™ 384-well plates were coated with 1 μg / ml albumin-fluorescein isothiocyanate conjugate (Sigma, #A9771) and incubated overnight at 4° C. After washing three times with 90 μl PBST buffer (PBST, double distilled water, 10xPBS+0.05% Tween 20) blocking buffer (1×PBS, 2% gelatin, 0.1% Tween-20) was added at 90 μl / well and incubated for 1 hour at room temperature. After washing three times with 90 μl PBST buffer, 25 μl of a 1:4 dilution of each reaction mixture was added to each well. After incubation for 1 hour at room temperature, the plate was again washed three times with 90 μl PBST buffer. 25 μl / well of biotin-Cy5 conjugate in 0.5% BSA, 0.025% Tween-20, 1×PBS was added to a final concentration of 0.1 μg / ml and the plate was incubated for 1 h at room temperature. After washing six times with 90 μl of PBST buffer, 25 μl of 1×PBS was added to each well. Cy5 fluorescence was measured in a Tecan Safire 2 Reader at an emission wavelength of 670 nm (excitation at 649 nm).

[0291] 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.

[0292] The preformed bispecific reference antibodies were analyzed by analytical size exclusion chromatography as described above.

[0293] Table 2. Monomer content of bispecific reference antibodies TIFF0007638900000026.tif15170

[0294] The absorbance signal from the reference antibody in the bridging ELISA setting was averaged from 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%. More than 100% polypeptide chain exchange reactions may be within this variability range. In addition, potential aggregates that may occur in the reaction mixture may result in an increase in the bridging signal.

[0295] The results are shown in Table 3.

[0296] Table 3. Formation of bispecific product polypeptides by polypeptide chain reaction from anti-bio and anti-fluo precursor polypeptides containing the indicated destabilizing mutations in the CH3 domain of the dummy chain. Columns indicate destabilizing mutations in the dummy hole polypeptide of the anti-bio precursor, and rows indicate destabilizing mutations in the dummy knob polypeptide of the anti-fluoro precursor. Relative absorbance detected by bridging ELISA is shown. Relative absorbance values ​​from pairs of CH3 mutations believed to favor polypeptide chain exchange are underlined. TIFF0007638900000027.tif185143TIFF0007638900000028.tif224143

[0297] Example 3: Analysis of polypeptide chain exchange efficiency by biochemical assay of bispecific product formation. A subset of anti-bio and anti-fluo precursors were reacted to form 86 bispecific product polypeptides. Equimolar amounts of precursor polypeptides described in Example 1 were combined for the reaction. Freshly prepared TCEP (60 eq., 0.5 mM TCEP pH 7.4 in 1×PBS, 0.05% Tween20) 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.

[0298] Bispecific product polypeptides were isolated as flow-through from a complete His-Tag column (Roche Diagnostics GmbH) equilibrated at 50 mM Na2HPo4, 300 mM NaCl, pH 8.0 and a flow rate of 1 ml / min. The product polypeptides, consisting of remaining unreacted precursor polypeptides and dummy chain heterodimers, were retained via their histidine tags and eluted for analytical purposes with 50 mM Na2HPo4, 300 mM NaCl, 250 mM imidazole at pH 8.0. Samples were concentrated to protein concentrations of 0.2 mg / ml–1.5 mg / ml via Amicon Ultra centrifuge tubes (Millipore) and analyzed by size-exclusion chromatography (SE-HPLC) using a BioSuite High Resolution SEC Column (250 Å, 5 μm, Waters, USA) using 200 mM K2HPo4 / KH2Po4, 250 mM KCl, pH 7.0 running buffer at a flow rate of 0.5 ml / min to determine the purity of bispecific product formation. CE-SDS (LabChip GXII (Perkin Elmer) was used to determine the quality of the bispecific product polypeptides under non-reducing (reformation of disulfide bridges) and reducing conditions (exact amount and presence of protein chains). Samples were prepared for CE-SDS as follows: 5 μl, c=0.1-1 mg / ml were combined with 35 μl 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 10-fold with HT Protein Express sample buffer (e.g., 100 μl reducing agent + 900 μl sample buffer). Afterwards, 70 μl of pure water was added to the sample and the sample plate was placed in the LAbChip system for analysis.

[0299] The results of 86 chain exchange reactions are shown in Table 4. Results from pairs of CH3 mutations with particularly high production yields are underlined and bolded. The total yield of bispecific product polypeptides after polypeptide chain exchange and purification steps is shown in mg. The production yield (%) is calculated as the relative amount of recombined product corrected to the maximum expected product mass. For this calculation, the amount of precursor polypeptides as analyzed by analytical size exclusion chromatography was corrected to the monomer content, since only monomers are expected to be effective for recombination. Furthermore, it was taken into account that the maximum expected production mass is limited by the less abundant precursor polypeptides.

[0300] (Table 4) Yields of bispecific product polypeptides after polypeptide chain exchange and purification from anti-bio and anti-fluo precursor polypeptides containing the indicated destabilizing mutations in the CH3 domain of the dummy chain. TIFF0007638900000029.tif174141TIFF0007638900000030.tif239141TIFF0007638900000031.tif246141

[0301] Example 4: Creation of monospecific precursor polypeptides to generate activatable binding sites upon polypeptide chain exchange This example is a proof-of-concept example for identifying destabilizing mutations to assess efficacy polypeptide chain exchange using a subset of destabilizing mutations identified in Examples 1-3, and evaluating activation of the antigen-binding site by polypeptide chain exchange via a cell-based T cell activation assay.

[0302] To assess the formation of bispecific anti-LeY / anti-CD3 antibodies from monospecific precursor polypeptides, monospecific precursor polypeptides were generated with domain arrangements as shown for the first and second heterodimeric precursor polypeptides shown in Figures 2 and 3.

[0303] Precursor polypeptide lacking the CH2 domain In a first set of experiments, a heterodimeric precursor polypeptide was provided having a domain sequence as shown in Figure 2. The precursor polypeptide lacked a CH2 domain and contained an antibody variable domain disposed N-terminal to the CH3 domain.

[0304] In a first alternative, the following precursor polypeptides were provided: - the first heterodimeric precursor polypeptide (also referred to as "anti-LeY-CD3(VH) knob precursor") contained a Fab fragment that specifically binds to LeY. The anti-LeY-CD3(VH) knob precursor contained 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") that contains a VH domain ("CD3(VH)") derived from an antibody that specifically binds to CD3, and a second heavy chain polypeptide according to SEQ ID NO: 13 (which represents a basic amino acid sequence without destabilizing mutations) that contained the destabilizing mutations and histidine tag shown below. The second heavy chain polypeptide (also referred to as "dummy-VL-hole" polypeptide) contained, from the N-terminus to the C-terminus, a hinge region, a VL domain derived from an antibody that specifically binds 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 that specifically binds to LeY. The anti-LeY-CD3(VL)-hole precursor contained a light chain polypeptide of SEQ ID NO: 11, i.e. a first heavy chain polypeptide of SEQ ID NO: 14 (also referred to as "LeY-CD3(VL)-holeHC") containing LeY LC, a VL domain ("CD3(VL)") derived from an antibody that specifically binds to CD3, and a second heavy chain polypeptide based on SEQ ID NO: 15 (representing a basic amino acid sequence without a destabilizing mutation) containing a histidine tag. The second heavy chain polypeptide (also referred to as "dummy-VH-knob" polypeptide) contained, from the N-terminus to the C-terminus, a hinge region, a VH domain derived from a non-binding antibody and a CH3 domain.

[0305] In a second option, the following precursor polypeptides were provided: - the first heterodimeric precursor polypeptide (also referred to as "anti-LeY-CD3(VL) knob precursor") contained a Fab fragment that specifically binds 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 (also referred to as "LeY-CD3(VL) knob HC") containing a CD3(VL) domain and a second heavy chain polypeptide based on SEQ ID NO: 17 (representing a basic amino acid sequence without destabilizing mutations) containing the destabilizing mutations and a histidine tag shown below. The second heavy chain polypeptide (also referred to as "dummy-VH-hole" polypeptide) contained, from the N-terminus to the C-terminus, a hinge region, a VH domain and a CH3 domain derived from a non-binding antibody. - the second heterodimeric precursor polypeptide (also referred to as "anti-LeY-CD3(VH)-hole precursor") contained a Fab fragment that specifically binds 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)-hole HC") containing a 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 a destabilizing mutation and a histidine tag as shown below. The second heavy chain polypeptide (also referred to as "dummy-VL-knob" polypeptide) contained, from the N-terminus to the C-terminus, a hinge region, a VL domain derived from an anti-dig antibody and a CH3 domain.

[0306] The polypeptide chain shown contains the following mutations:

[0307] Table 5 Amino acid substitutions in the CH3 domain of the precursor polypeptide TIFF0007638900000032.tif68163

[0308] Precursor Polypeptide Having an Fc Domain In a second set of experiments, a heterodimeric precursor polypeptide was provided having a domain sequence as shown in Figure 3. The precursor polypeptide contained a complete Fc domain and an antibody variable domain arranged N-terminal to the CH2 domain.

[0309] In a first alternative, the following precursor polypeptides were provided: - the first heterodimeric precursor polypeptide (also referred to as "anti-LeY-CD3(VH)-Fc(knob) precursor") contained a Fab fragment that specifically binds 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) containing the destabilizing mutations and histidine tag shown below. The second heavy chain polypeptide (also referred to as "dummy-VL-Fc(hole)" polypeptide) contained, from the N-terminus to the C-terminus, a hinge region, a VL domain ("dig") derived from an antibody that specifically binds to digoxigenin, 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 binds to LeY. The anti-LeY-CD3(VL)-Fc(hole) precursor contained a first heavy chain polypeptide of SEQ ID NO: 22 (also referred to as "LeY-CD3(VL)-Fc(hole)HC") containing LeY LC, CD3(VL) domains, and a second heavy chain polypeptide based on SEQ ID NO: 23 (representing a basic amino acid sequence without destabilizing mutations) with destabilizing mutations and a histidine tag as shown below. The second heavy chain polypeptide (also referred to as "dummy-VH-Fc(knob)" polypeptide) contained, from the N-terminus to the C-terminus, a hinge region, a VH domain from an anti-dig antibody, a CH2 domain and a CH3 domain.

[0310] In a second option, the following precursor polypeptides were provided: - the first heterodimeric precursor polypeptide (also referred to as "anti-LeY-CD3(VL)-Fc(knob) precursor") contained a Fab fragment that specifically binds to LeY. The anti-LeY-CD3(VL)-Fc(knob) precursor contained a first heavy chain polypeptide of SEQ ID NO: 24 (also referred to as "LeY-CD3(VL)-Fc(knob)HC") containing LeY LC, CD3(VL) domains and a second heavy chain polypeptide according to SEQ ID NO: 25 (representing a basic amino acid sequence without destabilizing mutations), containing the destabilizing mutations and histidine tag shown below. The second heavy chain polypeptide (also referred to as "dummy-VH-Fc(hole)" polypeptide) contained, from the N-terminus to the C-terminus, a hinge region, a VH domain 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 that specifically binds to LeY. The anti-LeY-CD3(VH)-Fc(hole) precursor contained a first heavy chain polypeptide of SEQ ID NO: 26 (also referred to as "LeY-CD3(VH)-Fc(hole)HC") containing 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 destabilizing mutations and a histidine tag as shown below. The second heavy chain polypeptide (also referred to as "dummy-VL-Fc(knob)" polypeptide) contained, 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.

[0311] The polypeptide chain shown contains the following mutations:

[0312] Table 6: Amino acid substitutions in the CH3 domain of the precursor polypeptide TIFF0007638900000033.tif68163

[0313] A heterodimeric precursor polypeptide was generated comprising the above dummy VL-hole polypeptide of SEQ ID NO: 13 and the above dummy VH-hole polypeptide of SEQ ID NO: 17, having the amino acid sequence of each of the above dummy polypeptides, and making one of the following amino acid substitutions: E357K, A368F, D399A, F405W, S364L, Y407W, or S354V.

[0314] A heterodimeric precursor polypeptide was generated comprising a dummy VH-knob polypeptide of SEQ ID NO: 15 and a dummy VL-knob polypeptide of SEQ ID NO: 19, each having the amino acid sequence of the respective dummy polypeptides described above, and one of the following amino acid substitutions was made: K370E, no destabilizing mutation, W366I K409D, V397Y or K392D.

[0315] A heterodimeric precursor polypeptide was generated comprising the above dummy-VL-Fc(hole) polypeptide of SEQ ID NO:21 and the above dummy-VH-Fc(hole) polypeptide of SEQ ID NO:25, having the amino acid sequence of each of the above dummy polypeptides, and making one of the following amino acid substitutions: E357K, A368F, D399A, F405W, S364L, D356K, or S354V.

[0316] A heterodimeric precursor polypeptide was generated comprising a dummy-VH-Fc(knob) polypeptide of SEQ ID NO:23 and a dummy-VL-Fc(knob) polypeptide of SEQ ID NO:27, as described above, having the amino acid sequence of each of the dummy polypeptides described above, and one of the following amino acid substitutions was made: K370E, no destabilizing mutation, W366I K409D, V397Y, K392D or K370E K439E.

[0317] Recombinant production of precursor polypeptides Expression was achieved by co-transfection of the three polypeptide chain plasmids of each precursor polypeptide according to the state of the art into mammalian cells (eg, HEK293 or Expi293F™).

[0318] For the expression of the precursor polypeptides described above, a transcription unit was used that contained the following functional elements: - the immediate early enhancer and promoter from human cytomegalovirus (P-CMV) including intron A, - a human heavy chain immunoglobulin 5' untranslated region (5'UTR), - mouse immunoglobulin heavy chain signal sequence, - a nucleic acid encoding each of the precursor polypeptides, and - a 3' untranslated region carrying a polyadenylation signal sequence.

[0319] In addition to the expression unit / cassette containing the desired gene to be expressed, a basic / standard mammalian expression plasmid is - an origin of replication allowing replication of this plasmid in E. coli, and - The beta-lactamase gene that confers ampicillin resistance to E. coli Includes.

[0320] Coding expression cassettes containing precursor polypeptide chains were generated by PCR and / or gene synthesis and assembled by known recombinant methods and techniques, e.g., by ligating the nucleic acid segments using unique restriction sites in each plasmid. Subcloned nucleic acid sequences were confirmed by DNA sequencing. For transient transfection, larger quantities of plasmid were prepared by plasmid preparation from transformed E. coli cultures (HiSpeed ​​Plasmid Maxi Kit, Qiagen).

[0321] Standard cell culture techniques were used as described in Current Protocols in Cell Biology (2000), Bonifacino, JS, Dasso, M., Harford, JB, Lippincott-Schwartz, J. and Yamada, KM (eds.), John Wiley & Sons, Inc.

[0322] Precursor polypeptide derivatives were produced by transient transfection with the respective plasmids using HEK293-F system (Invitrogen) or Expi293F™ system (Live Technologies) according to the manufacturer's instructions. Briefly, HEK293 F cells (Invitrogen) or Expi293F™ cells (Live Technologies) growing in suspension in either shake flasks or stirred fermenters in serum-free FreeStyle™ 293 expression medium (Invitrogen) or Expi293F™ expression medium (Life Technologies) were transfected with the respective expression plasmids and 293 fectin™, fectin (Invitrogen) or PEIpro (Polyplus) or the reagent mixture ExpiFectamine™ 293 Transfection Kit (Life Technologies). For 1-2 L shake flasks (Corning), HEK293-F cells or Expi293F™ cells were cultured at 1-1.3*10 in 250-600 mL. 6 The cells were seeded at a density of approximately 1.5 × 10 cells / mL and incubated at 120 rpm and 8% CO2. The day after the cells were transfected with the appropriate expression plasmids, HEK293-F cells were seeded at a density of approximately 1.5 × 10 6 At a cell density of 10 ... 6Cells were transfected at a cell density of 10000 cells / ml. Prior to transfection, 30 μg of plasmid DNA was diluted in pre-warmed (water bath, 37°C) opti-MEM (Gibco) to a final volume of 1.5 ml. The solution was mixed gently and incubated at room temperature for no more than 5 minutes. Then, 1.5 ml of a pre-incubated solution of ExpiFectamine™ reagent in opti-MEM was added to the DNA-optiMEM solution. The resulting solution was mixed gently and incubated at room temperature for 20-30 minutes. The entire volume of the mixture was added to a 100 ml shake flask containing 30 ml of Expi293F™ culture. The culture was incubated at 37°C, 7% Co2, 85% humidity, and 110 rpm for 7 days. For Expi293F™ cultures, 20 μl of ExpiFectamine™ Enhancer 1 and 200 μl of ExpiFectamine™ Enhancer 2 were added to 30 ml of cell culture 15-24 hours after transfection. Glucose solutions were added during the course of the fermentation according to glucose consumption. Correctly assembled split cytokine molecules were secreted into the culture supernatant like standard IgG. Supernatants containing the secreted antibodies were harvested after 5-10 days and antibodies were either purified directly from the supernatants or the supernatants were frozen and stored at -20°C.

[0323] Precursor polypeptides with the entire Fc region (CH2-CH3) bind to Protein A. These precursors were purified by Protein A chromatography followed by SEC.

[0324] The precursor polypeptides that lack CH2 domain contained kappa light chain. Therefore, these precursors were purified by applying standard kappa light chain affinity chromatography. The precursor polypeptides were purified from cell culture supernatant by affinity chromatography using KappaSelect (GE Healthcare, Sweden) and Superdex 200 size exclusion (GE Healthcare, Sweden) chromatography or ion exchange chromatography.

[0325] Briefly, sterile filtered cell culture supernatant was captured on KappaSelect resin equilibrated with PBS buffer (10 mM Na2HPo4, 1 mM KH2Po4, 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. Eluted precursor polypeptides were pooled and neutralized with 2 M Tris, pH 9.0. Precursor polypeptide pools were further purified by size exclusion or ion exchange chromatography. For size exclusion chromatography, a Superdex™ 200 pg HiLoad™ 16 / 600 (GE Healthcare, Sweden) column equilibrated with 20 mM histidine, 140 mM NaCl, pH 6.0 was used. For ion-exchange chromatography, the protein sample obtained from KappaSelect purification was diluted 1:10 in 20 mM histidine, pH 6.0 and loaded onto a HiTrap™ SP HP ion-exchange (GE Healthcare, Sweden) column equilibrated with buffer A (20 mM histidine, pH 6.0). A gradient of 0 to 100% buffer B (20 mM histidine, 1 M NaCl, pH 6.0) was applied to elute the different protein species.

[0326] Purity and integrity were analyzed after purification by SDS-PAGE. Protein solution (13 μl) was mixed with 5 μl of 4× NuPAGE LDS sample buffer (Invitrogen) and 2 μl of 10× NuPAGE sample reducing agent (Invitrogen) and heated to 95 °C for 5 min. Samples were loaded onto NuPAGE 4-12% Bis-Tris gels (Invitrogen) and run using Novex Mini-Cell (Invitrogen) and NuPAGE MES SDS running buffer (Life Technologies) according to the manufacturer's instructions. Gels were stained using InstantBlue™ Coomassie protein stain. Furthermore, protein integrity and homogeneity were analyzed using analytical size-exclusion chromatography.

[0327] (CE-)SDS-PAGE revealed that all expected polypeptide chains were present in the preparation; analytical size exclusion confirmed the purity of the preparation to be greater than 90%. For a review of methods for assessing antibody purity, see, e.g., Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0328] 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 generated in Example 4 as a proof-of-concept experiment. The structures of the expected 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. Polypeptide chain exchange leads to 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.

[0329] The effect of different CH3 interface mutations on the efficacy of this chain exchange reaction was evaluated in a cell-based reporter assay system consisting of LeY-expressing MCF7 cells and Jurkat reporter cell line (Promega J1621) according to the following principle: binding of the first and second heterodimeric polypeptides to MCF7 cells and polypeptide chain exchange leads to 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, which leads to luciferase expression from Jurkat cells. Luminescence was detected after addition of BioGlo substrate.

[0330] Briefly, cell-based assays were performed in 384-well plates as follows: RPMI1640 containing 10% FCS was used as the assay medium. 6×10 4 Jurkat effector cells were cultured at 2 x 10 in a total volume of 10 µl. 4MCF7 cells. Precursor polypeptides were applied alone or in combination at 200 nM and 2 nM in a final volume of 30 μl. Cells were incubated for 20 hours in cell culture conditions. 24 μl of Bioglo was added to each well and incubated for 5 minutes. Luminescence was measured in an Infinite® 200 PRo reader (TECAN).

[0331] (Table 7) Formation of bispecific product polypeptides by polypeptide chain reaction from a precursor polypeptide lacking the CH2 domain defined above in Example 3, containing the indicated destabilizing mutations in the CH3 domain of the dummy chain. Results are shown from an exchange reaction at a precursor polypeptide concentration of 200 nM. Luminescence efficiency is evaluated as follows: <10%..."-", 10-29%..."+", 30-50%..."++", >50%..."+++"). TIFF0007638900000034.tif148147

[0332] (Table 8) Formation of bispecific product polypeptides by polypeptide chain reaction from a precursor polypeptide lacking the CH2 domain defined above in Example 3, containing the indicated destabilizing mutations 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 evaluated as follows: <2%..."-", 2-4%..."+", 5-10%..."++", >10%..."+++"). TIFF0007638900000035.tif148147

[0333] (Table 9) Formation of bispecific product polypeptides by polypeptide chain reaction from precursor polypeptides having Fc domains as defined above in Example 3, including the destabilizing mutations shown 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 evaluated as follows: <10%... "-", 10-19%... "+", 20-50%... "++", >50%... "+++"). TIFF0007638900000036.tif78147

[0334] Example 5: Combined assessment of polypeptide chain exchange in solution and on cells as measured by a T cell activation assay In therapeutic applications, it is desirable to reduce undesirable off-target effects. Therefore, heterodimeric precursor polypeptides are therapeutically applied as prodrugs to form therapeutically active product polypeptides upon polypeptide chain exchange. Polypeptide chain exchange preferably occurs largely only after the precursor polypeptide binds to target cells, while spontaneous polypeptide chain exchange during circulation is desirably absent or only to a minor extent. Thus, precursor polypeptides that exhibit mild or low polypeptide chain exchange in solution while undergoing polypeptide chain exchange to activate antigen binding sites in target cells are particularly desirable for therapeutic applications. Thus, the results of Example 2 (polypeptide chain exchange in solution) and Example 4 (cell polypeptide chain exchange) are aligned.

[0335] Table 10. Formation of bispecific product polypeptides by polypeptide chain reaction from precursor polypeptides containing the indicated destabilizing mutations in the CH3 domain of the dummy chain. Columns indicate destabilizing mutations in the dummy knob polypeptide and lines indicate destabilizing mutations in the dummy hole polypeptide. Polypeptide chain exchange for each pair of destabilizing mutations in solution ("IS" as detected in Example 2) and cells ("oC" as detected in Example 4 for polypeptides lacking the CH2 domain) is shown. Polypeptide chain exchange efficacy is assessed as follows: low..."-", mild..."+", moderate..."++", high..."+++"). TIFF0007638900000037.tif147128

[0336] Table 11. Formation of bispecific product polypeptides by polypeptide chain reaction from precursor polypeptides containing the indicated destabilizing mutations in the CH3 domain of the dummy chain. Columns indicate destabilizing mutations in the dummy knob polypeptide and lines indicate destabilizing mutations in the dummy hole polypeptide. Polypeptide chain exchange for each pair of destabilizing mutations in solution ("IS" as detected in Example 2) and cells ("OC" as detected in Example 4 for polypeptides with Fc domains) is shown. Polypeptide chain exchange efficacy is assessed as follows: low..."-", mild..."+", moderate..."++", high..."+++"). TIFF0007638900000038.tif147132

[0337] Precursor polypeptides capable of mediating cellular activation of antigen binding sites and exhibiting low to mild polypeptide chain exchange in solution are believed to be particularly suitable for therapeutic applications.

[0338] Thus, from the different precursor polypeptides tested, precursor polypeptides containing the following pairs of destabilizing mutations are considered promising for use in the CH3 domain of heterodimeric precursor polypeptides for therapeutic applications.

[0339] Table 12. Destabilizing mutations in the CH3 domain with hole mutations and the CH3 domain with knob mutations of the heterodimeric precursor polypeptide of the present invention TIFF0007638900000039.tif74131

[0340] Among the above destabilizing mutation pairs deemed promising for use in the CH3 domain of heterodimeric precursor polypeptides for therapeutic applications, precursor polypeptides having the following destabilizing mutation pairs exhibit low to mild in solution polypeptide chain exchange, but highly mediated in cellular polypeptide chain exchange, as detected via T cell activation assays.

[0341] Table 13. Destabilizing mutations in the CH3 domain with hole mutations and the CH3 domain with knob mutations of the heterodimeric precursor polypeptide of the present invention TIFF0007638900000040.tif44131

[0342] Example 6: Generation of monospecific precursor polypeptides of the invention that bind FcRn to generate activatable binding sites upon polypeptide chain exchange To assess the formation of bispecific anti-LeY / anti-CD3 antibodies from monospecific precursor polypeptides, monospecific precursor polypeptides of domain sequences as shown for the first and second heterodimeric precursor polypeptides shown in FIG. 1 were generated.

[0343] The following precursor polypeptides were provided: - the first heterodimer precursor polypeptide (also referred to as "anti-LeY-CD3(VH) knob precursor") contained a Fab fragment that specifically binds to LeY. The anti-LeY-CD3(VH) knob precursor contained 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") that contains a VH domain ("CD3(VH)") from an antibody that specifically binds to CD3, and a second heavy chain polypeptide based on SEQ ID NO: 28 with a destabilizing mutation E357K and a histidine tag. The second heavy chain polypeptide (also referred to as "dummy hole" polypeptide) contained, from the N-terminus to the C-terminus, a mutated hinge region without cysteine, a CH2 domain and a CH3 domain. - the second heterodimeric precursor polypeptide (also referred to as "anti-LeY-CD3(VL)-hole precursor") contained a Fab fragment that specifically binds to LeY. The anti-LeY-CD3(VL)-hole precursor contained a light chain polypeptide of SEQ ID NO: 11, i.e. a first heavy chain polypeptide of SEQ ID NO: 14 (also referred to as "LeY-CD3(VL)-holeHC") that contains a VL domain ("CD3(VL)") derived from an antibody that specifically binds to CD3, and a second heavy chain polypeptide based on SEQ ID NO: 29 with a destabilizing mutation K370E and a histidine tag. The second heavy chain polypeptide (also referred to as "dummy knob" polypeptide) contained, from the N-terminus to the C-terminus, a mutated hinge region without cysteine, a CH2 domain and a CH3 domain.

[0344] The polypeptide chain shown contains the following mutations:

[0345] Table 14: Amino acid substitutions in the CH3 domain of the precursor polypeptide TIFF0007638900000041.tif30168

[0346] Recombinant production of precursor polypeptides Expression was performed by co-transfection of the three polypeptide chain plasmids of each precursor polypeptide into mammalian cells (e.g., HEK293 or Expi293F™) by state of the art techniques as described above in Example 1 (for dummy-hole and dummy-knob) and Example 4 (for LeY-CD3(VH)-knob HC and LeY-CD3(VL)-hole HC).

[0347] Purity and integrity were analyzed after purification by SDS-PAGE as described in Example 4 (Figure 5).

[0348] The precursor polypeptide was purified using Protein A chromatography followed by SEC as described in Example 4 (Figures 6 and 7).

[0349] FcRn binding was assessed by analytical FcRn affinity chromatography as described in Schlothauer T et al. (Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies. MAbs. 2013 Jul-Aug;5(4):576-86) (Figure 8). The results show that the heterodimeric precursor polypeptide bound to FcRn, whereas the product polypeptide, which contains an activating antigen binding site that specifically binds to CD3 but not to the CH2 domain, did not bind to FcRn.

[0350] To evaluate T cell activation mediated by bispecific anti-LeY / anti-CD3 antibodies formed from monospecific precursor polypeptides as provided in this example, monospecific precursor polypeptides that specifically bind to LeY were analyzed in a T cell activation assay as described in Example 5 (Figure 9). The results show that product polypeptides containing an activating antigen binding site that specifically binds to CD3 were able to activate T cells. As a comparison, precursor polypeptides that are not capable of activating T cells were analyzed individually.

[0351] Example 7: Generation of further 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 domain sequences as 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.

[0352] The first heterodimeric precursor polypeptide (also referred to as "anti-fluo precursor") comprised a Fab fragment that specifically bound the biotin derivative fluorescein ("fluo") and had 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: 31 (also referred to as "fluo HC") and a second heavy chain polypeptide based on SEQ ID NO: 05 (representing a basic amino acid sequence without destabilizing mutations) with destabilizing mutations and a C-tag as shown below. The second heavy chain polypeptide (also referred to as "dummy hole" polypeptide) comprised, from the N-terminus to the C-terminus, a hinge region, a CH2 domain and a CH3 domain.

[0353] The second heterodimeric precursor polypeptide (also referred to as "anti-bio precursor") comprised a Fab fragment that specifically binds 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: 30 (also referred to as "bio HC"), and a second heavy chain polypeptide based on SEQ ID NO: 10 (representing a basic amino acid sequence without destabilizing mutations) with a C-tag and destabilizing mutations as shown below. The second heavy chain polypeptide (also referred to as "dummy knob" polypeptide) comprised, from the N-terminus to the C-terminus, a hinge region, a CH2 domain, and a CH3 domain.

[0354] The first and second heterodimeric precursor polypeptides were produced according to the methods disclosed in Example 1.

[0355] The CH3 domain of the polypeptide chain shown contains the following mutations:

[0356] Table 15. Purification yield and monomer content of anti-fluoro precursor polypeptides with dummy hole chains with the indicated destabilizing mutations in the CH3 domain (purification yield [mg / ml] = amount of purified antibody per liter of expression volume, corrected for % monomer peak; monomer = desired heterodimeric precursor polypeptide). TIFF0007638900000042.tif105128

[0357] Table 16. Purification yield and monomer content of anti-bio precursor polypeptides with dummy knob chains with the indicated destabilizing mutations in the CH3 domain (purification yield [mg / ml] = amount of purified antibody per liter of expression volume, corrected for % monomer peak, monomer = desired heterodimeric precursor polypeptide). TIFF0007638900000043.tif105128

[0358] Example 8: Analysis of the polypeptide chain exchange efficiency of the precursor polypeptides from Example 7 To assess the effect of different destabilizing mutations on polypeptide chain exchange, exchange reactions between the precursor polypeptides produced in Example 7 were carried out. The experiments were carried out according to the methods described in Example 2. The structures of the predicted product polypeptides are shown in FIG.

[0359] Table 17. Formation of bispecific product polypeptides by polypeptide chain exchange reaction from anti-bio and anti-fluo precursor polypeptides containing the indicated destabilizing mutations 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 by production yield [%]. Experimentally obtained yields relate to the maximum possible yield of bispecific antibodies. The maximum possible yield of bispecific antibodies is corrected by the lowest % monomer peak SEC of the two respective input formats in each reaction, since only monomers are expected to be effective for recombination. TIFF0007638900000044.tif169143TIFF0007638900000045.tif239143TIFF0007638900000046.tif148143

[0360] Example 9: 2. Production of further monospecific precursor polypeptides comprising an intact Fc domain, the CH3 domain of the precursor polypeptide comprising knob-into-hole mutations but no cysteine ​​mutations. To assess the formation of bispecific anti-biocytinamide / anti-fluorescein antibodies from monospecific precursor polypeptides, monospecific precursor polypeptides were generated with domain sequences as 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.

[0361] The first and second heterodimeric precursor polypeptides described in Example 7 were produced according to the structures and methods disclosed therein.

[0362] Additionally, deviating from Example 1, bio HC is based on SEQ ID NO: 30 but has a serine residue at position 354, and fluo HC is based on SEQ ID NO: 31 but has a tyrosine residue at position 349. Thus, the mutations in the CH3 domain are summarized as follows:

[0363] Table 18: Amino acid substitutions in the CH3 domain of the precursor polypeptide TIFF0007638900000047.tif30167

[0364] The CH3 domain of the polypeptide chain shown contains the following mutations:

[0365] Table 19. Purification yield and monomer content of anti-bio precursor polypeptides with dummy knob chains with the indicated destabilizing mutations in the CH3 domain (purification yield [mg / ml] = amount of purified antibody per liter of expression volume, corrected for % monomer peak; monomer = desired heterodimeric precursor polypeptide). TIFF0007638900000048.tif105128

[0366] Table 20. Purification yield and monomer content of anti-fluoro precursor polypeptides with dummy hole chains with the indicated destabilizing mutations in the CH3 domain (purification yield [mg / ml] = amount of purified antibody per liter of expression volume, corrected for % monomer peak; monomer = desired heterodimeric precursor polypeptide). TIFF0007638900000049.tif112128

[0367] Example 10: Analysis of the polypeptide chain exchange efficiency of the precursor polypeptides from Example 9 To assess the effect of different destabilizing mutations on polypeptide chain exchange, exchange reactions were carried out between the precursor polypeptides produced in Example 9. The experiments were carried out according to the methods described in Example 2.

[0368] Table 21. Formation of bispecific product polypeptides by polypeptide chain exchange reaction from anti-bio and anti-fluo precursor polypeptides containing the indicated destabilizing mutations 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 by production yield [%]. Experimentally obtained yields relate to the maximum possible yield of bispecific antibodies. The maximum possible yield of bispecific antibodies is corrected by the lowest % monomer peak SEC of the two respective input formats in each reaction, since only monomers are expected to be effective for recombination. TIFF0007638900000050.tif150141TIFF0007638900000051.tif233141TIFF0007638900000052.tif84141

[0369] The results demonstrate that polypeptide chain exchange is detectable for heterodimeric precursor polypeptides, in which the knobs-into-holes mutations are not stabilized by additional cysteine ​​mutations.

[0370] Example 11: 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, monospecific precursor polypeptides were generated with domain sequences as 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.

[0371] The first and second heterodimeric precursor polypeptides described in Example 7 were produced according to the structures and methods disclosed therein, with the following differences.

[0372] Unlike Example 7, three precursor polypeptides were generated that specifically bind fluorescein, where the fluo HC was based on SEQ ID NO:31 and the dummy-hole polypeptide was based on SEQ ID NO:05 with the following CH3 mutations: TIFF0007638900000053.tif80165

[0373] - Deviating from Example 7, three precursor polypeptides were generated that specifically bind biocytinamide, where the bio HC is based on SEQ ID NO: 30 and the dummy-knob polypeptide has the following CH3 mutations and is based on SEQ ID NO: 10: TIFF0007638900000054.tif92162

[0374] Table 22: Purification yields and monomer content of the indicated precursor polypeptides (purification yield [mg / ml] = amount of purified antibody per liter of expression volume, corrected for % monomer peak; monomer = desired heterodimeric precursor polypeptide). TIFF0007638900000055.tif53128

[0375] Example 12: Analysis of the polypeptide chain exchange efficiency of the precursor polypeptide from Example 11 To assess the effect of different destabilizing mutations on polypeptide chain exchange, exchange reactions were carried out between the precursor polypeptides produced in Example 11. The experiments were carried out according to the methods described in Example 2.

[0376] Table 23: Formation of bispecific product polypeptides by polypeptide chain exchange reaction from the indicated anti-bio and anti-fluo precursor polypeptides. Values ​​indicate exchange efficiency by production yield [%]. Experimentally obtained yields relate to the maximum possible yield of bispecific antibodies. The maximum possible yield of bispecific antibodies is corrected by the lowest % monomer peak SEC of the two respective input formats in each reaction, since only monomers are expected to be available for recombination. TIFF0007638900000056.tif29128

[0377] The results show that the polypeptide chains arise independent of the placement of the cysteine ​​mutations in either the dummy polypeptide or the polypeptide chain containing the antigen-binding portion.

Claims

1. A set of heterodimeric precursor polypeptides comprising: a) a first heterodimeric precursor polypeptide, - a first heavy chain polypeptide comprising, from N-terminal to C-terminal direction, an antibody variable domain selected from a VH domain and a VL domain and a CH3 domain, the first heavy chain polypeptide comprising at least a portion of a first antigen-binding site; and a second heavy chain polypeptide comprising, from N-terminal to C-terminal, a CH2 domain and a CH3 domain Including, a first heterodimeric precursor polypeptide, wherein the first heavy chain polypeptide and the second heavy chain polypeptide 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; b) a second heterodimeric precursor polypeptide, - a third heavy chain polypeptide comprising, in N-terminal to C-terminal direction, an antibody variable domain selected from a VH domain and a VL domain and a CH3 domain, said antibody variable domain being capable of forming an antigen-binding site that specifically binds to a target antigen together with an antibody variable domain comprised in a first heavy chain polypeptide of said first heterodimeric precursor polypeptide, said third heavy chain polypeptide comprising at least a portion of a second antigen-binding site; and a fourth heavy chain polypeptide comprising, from N-terminal to C-terminal, a CH2 domain and a CH3 domain Including, a second heterodimeric precursor polypeptide, wherein the third heavy chain polypeptide and the fourth heavy chain polypeptide 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; Where: A) i) the first heavy chain polypeptide comprises a CH3 domain having the knob mutation and the third heavy chain polypeptide comprises a CH3 domain having the hole mutation; or ii) the first heavy chain polypeptide comprises a CH3 domain having the hole mutation and the third heavy chain polypeptide comprises a CH3 domain having the knob mutation; and B) i) the CH3 domain of the first heterodimeric precursor polypeptide comprising the knob mutation and the CH3 domain of the second heterodimeric precursor polypeptide comprising the hole mutation; or ii) the CH3 domain of the first heterodimeric precursor polypeptide comprising the hole mutation and the CH3 domain of the second heterodimeric precursor polypeptide comprising the knob mutation. any of the following amino acid substitutions selected from one or more of the following amino acid substitutions, wherein the numbering is according to the Kabat numbering system: - said CH3 domain carrying said hole mutation, substitution of E357 with a positively charged amino acid, substitution of S364 by a hydrophobic amino acid, substitution of A368 with a hydrophobic amino acid, and Substitution of V407 with a hydrophobic amino acid and comprising at least one amino acid substitution selected from the group consisting of - said CH3 domain carrying said knob mutation substitution of K370 with a negatively charged amino acid, substitution of K370 by a negatively charged amino acid and substitution of K439 by a negatively charged amino acid, substitution of K392 with a negatively charged amino acid, and Substitution of V397 with a hydrophobic amino acid The amino acid sequence includes at least one amino acid substitution selected from the group consisting of:

2. The set of heterodimeric precursor polypeptides described in claim 1, wherein the first antigen-binding site and / or the second antigen-binding site are antibody fragments.

3. a) the first heterodimeric precursor polypeptide comprises: - a further antibody variable domain (first antibody variable domain) within said first heavy chain polypeptide comprising a CH3 domain, and a further polypeptide chain which is a light chain polypeptide comprising a second antibody variable domain wherein said first antibody variable domain and said second antibody variable domain together form a first antigen-binding site that specifically binds to a target antigen; and b) the second heterodimeric precursor polypeptide comprises: - a further antibody variable domain (a third antibody variable domain) within said third heavy chain polypeptide comprising a CH3 domain, and a further polypeptide chain which is a light chain polypeptide comprising a fourth antibody variable domain wherein the third antibody variable domain and the fourth antibody variable domain together form a second antigen-binding site that specifically binds to a target antigen. A set of heterodimeric precursor polypeptides according to claim 1 or 2.

4. In the first heterodimeric polypeptide, no interchain disulfide bond is formed between the two polypeptide chains comprising the CH3 domain, and in the second heterodimeric polypeptide, no interchain disulfide bond is formed between the two polypeptide chains comprising the CH3 domain. A set of heterodimeric precursor polypeptides according to any one of claims 1 to 3.

5. The set of heterodimeric precursor polypeptides according to any one of claims 1 to 4, wherein the antigen-binding site of the first heterodimeric precursor polypeptide and the antigen-binding site of the second heterodimeric precursor polypeptide bind to the same antigen.

6. The set of heterodimeric precursor polypeptides according to any one of claims 1 to 5, wherein the antibody variable domains contained in the first heavy chain polypeptide and the third heavy chain polypeptide are capable of forming an antigen-binding site that specifically binds to CD3.

7. 7. A method for making a heterodimeric precursor polypeptide comprising contacting a first heterodimeric precursor polypeptide of any one of claims 1 to 6 with a second heterodimeric precursor polypeptide to form a third heterodimeric polypeptide comprising the first heavy chain polypeptide and the third heavy chain polypeptide.

8. 8. The method of claim 7, comprising contacting the first heterodimeric precursor polypeptide with the second heterodimeric precursor polypeptide to form a fourth heterodimeric polypeptide comprising the second heavy chain polypeptide and the fourth heavy chain polypeptide.

9. 9. The method of claim 7 or 8, wherein in the first heterodimeric precursor polypeptide, no interchain disulfide bond is formed between the two polypeptide chains comprising the CH3 domain, and in the second heterodimeric precursor polypeptide, no interchain disulfide bond is formed between the two polypeptide chains comprising the CH3 domain, and the contacting is performed in the absence of a reducing agent.

10. A set of heterodimeric precursor polypeptides according to any one of claims 1 to 6 for use as a medicament.

11. A pharmaceutical composition comprising a set of heterodimeric precursor polypeptides according to any one of claims 1 to 6 and a pharma- ceutically acceptable carrier.

12. The set of heterodimeric precursor polypeptides according to any one of claims 1 to 6, wherein in the first and second heterodimeric precursor polypeptides, the antibody variable domains comprised in the first and third heavy chain polypeptides are capable of forming an antigen binding site that specifically binds to CD3 for use in the treatment of cancer.

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